# BactoBox®
BactoBox® is a benchtop instrument that gives direct cell counts in minutes. Use BactoBox® to optimize your starter culture, track growth, or find the best harvest time.
Get an answer right away from our help center AI agent
Cover image
:graduation-cap:BactoBox® first steps
We'll get you up and running in no time! See our get getting started section.
## Step by step
{% stepper %}
{% step %}
#### Open the packaging
Use the right tool to open the cardboard boxes. You may have multiple boxes.
{% endstep %}
{% step %}
#### Retrieve items included in the packaging
Make sure that you find all of these items in the packaging:
1 × BactoBox®
1 × power supply
1 × flow cell
1 × USB cable
1 × vial rack
1 × tubing kit 1 × external filter
2 × dilution vial
1 × disinfection vial
1 × QC stock
{% hint style="warning" %}
The supplied items are designed only for BactoBox® and may not be compatible with other devices.
{% endhint %}
{% endstep %}
{% step %}
#### Check included items
Check for missing or damaged items. If something is not right, contact support at .
{% endstep %}
{% step %}
#### Retrieve items NOT included in the packaging
These items are required for the [installation](/getting-started/installation) and [qualification](/getting-started/qualification). Find them before you proceed or you will get stuck later.
1 × P200 pipette
1 × vortex mixer
{% endstep %}
{% step %}
#### Remove display protection
Remove the glass protection film from the display of your BactoBox®.
{% endstep %}
{% step %}
#### That is it!
Now that you have all the required items at hand, let's [install](/getting-started/installation) your BactoBox®.
{% endstep %}
{% endstepper %}
# Installation
Power on your BactoBox®, install the tubing kit, and install the flow cell.
# Power on
Assemble power supply, insert power cable, and flip the rocker switch
## Step by step
{% stepper %}
{% step %}
#### Assemble power supply
Slide the socket adapter onto the power supply until you hear a distinct click. Plug the power supply into a wall socket.
{% endstep %}
{% step %}
#### Insert power cable
*Firmly* insert the round plug into the power port on the right side of BactoBox®.
{% hint style="info" %}
Ensure it is fully inserted to avoid unexpected restarts.
{% endhint %}
{% endstep %}
{% step %}
#### Flip the rocker switch
Flip the rocker switch to the *On* position. The green LED next to the rocker switch lights up immediately. Wait 2–3 min for your BactoBox® to start.
{% endstep %}
{% step %}
#### That is it!
You should now see the home screen.
{% hint style="info" %}
You may see messages on the home screen. For example: “Insert flow cell”, “Clean the device”, or “Perform a QC test”. This is normal.
{% endhint %}
{% endstep %}
{% endstepper %}
# Install tubing kit
Connect tubing kit to BactoBox®, remember to use an external filter, and place the vial in the vial rack.
## Get things ready
You need these items:
* 1 × [BactoBox®](/item-register/bactobox-r/bactobox-r)
* 1 × [tubing kit](/item-register/accessories/tubing-kit)
* 1 × [external filter](/item-register/consumables/external-filter)
## Step by step
{% stepper %}
{% step %}
#### Connect tubing kit to BactoBox®
Use the color-coded connectors as a guide. Connect the white inlet to the white panel mount on your BactoBox®. Connect the black outlet to the black panel mount.
{% hint style="success" %}
## Always use an external filter
Always place an [external filter](https://github.com/sbtinstruments/docsites/tree/main/items/consumables/external-filter.md) between BactoBox® and the [tubing kit](https://github.com/sbtinstruments/docsites/tree/main/items/accessories/tubing-kit.md). Connect the external filter to the inlet (white connector).
The external filter protects your BactoBox® setup against clogging.
{% endhint %}
{% endstep %}
{% step %}
#### Place the vial in the vial rack
Tilt the [vial rack](/item-register/accessories/vial-rack) at a 45° angle to make it easier to transfer the tubing kit between vials.
{% endstep %}
{% step %}
#### That is it!
Now the tubing kit installation is done.
{% endstep %}
{% endstepper %}
# Install flow cell
Remove flow cell cover, place the flow cell, press the flow cell into place, insert the flow cell cover.
## Get things ready
You need these items:
* 1 × [BactoBox®](/item-register/bactobox-r/bactobox-r)
* 1 × [flow cell](/item-register/consumables/flow-cell)
## Step by step
{% stepper %}
{% step %}
#### Remove flow cell cover
Remove the [flow cell cover](/item-register/accessories/flow-cell-cover) (the grey lid). This reveals the flow cell compartment underneath.
{% endstep %}
{% step %}
#### Remove existing flow cell (optional)
If a flow cell is already installed, remove it. Press the clamps together. Lift the flow cell out.
{% hint style="success" %}
[Clean](/maintenance/clean-the-setup) the flow cell before you remove it.
{% endhint %}
{% endstep %}
{% step %}
#### Place the flow cell
Place the flow cell loosely onto the metal bracket.
{% endstep %}
{% step %}
#### Press flow cell into place
First, press *firmly* on the top section labeled (1) until you hear a distinct click.
{% hint style="warning" %}
Make sure to press the cartridge itself and not the clamp.
{% endhint %}
Second, press *firmly* on the lower section labeled (2) until you hear a second click.
{% hint style="success" %}
**Ensure that everything works**
* [ ] The user interface no longer displays the "Insert flow cell" animation.
* [ ] The flow cell has a slight springy feel when you press the center section.
{% endhint %}
{% endstep %}
{% step %}
#### Insert flow cell cover
Insert the flow cell cover (grey lid) on top of the flow cell compartment.
{% hint style="info" %}
The flow cell cover protects the flow cell compartment against dust and liquid.
{% endhint %}
{% endstep %}
{% step %}
#### That is it!
Flow cell installation is done.
{% hint style="success" %}
### Replace the external filter when you replace the flow cell
It is good practice to replace the [external filter](https://github.com/sbtinstruments/docsites/tree/main/items/consumables/external-filter.md) whenever you replace the flow cell. See [Install tubing kit](https://help.sbtinstruments.com/getting-started/installation/install-tubing-kit) for details.
**Run a** [QC test](/getting-started/qc-test) **when you replace the external filter.**
{% endhint %}
{% hint style="info" %}
### The flow cell is worn out after 250 measurements
After 250 measurements, the flow cell reaches its usage limit and is automatically locked. This limit is set to guarantee the factory calibration of the flow cell.
{% endhint %}
{% endstep %}
{% endstepper %}
# Qualification
Run a QC test. Use only SBT dilution vials (bundled as DV01).
Cover image
:vial-circle-check:Make QC vial
Always use SBT dilution vials (bundled as DV01) when you qualify.
{% hint style="success" %}
### Always use SBT dilution vials (bundled as `DV01`) when you qualify
This is different from a regular (weekly) QC test where the choice of dilution vial depends on your use case.
SBT dilution vials maintain a high quality standard, as these vials undergo rigorous quality checks.
{% endhint %}
## When to qualify
[Qualify](/getting-started/qualification) your BactoBox® in these cases:
* **At least once a year.**
* When you relocate your BactoBox® setup.\ Example: You install your BactoBox® for the very first time.\ Example: You ship your BactoBox® to another physical location.\ Example: You get your BactoBox® back from service/repair at SBT.
See [Maintenance](/maintenance/maintenance) for more information.
# QC test
Make a QC vial and run the QC test program.
## When to perform a QC test
Perform a [QC test](/getting-started/qc-test) in these cases:
* **At least once a week.**
* After a [deep clean](/maintenance/deep-clean-1) (of any length).
* When you change your BactoBox® setup.\ Example: You replace the flow cell.\ Example: You replace the external filter.\ Example: You replace the tubing kit.
* When you switch from the SBT dilution vial to the [DIY dilution vial](https://github.com/sbtinstruments/docsites/tree/main/items/do-it-yourself-diy/diy-dilution-dispenser/README.md).
* As part of [qualification](/getting-started/qualification).
See [Maintenance](/maintenance/maintenance) for more information.
# Make QC vial
Simply put, a QC vial contains diluted QC stock
You mix QC stock and dilution liquid to make a QC vial. The components are simple but the workflow still requires attention to detail.
## Get things ready
You need these items:
* 1 × SBT [dilution vial](/item-register/vials-flasks-and-liquids/dilution-vial) (bundled as `DV01`)
* 1 × [QC stock](/item-register/vials-flasks-and-liquids/qc-stock-1-ml)
* 1 × vortex mixer (not part of packaging)
* 1 × P200 pipette (not part of packaging)
{% hint style="success" %}
### Always use an SBT dilution vial to make a QC vial
It may be tempting to use in-house or third-party diluent or vials. However, this introduces an element of uncertainty that you don't want for the qualification procedure.
{% endhint %}
{% hint style="danger" %}
### Do *not* use a DIY[^1] dilution vial to make a QC vial
That is, do *not* use a [dilution vial made with the dilution dispenser](/item-register/do-it-yourself-diy/diy-dilution-dispenser/make-diy-dilution-vial).
{% endhint %}
## Step by step
{% stepper %}
{% step %}
#### Shake QC stock
Shake QC stock by hand for at least 30 seconds.
Shake the QC stock vigorously for at least **30 seconds** to uniformly suspend the microspheres[^2] within.
{% hint style="success" %}
### Shake the QC stock by hand
Shake the QC stock by hand for at least **30 seconds**.
{% endhint %}
{% hint style="danger" %}
### Do not use a vortex mixer on the QC stock
A vortex mixer does not properly suspend the microspheres[^2] within as they may stick to the inner lid of the cap.\
\
{% endhint %}
{% endstep %}
{% step %}
#### Transfer QC stock
Transfer from QC stock to dilution vial. For visualization purposes, this demonstration uses milk instead of QC stock.
* Aspirate twice in the QC stock. This provides a more precise volume than a single aspirate.
* Use a P200 pipette to transfer 101 µL of QC stock to the dilution vial.
* Aspirate the liquid tip ten times in the dilution vial. This rinses the pipette tip for silica beads that sticks to the plastic surfaces.
* Label the vial “QC”.
{% hint style="info" %}
The P200 pipette is *not* part of the packaging. You must acquire a P200 pipette yourself.
{% endhint %}
{% endstep %}
{% step %}
#### Mix QC vial
Mix the QC vial to suspend the microspheres[^2]. Place the QC vial on a vortex mixer and mix for **30 seconds** at max RPM[^3].
{% hint style="success" %}
We recommend that you use a vortex mixer to mix the QC vial.
{% endhint %}
{% hint style="success" %}
Ensure that a vortex/cyclone forms in the liquid.
{% endhint %}
{% hint style="info" %}
The vortex mixer is *not* part of the packaging. You must acquire a vortex mixer yourself.
{% endhint %}
{% hint style="warning" %}
### Mix by shaking as a last resort
Only shake the QC vial by hand if you don't have a vortex mixer. Shake the QC vial vigorously for at least **30 seconds**.
{% endhint %}
{% endstep %}
{% step %}
#### That is it!
You now have a QC vial. Use it to [run a QC test](/getting-started/qc-test/run-qc-test).
{% endstep %}
{% endstepper %}
{% hint style="warning" %}
## The QC vial is for *single use* only
Once the liquid passes through BactoBox®, its properties may change. Trace residues of disinfection liquid from the previous cleaning cycle can lower both the conductivity and the concentration of microspheres[^4].
**Always discard the QC vial after use!**
{% endhint %}
[^1]: Do it yourself
[^2]: Microspheres are spherical (1 µm diameter) silica particles.
[^3]: Rotations per minute
[^4]: Microspheres are spherical (1 μm diameter) silica particles.
# Run QC test
## Get things ready
You need these items:
* 1 × [BactoBox® setup](/item-register/bactobox-r/bactobox-r)
* 1 × [disinfection vial](/item-register/vials-flasks-and-liquids/disinfection-vial)
* 1 × [QC vial](/item-register/vials-flasks-and-liquids/qc-vial) (see [Make QC vial](/getting-started/qc-test/make-qc-vial))
* 1 × certificate of analysis for the QC vial
* 1 × [dilution vial](/item-register/vials-flasks-and-liquids/dilution-vial) (see the info box below)
{% hint style="info" %}
## Difference between QC test and qualification
**When you** [***qualify***](/getting-started/qualification) **your BactoBox® setup, always use an SBT dilution vial (bundled as `DV01`).**
For the regular (weekly) QC test, use the dilution vial that you intend to use going forward for your own measurements. This depends on your use case and may be either of:
* SBT dilution vial (bundled as `DV01`)
* DIY[^1] dilution vial ([made with the dilution dispenser](/item-register/do-it-yourself-diy/diy-dilution-dispenser/make-diy-dilution-vial))
Besides the choice of dilution vial, qualification and QC test are the same.
{% endhint %}
## Step by step
{% stepper %}
{% step %}
#### Start the *QC test* program
You do this with three button presses.
**Enter the *****Programs***** menu**
First, press and hold Measure until the *Programs* menu appears.
Press and hold Measure until the Programs menu appears.
**Enter the *****QC test***** screen**
Second, press Measure again briefly to select the *QC test* program.
Press Measure again briefly to select QC test program.
{% endstep %}
{% step %}
#### Set the target concentration
Check the sticker on the [QC stock](/item-register/vials-flasks-and-liquids/qc-stock-1-ml) or the certificate of analysis and find the value for *Concentration in prepared 1x QC test sample.*
Input the target concentration in million particles/mL.
{% hint style="success" %}
### Enter the value in million particles/mL
Convert the particle concentration from *particles/mL* to *million particles/mL*.
For example, 2 080 000 particles/mL becomes 2.08 million particles/mL.
{% endhint %}
Press OK to submit the target concentration.
{% hint style="info" %}
### Does your *QC test* program look different?
[Update your BactoBox® software](/best-practices/update-bactobox-r-software). We introduced the *Input target concentration* screen in software v2025.10.
{% endhint %}
{% endstep %}
{% step %}
#### Clean the setup
Transfer the tubing kit to the disinfection vial.
Press OK to start the *Clean* program.
{% hint style="info" %}
If you already followed [Clean](/maintenance/clean-the-setup), BactoBox® automatically skips this step.
{% endhint %}
{% endstep %}
{% step %}
#### Measure on QC vial
Transfer the tubing kit to the QC vial.
Press OK to start the measurement.
{% hint style="success" %}
### Status: Pass
BactoBox® checks if **total/mL** is within **±15 %** of the certificate value. If it is, the program moves to the next step.
{% endhint %}
{% hint style="danger" %}
### Status: Fail
If an error occurs, or if **total/mL** is outside the limits, the program returns to the previous step.
See [QC vial fails](/troubleshooting/qc-test-fails/i-get-error-40xx) for a troubleshooting guide.
{% endhint %}
{% endstep %}
{% step %}
#### Clean the setup
Transfer the tubing kit to the disinfection vial and press OK to clean.
{% endstep %}
{% step %}
#### Measure on dilution vial
Transfer the tubing kit to the dilution vial and press OK to measure.
{% hint style="success" %}
### Status: Pass
The concentrations are *below* **30 000 cells/mL** and *below* **100 000 total/mL**.
{% endhint %}
{% hint style="danger" %}
### Status: Fail
If any error occurs, or, if the concentrations are outside the requirements, BactoBox® moves back to previous step.
See [Dilution vial fails](/troubleshooting/qc-test-fails/dilution-vial-is-outside..) for a troubleshooting guide.
{% endhint %}
{% endstep %}
{% step %}
#### That is it!
**Discard the QC vial and the dilution vial**. They are for *single use* only.
{% endstep %}
{% endstepper %}
## When to perform a QC test
Perform a [QC test](/getting-started/qc-test) in these cases:
* **At least once a week.**
* After a [deep clean](/maintenance/deep-clean-1) (of any length).
* When you change your BactoBox® setup.\ Example: You replace the flow cell.\ Example: You replace the external filter.\ Example: You replace the tubing kit.
* When you switch from the SBT dilution vial to the [DIY dilution vial](https://github.com/sbtinstruments/docsites/tree/main/items/do-it-yourself-diy/diy-dilution-dispenser/README.md).
* As part of [qualification](/getting-started/qualification).
See [Maintenance](/maintenance/maintenance) for more information.
[^1]: Do it yourself
# Your BactoBox® journey
Now you're done with the Getting started section. At this point, you have a qualified BactoBox® setup.
Congratulations, this means you are now at the *Beginner* skill level. In turn, you are ready for the [Do a simple measurement](/tutorials/measure-vitroid-tm-disc) tutorial.
Each step comes in two versions: a detailed **tutorial** or **how-to guide** (the proven path), and a condensed **mini course** (the fast path). Pick whichever fits your time.
## Measure on cells
# Always disaggregate
Mini course: A single cell suspension is essential for reliable BactoBox® measurements.
BactoBox® is a flow cytometer. This means that cells are counted individually as they pass through the detector. For the most reliable results, disaggregate cell clumps into a single-cell suspension before measurement.
Single cells can readily enter the measurement channel, while larger clumps of cells must be disaggregated prior to dilution and measurement. Otherwise underestimation of cells may occur.
## How to get a single-cell suspension
The best way to disaggregate cells is to use a mechanical method when the cells are still in the growth medium.
Vortex at maximum speed for 1 minute in a small vial like a 2 mL or 5 mL Eppendorf tube.
## Master the art of vortexing
Vortexing may look simple. It isn't. Especially not for larger vials. Proper vortexing requires a suitable vortex mixer attachment and a proper hold of the vial.
Slow-motion vortexing of a 15 mL vial. Left: correct grip and correct attachment. Center: too tight grip. Right: wrong attachment.
{% columns %}
{% column %}
Correct:check:
The cap is initially held with three fingers to start the motion, after which a single finger applies light downward pressure while allowing the tube to vortex freely.
{% endcolumn %}
{% column %}
Incorrect ✘
The vial is held too tightly using a central grip. The liquid bounces slightly, but the vortex does not form.
{% endcolumn %}
{% column %}
Incorrect ✘
A 50 mL vortex mixer attachment is used for the 15 mL tube. The vortex does not start even though the grip is correct.
{% endcolumn %}
{% endcolumns %}
## Learn the ropes
This mini course provided you with the essentials on how to disaggregate cell clumps prior to BactoBox® measurements.
Try our first tutorial for a quick, hands-on example on how to disaggregate cell clumps.
{% content-ref url="/pages/yOiWxVnPgOuX7cibH9R9" %}
[Do a simple measurement](/tutorials/measure-vitroid-tm-disc)
{% endcontent-ref %}
Proceed to the next mini-course [Hit the right concentration](/measurement-essentials/hit-the-right-concentration) to learn why cultures must be diluted prior to measurements.
# Hit the right concentration
Mini course: How to dilute samples prior to BactoBox® measurements.
BactoBox® has a working range from 30,000 to 5,000,000 objects/mL. Cultures may be as concentrated as 1×1011 cells/mL, that is 100,000,000,000 cells/mL. Therefore, cultures must be diluted prior to BactoBox® measurements.
## Dilutions of bacterial cultures
To get conductivity right, the minimal dilution factor when using BactoBox® diluent is typically 1:100. This dilution is done by a single pipetting step and works well for the early growth stages.
{% hint style="success" icon="square-bolt" %}
## Dilute at least 1:100 to hit the right conductivity.
Most growth media must be diluted at least 1:100 to reach the conductivity range required for BactoBox® measurements (1,500–2,200 µS/cm).
{% endhint %}
As the cell concentration of a culture changes so does the required dilution. The table below summarizes the dilution factors used for various growth stages.
## Dilution schemes
Below dilution schemes provide an overview of how you obtain the different dilutions. The expandable[^1] (:chevron-right:) in each section reveals step by step videos.
### 1:100 dilutions
1:100 dilution is done by a single transfer of 101 µL culture of 10 mL of diluent.
Hands-on demonstration of 1:100 dilutions (click:chevron-right:to show more)
{% stepper %}
{% step %}
### **Dilute 1:100 in BactoBox® diluent**
The below video demonstrates how to do a 1:100 dilution.
* Transfer 101 µL of your sample to 10 mL of diluent.
* Suspend ×10 to rinse the pipet tip for residual bacteria
* Vortex 10 seconds at maximum speed.
{% endstep %}
{% step %}
### **Measure the diluted sample on BactoBox®**
The below video demonstrates how to do the bacterial measurement
* Transfer the tubing kit to the 1:100 vial.
* Press Measure to start a new measurement and wait for the results.
{% endstep %}
{% endstepper %}
### 1:1,000 dilutions
1:1,000 dilution is done by two consecutive dilution steps. First a 1:100 and then a sequential 1:1,000 dilution. Note that 1 mL is removed from the second dilution vial before transferring the sample from the 1:100 vial.
Hands-on demonstration of 1:1,000 dilutions (click:chevron-right:to show more)
{% stepper %}
{% step %}
### Dilute 1:100
Label a dilution vial 1:100 .
Vortex sample vial briefly (it has already been thoroughly vortexed when sampling).
Transfer 101 µL of your sample to the 1:100 vial. Pipette up and down ×10 in the 1:100 vial to rinse the pipette tip for residual bacteria.
Cap tightly and vortex 10 seconds at maximum speed.
{% endstep %}
{% step %}
### Dilute 1:1,000
Label a second dilution vial 1:1,000.
Aspirate and discard 1 mL diluent from the vial labeled 1:1,000
Transfer 1 mL of the 1:100 vial to the 1:1,000 vial. Pipette up and down ×10 in the 1:1,000 vial to rinse the pipette tip for residual bacteria. Cap tightly and vortex 10 seconds at maximum speed.
{% endstep %}
{% step %}
### Measure the diluted sample on BactoBox®
Transfer the tubing kit to the 1:1,000 vial.
Press Measure to start a new measurement and wait for the results.
{% endstep %}
{% endstepper %}
### 1:10,000 dilutions
1:10,000 dilution is done by two consecutive dilution steps. First a 1:100 dilution and then a sequential 1:10,000 dilution.
Hands-on demonstration of 1:10,000 dilutions (click:chevron-right:to show more)
{% stepper %}
{% step %}
### Label two dilution vials
Label first dilution vial 1:100.
Label second dilution vial 1:10,000.
{% endstep %}
{% step %}
### Dilute 1:100
Transfer 101 µL of your sample to the 1:100 vial.
Suspend ×10 to rinse the pipette tip for residual bacteria.
Vortex 10 seconds at maximum speed.
{% endstep %}
{% step %}
### Dilute 1:10,000
Transfer 101 µL of the 1:100 vial to the 1:10,000 vial.
Suspend ×10 to rinse the pipette tip for residual bacteria.
Cap tightly and vortex 10 seconds at maximum speed.
{% endstep %}
{% step %}
### Measure the diluted sample on BactoBox®
Transfer the tubing kit to the 1:10,000 vial.
Press Measure to start a new measurement and wait for the results.
{% endstep %}
{% endstepper %}
## Learn the ropes
This mini course provided you with the essentials on how to dilute bacterial cultures prior to BactoBox® measurements. Try tutorial 3 for a hands-on example on how to adjust the dilution factors when tracking a growth curve.
{% content-ref url="/pages/Kig8Uq7Hw23ovBr65Us0" %}
[Track growth curve](/tutorials/measure-vitroid-tm-growth-curve)
{% endcontent-ref %}
[^1]: Expandable hides optional details. Click each expandable to reveal additional information about sample preparation.
# Do measurements in Access
Mini-course: Connect BactoBox® to a computer and use Access to annotate your data
Did you know that you can connect BactoBox® to a computer and do the measuremnets via **Access** . Access allows you to store essential information such as sample description, sampling time, and dilution factor alongside your measurements.
The video below shows a growth curve experiment monitored and recorded in Access.
In this mini course we will show you the essentials of how to run measurements in Access.
{% stepper %}
{% step %}
### Open Access
Connect your BactoBox® to your computer and open Access in your browser. The familiar BactoBox® buttons move onto your screen.
Use the USB cable to connect your BactoBox® to your computer.
{% endstep %}
{% step %}
### Create a measurement group
Start a *basic* measurement group as shown in the video. Basic is the simplest measurements view. It merely groups the measurements together. Doesn't offer additional analytics or plotting.
Create a measurement group.
{% endstep %}
{% step %}
### Measure and annotate each sample
Set the sampling time, dilution factor and a label. Then click Measure to run the measurement.
When the measurement is completed, Access automatically works out the concentration in the non-diluted culture and adds it to the table.
Add information on your sample and click Measure .
{% endstep %}
{% endstepper %}
## Learn the ropes
This mini course showed you how to do a basic BactoBox® measurement via Access.
There are is a more advanced feature in Access that allows you to visualize a growth curve in real time. Click the link below to learn more.
{% content-ref url="/spaces/seAjVXi9YrPNSfdp1OXS/pages/w3Mb1dexrs8DmLioC8vu" %}
[Track growth curve](/mpd/mini-course/track-growth-curve)
{% endcontent-ref %}
## Next steps
With these 3 measurement essentials mini courses, you now have a foundation for testing your own cultures. In principle you can now proceed to our [Workflows](/mpd/workflows/bioprocess-workflows) in [Microbial process development](https://help.sbtinstruments.com/mpd/)
Before doing so, we highly recommend that you complete tutorials 1-3 or at least consider these as a fall-back option if you run into technical difficulties with your own cultures.
In any case, we are always happy to help. If you find yourself stuck, feel free to [Contact our support team](/troubleshooting/contact-our-support-team/contact-our-support-team) for assistance.
# Do a simple measurement
TUT-1: Your first BactoBox® measurement in a 30 min tutorial
This tutorial is for your first bacterial measurement on BactoBox®. The tutorial is simple, fast, and predictable. You will learn how to prepare bacterial suspensions for BactoBox® measurements.
For this tutorial, the success criterion is to get a BactoBox® measurement within 30,000 to 5,000,000 cells/mL.
The below table lists the overall considerations for this tutorial. We use similar tables for our microbial process development [Workflows](/mpd/workflows/bioprocess-workflows) so you know if the workflow is right for you and what time to set aside.
You can complete this tutorial as a :school\_satchel:Beginner to BactoBox® measurements.
## Soluble *E. coli* Vitroids™ disc
For this tutorial you need to procure [*Escherichia coli* WDCM 00013 Vitroids™](https://www.sigmaaldrich.com/DK/en/product/sial/vt000137) ATCC® 25922 from Sigma/Merck. Lead time is usually 1-5 days. Price is \~200 USD including shipping. 10 vials are included in each shipment. Concentration per disc is stated as 50,000-150,000 CFU mean value range.
The discs are convenient for this tutorial as they dissolve easily in water and have a well-defined concentration of bacteria within the working range of BactoBox® measurements.
A soluble Vitroid™ disc containing 50,000–150,000 CFU of E. coli ATCC® 25922 .
## How to get a successful measurement on BactoBox®
Three criteria are essential to get a reliable cell concentration on BactoBox®. Below is the checklist.
:bacteria: The sample should ideally be a single-cell suspension, i.e. without cell clumps.
:square-bolt: Conductivity must be within 1,500–2,200 µS/cm.
:xmarks-lines: Concentration of the diluted sample must be between 30,000 - 5,000,000 objects/mL
### Disaggregation of cell clumps
The most important learning of this tutorial is how to get a single-cell suspension. The below video explains why large clumps must be disaggregated before the individual cells can enter the measurement channel.
## Overview
In this tutorial we will demonstrate how to solubilize the disc and get the bacteria ready for BactoBox® analyses. The overall steps in the tutorial are given below.
1. [Get things ready](/tutorials/measure-vitroid-tm-disc/get-things-ready)
2. [Transfer disc](/tutorials/measure-vitroid-tm-disc/transfer-vitroid-tm-disc)
3. [Prepare sample](/tutorials/measure-vitroid-tm-disc/disaggregate-bacteria)
4. [Dilute and measure](/tutorials/measure-vitroid-tm-disc/dilute-and-measure)
5. [Summary](/tutorials/measure-vitroid-tm-disc/summary)
## Summary
After this introduction you can proceed to [Get things ready](/tutorials/measure-vitroid-tm-disc/get-things-ready).
# Get things ready
All the below items are required for the tutorial. Most items are supplied by SBT in a bundle.
Note that the Vitroids**™** discs must be procured from Sigma/Merck. The additional items are standard consumables in a microbiological lab.
## Hardware
* [BactoBox® setup](https://help.sbtinstruments.com/items/bactobox-r#bactobox-r-setup) including a [flow cell](https://help.sbtinstruments.com/items/consumables/flow-cell) with at least one remaining measurements.
* HW: [7.6a](/item-register/bactobox-r/bactobox-r-hardware-changelog)
* SW: ≥ v2026.02a
* Marker (not shown; not supplied)
* Vortex mixer (not supplied)
* Rack for holding 15 mL centrifuge vials (not supplied)
* Small vial, e.g. 1.5 mL Eppendorf tube (not supplied)
* P200 pipette (not supplied)
* Sterile pipette tips (not supplied).
## Reagents
* Certified reference material (CRM): [*Escherichia coli* WDCM 00013 Vitroids™](https://www.sigmaaldrich.com/DK/en/product/sial/vt000137) (not supplied).
* 1 × [dilution vial](https://help.sbtinstruments.com/items/vials-flasks-and-liquids/dilution-vial) ready-to-use.
* 1 × [disinfection vial](https://help.sbtinstruments.com/items/vials-flasks-and-liquids/disinfection-vial).
## Summary
With these things in hand, you are ready to for the next step: [Transfer disc](/tutorials/measure-vitroid-tm-disc/transfer-vitroid-tm-disc).
# Transfer disc
BactoBox® measures bacteria in liquid suspension. A Vitroids**™** disc is solid. Therefore you must first solubilize the disc. The below video demonstrates the procedure and all the details are provided in the [#step-by-step](#step-by-step "mention") instructions.
{% hint style="success" %}
## Bacteria can only be in BactoBox® diluent for short durations.
In this tutorial we use diluent from a [Dilution vial](/item-register/vials-flasks-and-liquids/dilution-vial) to solubilize the bacteria in the disc. The diluent provides excellent differentiation of cells from other objects. On the flip side it is [hypotonic ](#user-content-fn-1)[^1]and cells may lyse if stored in the diluent. Always complete measurements within 15 minutes after dilution in BactoBox® diluent.
{% endhint %}
Transfer of Vitroid™ disc to diluent for initial solubilization.
## Step by step
{% stepper %}
{% step %}
### Label a vial
Find a small vial, for example a 1.5 mL Eppendorf vial.
Use a marker to label the vial *E. coli*.
{% endstep %}
{% step %}
### Prepare a solubilization vial
Use a P200 pipette to transfer 110 µL of [Dilution vial](/item-register/vials-flasks-and-liquids/dilution-vial) to the small vial.
{% hint style="success" %}
Preferably use the gamma-sterilized ready-to-use [Dilution vial](/item-register/vials-flasks-and-liquids/dilution-vial) instead of DIY versions of the BactoBox® diluent.
{% endhint %}
{% endstep %}
{% step %}
### Transfer Vitroids**™** disc to vial
Uncap the Vitroids**™** vial and hold it over the mound of the small vial.\
Tap the Vitroids**™** vial until the disc is transferred to the small vial.\
Cap the small vial tightly.
{% endstep %}
{% step %}
### Discard the opened dilution vial
Discard the dilution vial that you used to retrieve 110 µL BactoBox® diluent.
The [Dilution vial](/item-register/vials-flasks-and-liquids/dilution-vial) is no longer sterile and does not contain the required 10 mL volume.
{% endstep %}
{% endstepper %}
## Summary
You have now prepared for the solubilization of the Vitroid**™** disc. The disc would dissolve by itself within 5 minutes, but in the next page we will speed up this process by vortexing. This will also disaggregate clumps of bacteria. Proceed to [Prepare sample](/tutorials/measure-vitroid-tm-disc/disaggregate-bacteria) for further instructions.
[^1]: A solution with a lower concentration of solutes compared to another solution (like the fluid inside a cell)
# Prepare sample
The Vitroids**™** disc is now dissolving in the diluent. You will speed up the dissolution process by vortexing the disc for one minute at maximum speed. The below video demonstrates the procedure and the details are provided in the [#step-by-step](#step-by-step "mention") instructions.
{% hint style="success" icon="bacteria" %}
## Vortex thoroughly and vigorously
BactoBox is a flow cytometer and works best when cells are present in a single-cell suspension.
Cells stick together and you need thorough disaggregation to prepare a single-cell suspension.
One minute of vortexing may sound excessive, but trust us. You need it!
{% endhint %}
## Step by step
{% stepper %}
{% step %}
### Vortex vial
Transfer the small vial to a vortex mixer and vortex at maximum speed for 1 min.\
Ensure you see a proper vortex forming in the tube.
Use a vortex mixer to solubilize Vitroids™ disc and disaggregate bacterial clumps.
{% endstep %}
{% step %}
### Inspect the vial for complete solubilization
Visually inspect the vial for residual clumps of the Vitroids**™** disc.\
Increase vortexing time if you see clumps.\
\
The disc must be completely solubilized before proceeding to next page.
{% endstep %}
{% endstepper %}
## Summary
You have now dissolved the disc and disaggregated bacterial clumps. Proceed to [Dilute and measure](/tutorials/measure-vitroid-tm-disc/dilute-and-measure) for your first BactoBox® measurement on cells.
# Dilute and measure
By now the Vitroids™ disc is solubilized and a single-cell suspension is ready for BactoBox® measurements. The bacterial concentration is low so we use as little dilution factor as possible to hit the right conductivity. This is typically 1:100.
## How to make 1:100 dilutions
1:100 dilutions are done by transferring 101 µL sample to 10 mL of diluent.
### Proper vortexing of the 15 mL vial is important for reliable results
Proper vortexing of the 15 mL vials is critical. It isn't trivial. It requires a suitable vortex mixer attachment and a proper hold of the vial.
When vortexing is done correctly, a strong vortex forms in the vial within a few seconds. Follow the recommendations in the green information box and avoid the common mistakes demonstrated in the below slow-motion video (✔ demonstrates correct. ✘ demonstrates incorrect).
{% hint style="success" %}
## Good vortexing technique
:hashnode: Choose a mixer platform that is compatible with your vial. [Some platforms](https://www.scientificindustries.com/collections/vortex-mixers-shakers-accessories/products/3-inch-platform-with-rubber-cover) can accommodate multiple vial types and sizes.
:hand-point-down: Use a gentle grip on the vial cap while vortexing the tube. Start with a firm 3-finger hold on the cap and switch to a gentle press with one finger once the motion starts.
{% endhint %}
* **The left panel demonstrates proper vortexing technique:** The cap is initially held with three fingers to start the motion, after which a single finger applies light downward pressure while allowing the tube to vortex freely.
* **The center panel demonstrates improper technique:** The vial is held too tightly using a central grip. The liquid bounces slightly, but the vortex does not form.
* **The right panel demonstrates improper technique:** A 50 mL vortex mixer attachment is used for the 15 mL tube. The vortex does not start even though the grip is correct.
## Step by step
{% stepper %}
{% step %}
### **Dilute 1:100 in BactoBox® diluent**
The below video demonstrates how to do a 1:100 dilution.
* Transfer 101 µL of your sample to 10 mL of diluent.
* Suspend ×10 to rinse the pipet tip for residual bacteria
* Vortex 10 seconds at maximum speed.
{% endstep %}
{% step %}
### **Measure the diluted sample on BactoBox®**
The below video demonstrates how to do the bacterial measurement
* Transfer the tubing kit to the 1:100 vial.
* Press Measure to start a new measurement and wait for the results.
{% endstep %}
{% step %}
### Create a table of the results
Note down your results in a table.
In this example, the concentration is 89 000 cells/mL out of 130 000 total/mL. The first number is the bacterial concentration; the latter is the total objects detected in the measurement. Bacterial concentration is reported as a subset of the total objects and therefore 41 000 objects were something *other* than bacterial cells. The concentration may differ between different Vitroids™ discs.
Measurement ID
Dilution
Cells/mL
Total/mL
Q17
100
89 000
130 000
{% hint style="info" %}
## Full measurement cycle is used for samples with low bacterial concentration
Because the bacterial concentration is in the low end of the [BactoBox® working range](/advanced/advanced-sample-preparation/hit-the-right-concentration), the full measurement time is used.
{% endhint %}
{% endstep %}
{% step %}
### Clean the device
Transfer the tubing kit to the disinfection vial and press Clean .
Always run a Clean after running a measurement.
{% endstep %}
{% endstepper %}
## Summary
You have now measured the cell concentration in the liquid derived from the solubilized Vitroid™ disc. In the next page, [Summary](/tutorials/measure-vitroid-tm-disc/summary), we will give an overview of the procedure and the obtained results.
{% hint style="success" icon="laptop" %}
## Run your measurements in [Access](/software/access/access)
In this tutorial we keep things simple and demonstrate BactoBox® as a stand-alone-device.
But a manual log like the above table belongs to last century. Manual logs may get lost and it is common to make typing mistakes when you transfer data to spreadsheets.
[Access](/software/access/access) is a powerful tool to keep track of dilution factors, sample IDs and results. Data are stored on the device and can be exported easily as a csv file. See an example in [Track growth curve](/mpd/workflows/track-growth-curve).
{% endhint %}
# Summary
In this tutorial you learned how to dissolve a Vitroids™ disc and prepare the bacterial suspension for BactoBox® analyses.
## Calculate cell concentration of non-diluted suspension
You just did your first BactoBox® measurement. The result on the BactoBox® is for the diluted sample. Always remember to multiply the obtained result with the dilution factor to get the concentration of the non-diluted suspension.
BactoBox® dilution
BactoBox® concentration
Non-diluted suspension
100
89 000 cells/mL
100 × 89 000 cells/mL = 8 900 000 cells/mL
## Comparison between CFU and BactoBox® results
We can estimate the number of cells in the Vitroids™ disc from the measured cell concentration after dissolution. We assume that the total volume remains 110 µL (0.110 ml) after the disc is added. That gives a volume of 0.110 ml/disc.
1. Measure the cell concentration in the dissolved sample:\
**8,900,000 cells/mL**
2. Multiply by the dissolution volume:\
**8,900,000 cells/mL × 0.110 mL/disc = 979,000 cells/disc**
To develop this tutorial, we repeated the procedure with **three Vitroids™ discs from the same lot**. The table below summarizes the results (all values are reported as **cells/disc**).
#1
#2
#3
Arithmetic mean ± Standard deviation
979 000
970 000
1 100 000
1 000 000 ± 90 000 cells/disc
The concentration is stated by Sigma/Merck as 50,000-150,000 CFU mean value range. This is \~1 log below the results obtained with the BactoBox® measurements. It is normal to see cells ≠ CFU for formulated, stored products, see the box for more information.
{% hint style="info" %}
## BactoBox detects all forms of bacteria. Plate counts only detects culturable cells.
A plate count only measures the culturable cells and does not detect VBNC forms of bacteria. BactoBox® detects viable, dead and VBNC forms of bacteria. BactoBox® shows [great correlation](/mpd/correlation-and-linearity/correlation-with-plate-counts) for most [Active cultures](/mpd/cell-growth/active-cultures) while BactoBox® measurements on bacteria in dormant and stressed states may show [https://app.gitbook.com/s/seAjVXi9YrPNSfdp1OXS/cell-growth/divergence-from-plate-counts](https://app.gitbook.com/s/seAjVXi9YrPNSfdp1OXS/cell-growth/divergence-from-plate-counts "mention").
{% endhint %}
## BactoBox® skills
After successfully completing this tutorial you have acquired the following skills:
:bacteria: Disaggregrate bacterial clumps by vortexing
:boxing-glove: Make a 1:100 dilution of a dilute bacterial sample and measure on BactoBox®
:calculator-simple: Calculate cell concentrations of the non-diluted bacterial suspension
## Next steps
This, first tutorial was a quick and simple introduction to a bacterial measurements.
We have another great tutorial lined up for you: [Measure a dense culture](/tutorials/measure-overnight-culture). You will prepare a dense culture using a Vitroids™ disc (or an in-house *E. coli* cryo stock). After overnight incubation you will learn how to dilute the sample 1:10,000 to hit the right concentration.
# Measure a dense culture
TUT-2: Your first BactoBox® measurement on an overnight culture with high bacterial concentration
This tutorial is for your second bacterial measurement on BactoBox®. It is slightly more advanced than the first tutorial [Do a simple measurement](/tutorials/measure-vitroid-tm-disc) but still simple and predictable with short hands-on time. You will make an overnight culture and learn how to dilute to a suitable working range.
The success criterion for this tutorial is to get a BactoBox® measurement within 30,000 to 5,000,000 cells/mL. You can complete this tutorial as a :school\_satchel:Beginner to BactoBox® measurements.
## Soluble *E. coli* Vitroids™ disc
For this tutorial we recommend [*Escherichia coli* WDCM 00013 Vitroids™](https://www.sigmaaldrich.com/DK/en/product/sial/vt000137) ATCC® 25922 from Sigma/Merck. Lead time is usually 1-5 days. Price is \~200 USD including shipping. 10 vials are included in each shipment. Concentration per disc is stated as 50,000-150,000 CFU mean value range.
A soluble Vitroid™ disc containing 50,000–150,000 CFU of E. coli ATCC® 25922 .
{% hint style="info" icon="snowflakes" %}
## Alternative: Use an in-house *E.coli* cryo stock instead of a Vitroids™ disc
You will get the best experience if you procure [*Escherichia coli* WDCM 00013 Vitroids™](https://www.sigmaaldrich.com/DK/en/product/sial/vt000137) from Sigma/Merck. Alternatively, you can use an in-house *E. coli* glycerol stock to prepare an overnight culture. See more details in [Inoculate LB medium](/tutorials/measure-overnight-culture/inoculate-lb-medium).
{% endhint %}
## Hit the right concentration
An *E. coli* shake flask overnight culture typically has \~1×1010 cells/mL, i.e. 10,000,000,000 cells/mL. Max concentration for a BactoBox® measurement is 5×106 total/mL, i.e. 5,000,000 total/mL.
A typical overnight culture must be diluted 1:10,000 to hit the right concentration.
Use two consecutive 1:100 dilution steps to prepare a 1:10,000 dilution.
A 1:10,000 dilution is made by two consecutive 1:100 dilutions.
{% hint style="success" icon="foot-wing" %}
## Save time and reagents by using 1:100 dilutions instead of 1:10 dilutions
BactoBox® has a \~100-fold [dynamic range](#user-content-fn-1)[^1]. You save time and reagents by using 1:100 dilutions.
This is different from the 1:10 dilution series used in microbiology. The 1:10 dilutions are needed for plate counts where the dynamic range is only 10-fold (25–250 colonies are typically counted on an agar plate).
{% endhint %}
## Overview
In this tutorial we will demonstrate how to solubilize the disc and get the bacteria ready for BactoBox® analyses. The overall steps in the tutorial are given below.
1. [Get things ready](/tutorials/measure-overnight-culture/get-things-ready)
2. [Inoculate LB medium](/tutorials/measure-overnight-culture/inoculate-lb-medium)
3. [Prepare sample](/tutorials/measure-overnight-culture/disaggregate-bacteria)
4. [Dilute and measure](/tutorials/measure-overnight-culture/dilute-and-measure)
5. [Summary](/tutorials/measure-overnight-culture/summary)
## Summary
After this introduction you can proceed to [Get things ready](/tutorials/measure-overnight-culture/get-things-ready).
[^1]: Dynamic range is the interval over which a method provides reliable quantitative measurements.
# Get things ready
All the below items are required for the tutorial. Most items are supplied by SBT in a bundle.
Note that the Vitroids**™** discs must be procured from Sigma/Merck. The additional items are commonly used in microbiology laboratories.
## Hardware
* [BactoBox® setup](/item-register/bactobox-r/bactobox-r) including a [flow cell](/item-register/consumables/flow-cell) with at least one remaining measurements.
* HW: [7.6a](https://help.sbtinstruments.com/tutorials/measure-overnight-culture/spaces/AjOYV4v43T0TFlSRqgAp/pages/3JJDRTohjXVJqe8Q6JDr#v7.6a)
* SW: ≥ v2026.02a
* Marker (not supplied)
* Vortex mixer (not supplied)
* Rack for holding 15 mL centrifuge vials (not supplied)
* Centrifuge vial, 15 mL (supplied) or 5 mL (not supplied)
* P200 pipette (not supplied)
* Sterile pipette tips (not supplied).
* Serological pipette, preferably 2 or 5 mL (not supplied).
* Serological pipette controller (not supplied).
* Shaking incubator (not supplied).
## Reagents
* Certified reference material (CRM): [*Escherichia coli* WDCM 00013 Vitroids™](https://www.sigmaaldrich.com/DK/en/product/sial/vt000137) (not supplied).
* 250 mL shake flask with 50 mL autoclaved LB medium. Preferably [baffled shake flask with vented cap](https://pipette.com/dwk-life-sciences-llc-354239.html).
* 2 × [dilution vials](/item-register/vials-flasks-and-liquids/dilution-vial) ready-to-use.
* 1 × [disinfection vial](/item-register/vials-flasks-and-liquids/disinfection-vial).
## Summary
With these things in hand, you are ready to for the next step: [Inoculate LB medium](/tutorials/measure-overnight-culture/inoculate-lb-medium).
# Inoculate LB medium
First objective is to start an overnight culture. We provide two options in the below step by step procedure. Click the tabs in the upper left corner to toggle between the two options.
The preferred route is to use a Vitroids™ disc for the inoculation. We have tested the robustness of this workflow and we know it works.
The alternative route is to use an in-house glycerol stock for the inoculation. We can't guarantee a successful outcome because we have no information on your glycerol stocks. If you have other guidelines for *E. coli* overnight cultures, feel free to use these.
## Step by step
{% tabs %}
{% tab title="Preferred: Vitroids™ disc" icon="flying-disc" %}
{% stepper %}
{% step %}
### Start the culture in the afternoon
Wait until the end of the workday before inoculating the flask. Inoculate at 4:00 PM to ensure the overnight culture is ready by 8.00 AM the following morning.
{% endstep %}
{% step %}
### Label the shake flask
Use a marker to write *E. coli*ATCC®25922 and inoculation time on the 250 mL baffled shake flask.
{% endstep %}
{% step %}
### Transfer Vitroids**™** disc
Use proper aseptic technique and drop the disc in the shake flask that already contains 50 mL autoclaved LB medium.
Demonstration of the dissolution of a Vitroids™ disc in growth medium. You can proceed directly step 4 after transferring the disc (You don't need to wait for the dissolution to happen).
{% endstep %}
{% step %}
### Start the incubation
Transfer the shake flask to the shaking incubator. Incubate 16 hours at 37 °C, 200 RPM.
{% endstep %}
{% endstepper %}
{% endtab %}
{% tab title="Alternative: In-house glycerol stock" icon="snowflakes" %}
{% stepper %}
{% step %}
### Start the culture in the afternoon
Wait until the end of the workday before inoculating the flask. Inoculate at 4:00 PM to ensure the overnight culture is ready by 8.00 AM the following morning.
{% endstep %}
{% step %}
### Label the shake flask
Use a marker to write *E. coli,* in-house strain designation, and inoculation time directly on the 250 mL baffled shake flask.
{% endstep %}
{% step %}
### Inoculate using 0.1 % (V/V)
Transfer 50 µL *E. coli* glycerol stock directly to the shake flask. The flask already contains 50 mL LB medium and therefore this corresponds to \~0.1% volumetric seeding
{% hint style="info" %}
## How to get great overnight cultures
An *E. coli* culture inoculated at 0.1 % (V/V) is likely to be within stationary or even decline phase after 16 hours of incubation. The culture will be fine for this tutorial, but bad for e.g. bioprocess seed trains.
Check our workflow [Optimize starter culture](/mpd/workflows/identify-optimal-inoculum-size) for how to get a [High-quality culture](/mpd/cell-growth/high-quality-culture).
{% endhint %}
{% endstep %}
{% step %}
### Start the incubation
Transfer the shake flask to the shaking incubator. Incubate 16 hours at 37 °C, 200 RPM.
{% endstep %}
{% endstepper %}
{% endtab %}
{% endtabs %}
## Summary
You inoculated the LB growth medium to get an overnight *E. coli* culture. Wait for \~16 hours and proceed to the next step [Prepare sample](/tutorials/measure-overnight-culture/disaggregate-bacteria).
# Prepare sample
After \~16 hours of incubation, the overnight culture is at a high cell concentration and we can use it to demonstrate BactoBox® measurements on concentrated bacterial suspensions.
{% hint style="success" icon="bacteria" %}
## Vortex thoroughly and vigorously
BactoBox is a flow cytometer and works best when cells are present in a single-cell suspension.
Cells stick together and you need thorough disaggregation to prepare a single-cell suspension.
One minute of vortexing may sound excessive, but trust us. You need it!
{% endhint %}
## Step by step
{% stepper %}
{% step %}
### Get a sample of the culture
Make figure-eight swirls to suspend the culture
Use a serological pipette to aspirate 2 mL culture.
Transfer to a 5 or 15 mL centrifuge vial.
{% endstep %}
{% step %}
### Disaggregate clumps of bacteria
Vortex the sample 1 minute at max speed.
Ensure that a proper vortex is seen inside the tube when vortexing.
{% endstep %}
{% endstepper %}
## Summary
You retrieved a sample of the overnight culture and used vortex mixing to disaggregate clumps of bacteria. Proceed to [Dilute and measure](/tutorials/measure-overnight-culture/dilute-and-measure) to determine cell concentration with BactoBox®.
# Dilute and measure
The overnight culture sample is ready for BactoBox® measurements.
An *E. coli* shake flask overnight culture typically has \~1×1010 cells/mL. A 1:10,000 dilution is suitable to hit the right concentration for BactoBox® measurements. Dilution is done by two consecutive dilution steps. First a 1:100 dilution and then a 1:10 000 dilution.
## How to make 1;10,000 dilutions
Dilution is done by two consecutive dilution steps. First a 1:100 dilution and then a sequential 1:10,000 dilution.
Tutorials 1 explained the importance of using the correct vortexing technique for 15 mL vials. If you need a refresher, expand the section below. Skip ahead if you've already mastered the technique.
Proper vortex technique
When vortexing is done correctly, a strong vortex forms in the vial within a few seconds. Follow the recommendations in the green information box and avoid the common mistakes demonstrated in the below slow-motion video (✔ demonstrates correct. ✘ demonstrates incorrect).
{% hint style="success" %}
## Good vortexing technique
:hashnode: Choose a mixer platform that is compatible with your vial. [Some platforms](https://www.scientificindustries.com/collections/vortex-mixers-shakers-accessories/products/3-inch-platform-with-rubber-cover) can accommodate multiple vial types and sizes.
:hand-point-down: Use a gentle grip on the vial cap while vortexing the tube. Start with a firm 3-finger hold on the cap and switch to a gentle press with one finger once the motion starts.
{% endhint %}
* **The left panel demonstrates proper vortexing technique:** The cap is initially held with three fingers to start the motion, after which a single finger applies light downward pressure while allowing the tube to vortex freely.
* **The center panel demonstrates improper technique:** The vial is held too tightly using a central grip. The liquid bounces slightly, but the vortex does not form.
* **The right panel demonstrates improper technique:** A 50 mL vortex mixer attachment is used for the 15 mL tube. The vortex does not start even though the grip is correct.
## Step by step
{% stepper %}
{% step %}
### Label two dilution vials
Label first dilution vial 1:100.
Label second dilution vial 1:10,000.
{% endstep %}
{% step %}
### Dilute 1:100
Transfer 101 µL of your sample to the 1:100 vial.
Suspend ×10 to rinse the pipette tip for residual bacteria.
Vortex 10 seconds at maximum speed.
{% endstep %}
{% step %}
### Dilute 1:10,000
Transfer 101 µL of the 1:100 vial to the 1:10,000 vial.
Suspend ×10 to rinse the pipette tip for residual bacteria.
Vortex 10 seconds at maximum speed.
{% endstep %}
{% step %}
### Measure the diluted sample on BactoBox®
The below video demonstrates how to do the bacterial measurement
* Transfer the tubing kit to the 1:10,000 vial.
* Press Measure to start a new measurement and wait for the results.
{% hint style="info" icon="timeline-arrow" %}
## A measurement consists of three distinct stages
:square-1: First stage is **Starting up**: Conductivity and flow is checked
:square-2: Second stage is **Measuring**: Electric fields in the flow cell are turned on and cells are counted
:square-3: Third stage is **Emptying**: Pump direction is reversed and the flow path is drained.
{% endhint %}
{% endstep %}
{% step %}
### Create a table of the results
Note down your results in a table.
In this example, the concentration is 540 000 cells/mL out of 540 000 total/mL. The first number is the cell concentration; the latter is the total objects detected in the measurement. Cell concentration is reported as a subset of the total objects. The numbers are identical, i.e. all detected objects are cells.
Measurement ID
Dilution
Cells/mL
Total/mL
M77
10,000
540 000
540 000
{% endstep %}
{% step %}
### Clean the device
Transfer the tubing kit to the disinfection vial and press Clean .
Always run a Clean after a measurement.
{% endstep %}
{% endstepper %}
## Summary
You have now measured the cell concentration in the *E. coli* overnight culture. Notice that the BactoBox® measurement was faster here than in the first tutorial [Do a simple measurement](/tutorials/measure-vitroid-tm-disc). The :forward: **fast results** feature is triggered because the bacterial concentration in the diluted sample was higher.
In the next page, [Summary](/tutorials/measure-overnight-culture/summary), we will give an overview of the procedure and the obtained results.
{% hint style="success" icon="laptop" %}
## Run your measurements in [Access](/software/access/access)
In this tutorial we keep things simple and demonstrate BactoBox® as a stand-alone-device.
But a manual log like the above table belongs to last century. Manual logs may get lost and it is common to make typing mistakes when you transfer data to spreadsheets.
[Access](/software/access/access) is a powerful tool to keep track of dilution factors, sample IDs and results. Data are stored on the device and can be exported easily as a csv file. See an example in [Track growth curve](/mpd/workflows/track-growth-curve).
{% endhint %}
# Summary
In this tutorial you learned how to prepare a dense bacterial culture for BactoBox® measurements. After thorough disaggregation and 1:10,000 dilution, you got a :forward: **fast result**.
## Calculate cell concentration of non-diluted suspension
The BactoBox® result was for the diluted sample. Always remember to multiply the obtained result with the dilution factor to get the concentration of the non-diluted suspension.
BactoBox® dilution
BactoBox® concentration
Non-diluted suspension
10 000
540 000 cells/mL
10 000 × 540 000 cells/mL = 5.4 × 109 cells/mL
## BactoBox® skills
After successfully completing this tutorial you have acquired the following skills:
:bacteria: Disaggregrate bacterial clumps by vortexing
:eye-dropper: Make a 1:10 000 dilution of a dense bacterial culture and measure on BactoBox®
:calculator-simple: Calculate cell concentrations of the non-diluted bacterial suspension
## Proper disaggregation is extremely important
Disaggregation of cell clumps is important for any bacterial enumeration method. But it is especially important for BactoBox® measurements. The measurement channel in the flow cell will only accommodate objects smaller than 5 µm spherical diameter. Larger objects will stay in the bypass channel. Clumpy, bacterial suspensions will be severely underestimated on BactoBox® unless proper disaggregation is done.
Vortex works well for most liquid cultures of bacteria, but sometimes you need more advanced sample workup to [Break up clumps and chains](/advanced/advanced-sample-preparation/break-up-clumps-and-chains).
## Next steps
This, second tutorial was a simple introduction on how to measure a concentrated bacterial culture on BactoBox®.
We have a third and last tutorial on cell measurements for you: [Track growth curve](/tutorials/measure-vitroid-tm-growth-curve). In this tutorial you will inoculate LB medium using a Vitroids™ disc (or an in-house *E. coli* cryo stock). After a few hours of incubation you will use BactoBox® to track a beautiful growth curve.
We will soon have a third and last tutorial on cell measurements for you. In this tutorial you will inoculate LB medium using a Vitroids™ disc (or an in-house *E. coli* cryo stock). After a few hours of incubation you will use BactoBox® to track a beautiful growth curve.
# Track growth curve
TUT-3: Your first growth curve tracked with BactoBox®
This tutorial will teach you how to track a bacterial growth curve using BactoBox® as a stand-alone device.
You should plan for approximately 4 hours of hands-on work and a total duration of 12 hours to complete the full growth curve experiment. During the first 5 hours, most of the time will be spent waiting for the culture to reach a suitable concentration.
The first step is straightforward. To make your workday more efficient and avoid a long day in the lab, consider asking a colleague who arrives early to inoculate the culture before you begin.
{% hint style="success" %}
## Complete tutorial :square-1: and :square-2: first
Before starting this tutorial, first complete the [Do a simple measurement](/tutorials/measure-vitroid-tm-disc) and [Measure a dense culture](/tutorials/measure-overnight-culture). They are simpler and faster. The present tutorial is slightly more complex.
{% endhint %}
The goal of this tutorial is to generate a high-quality BactoBox® growth curve that enables reliable determination of the growth rate.
This tutorial is suitable for :school\_satchel:Beginners and can be completed without prior experience using BactoBox® for growth measurements.
## *E. coli* Vitroids™ disc
We recommend [*Escherichia coli* WDCM 00013 Vitroids™](https://www.sigmaaldrich.com/DK/en/product/sial/vt000137) ATCC® 25922 from Sigma/Merck. Lead time is usually 1-5 days. Price is \~200 USD including shipping. 10 vials are included in each shipment. Concentration per disc is stated as 50,000-150,000 CFU mean value range.
A soluble Vitroid™ disc containing 50,000–150,000 CFU of E. coli ATCC® 25922 .
{% hint style="info" icon="snowflakes" %}
## Alternative: Use an in-house *E.coli* glycerol stock instead of a Vitroids™ disc
For the best experience, we recommend using [*Escherichia coli* WDCM 00013 Vitroids™](https://www.sigmaaldrich.com/DK/en/product/sial/vt000137) from Sigma/Merck. Alternatively, you can prepare an overnight culture from an in-house *E. coli* glycerol stock. For detailed preparation instructions, see [Inoculate LB medium](/tutorials/measure-vitroid-tm-growth-curve/inoculate-lb-medium).
{% endhint %}
## Hit the right conductivity
The conductivity must be within the range of 1,500–2,200 µS/cm to be measured with BactoBox®. A 1:100 dilution is typically sufficient to achieve the required conductivity.
For most growth media, undiluted cultures and 1:10 dilutions have a conductivity that exceeds the acceptable range. If such samples are measured, BactoBox® will display a conductivity error.
Conductivity measurements for a dilution series of the growth medium. Dilutions of 1:100, 1:1,000, 1:10,000, and 1:100,000 all fall within the accepted conductivity range of 1,500–2,200 µS/cm. In contrast, the undiluted growth medium and the 1:10 dilution exceed the acceptable conductivity range.
{% hint style="success" icon="square-bolt" %}
## Dilute at least 1:100 to hit the right conductivity.
Most growth media must be diluted at least 1:100 to reach the conductivity range required for BactoBox® measurements (1,500–2,200 µS/cm).
{% endhint %}
## Pre-incubate culture 5 hours before measurements
The culture must be incubated before its cell concentration can be measured using BactoBox®.
Once dissolved in 50 mL of growth medium, the Vitroids™ disc yields a cell concentration of approximately 1,000 CFU/mL. Based on experience, the culture typically requires around 5 hours of incubation before the cell concentration becomes detectable with a 1:100 dilution.
## Change dilution factors as the concentration increases
Begin the measurements using a 1:100 dilution. As the cell concentration increases, use higher dilution factors, such as 1:1,000 and 1:10,000. The following subpages will guide you through each step.
## Overview
The overall steps in the tutorial are given below.
1. [Get things ready](/tutorials/measure-vitroid-tm-growth-curve/get-things-ready)
2. [Inoculate LB medium](/tutorials/measure-vitroid-tm-growth-curve/inoculate-lb-medium)
3. [Prepare sample](/tutorials/measure-vitroid-tm-growth-curve/disaggregate-bacteria)
4. [1:100 dilutions](/tutorials/measure-vitroid-tm-growth-curve/1-100-dilutions)
5. [1:1,000 dilutions](/tutorials/measure-vitroid-tm-growth-curve/1-1-000-dilutions)
6. [1:10,000 dilutions](/tutorials/measure-vitroid-tm-growth-curve/1-10-000-dilutions)
7. [Data analysis](/tutorials/measure-vitroid-tm-growth-curve/data-analysis)
8. [Summary](/tutorials/measure-vitroid-tm-growth-curve/summary)
## Summary
After this introduction you can proceed to [Get things ready](/tutorials/measure-vitroid-tm-growth-curve/get-things-ready).
# Get things ready
The below items are required for the tutorial. Most items are supplied by a SBT in a standard BactoBox® bundle.
Note that the Vitroids**™** discs must be procured from Sigma/Merck. The additional items are commonly used in microbiology laboratories.
## Hardware
* [BactoBox® setup](/item-register/bactobox-r/bactobox-r) including a [flow cell](/item-register/consumables/flow-cell) with at least one remaining measurements.
* HW: [7.6a](https://help.sbtinstruments.com/tutorials/measure-vitroid-tm-growth-curve/spaces/AjOYV4v43T0TFlSRqgAp/pages/3JJDRTohjXVJqe8Q6JDr#v7.6a)
* SW: ≥ v2026.02a
* Marker (not supplied)
* Vortex mixer (not supplied)
* Rack for holding 15 mL centrifuge vials (not supplied)
* Centrifuge vial, 15 mL (supplied) or 5 mL (not supplied)
* P200 pipette (not supplied)
* Sterile pipette tips (not supplied).
* Serological pipette, preferably 2 or 5 mL (not supplied).
* Serological pipette controller (not supplied).
* Shaking incubator (not supplied).
* Countdown timer (not supplied).
## Reagents
* Certified reference material (CRM): [*Escherichia coli* WDCM 00013 Vitroids™](https://www.sigmaaldrich.com/DK/en/product/sial/vt000137) (not supplied).
* 250 mL shake flask with 50 mL autoclaved LB medium. Preferably [baffled shake flask with vented cap](https://pipette.com/dwk-life-sciences-llc-354239.html).
* [Dilution vials](/item-register/vials-flasks-and-liquids/dilution-vial) ready-to-use.
* [Disinfection vial](/item-register/vials-flasks-and-liquids/disinfection-vial).
## Summary
With these things in hand, you are ready to for the next step: [Inoculate LB medium](/tutorials/measure-vitroid-tm-growth-curve/inoculate-lb-medium).
# Inoculate LB medium
The first objective is to inoculate the growth medium. The step-by-step procedure below provides two options. Click the tab headers to switch between them.
Our recommended approach is to inoculate the culture using a Vitroids™ disc. This method has been thoroughly tested, and the expected timeline is well established.
Alternatively, you can inoculate the culture using an in-house glycerol stock. Because we have no information about the characteristics or storage conditions of your glycerol stocks, we cannot guarantee either the timeline or the outcome. If you already have established protocols for preparing *E. coli* overnight cultures, feel free to follow those and use our tutorials as inspiration.
## Step by step
{% tabs %}
{% tab title="Preferred: Vitroids™ disc" icon="flying-disc" %}
{% stepper %}
{% step %}
### Start the inoculation early
Start the inoculation procedure below as early as possible during your workday.
Alternatively, have an :earlybirds: early bird colleague to complete these steps before you arrive.
{% endstep %}
{% step %}
### Label the shake flask
Use a marker to write *E. coli*ATCC®25922 and inoculation time on the 250 mL baffled shake flask.
{% endstep %}
{% step %}
### Transfer Vitroids**™** disc
Use proper aseptic technique and transfer the disc to the shake flask. The flask already contains 50 mL of autoclaved LB medium.
The video below demonstrates manual shaking and a 5-minute dissolution step. You may skip these steps and allow the incubator shaker to dissolve the disc during the next step.
Demonstration of the dissolution of a Vitroids™ disc in growth medium. You can proceed directly step 4 after transferring the disc (You don't need to wait for the dissolution to happen).
{% endstep %}
{% step %}
### Start the incubation
Transfer the shake flask to the shaking incubator. Incubate at 37 °C, 200 RPM.
{% endstep %}
{% endstepper %}
{% endtab %}
{% tab title="Alternative: In-house glycerol stock" icon="snowflakes" %}
{% stepper %}
{% step %}
### Label the shake flask
Use a marker to write *E. coli,* in-house strain designation, and inoculation time directly on the 250 mL baffled shake flask containing 50 mL of LB medium.
{% endstep %}
{% step %}
### Thaw glycerol stock
Collect an *E. coli* glycerol stock from your -80 °C freezer and allow it to thaw for \~30 minutes at room temperature.
{% endstep %}
{% step %}
### Inoculate using \~0.1 % (V/V)
Transfer 50 µL glycerol stock to the flask.
{% endstep %}
{% step %}
### Start the incubation
Transfer the shake flask to the shaking incubator. Incubate at 37 °C, 200 RPM.
{% endstep %}
{% step %}
### Run BactoBox® measurements every 30 min
In the preferred version of this tutorial (Vitroids™ disc), the culture is incubated for \~5 hours to reach concentrations detectable by BactoBox® measurements.
**When using the in-house alternative (glycerol stock), perform measurements every 30 minutes.**
{% endstep %}
{% endstepper %}
{% endtab %}
{% endtabs %}
## Summary
You have now inoculated the LB growth medium. For the Vitroids™ disc procedure \~5 hours of incubation are required before the bacterial concentration is high enough for BactoBox® measurements. Proceed to [Prepare sample](/tutorials/measure-vitroid-tm-growth-curve/disaggregate-bacteria) for how to sample and disaggregate.
# Prepare sample
After \~5 hours of incubation, the cell concentration should be sufficient for BactoBox® measurements. The step-by-step guide below describes the sample preparation for all growth curve measurements.
{% hint style="success" icon="bacteria" %}
## Vortex thoroughly and vigorously
BactoBox® is a flow cytometer and performs best when cells are present as a single-cell suspension.
Because bacterial cells can stick together, thorough disaggregation is essential. One minute of vortexing may seem excessive, but it is necessary to achieve reliable measurements.
{% endhint %}
## Step by step
{% stepper %}
{% step %}
### Get a sample of the culture
Make 10 figure-eight swirls to resuspend the culture prior to sampling.
Use a serological pipette to aspirate 2 mL culture and transfer it to a 5 or 15 mL centrifuge vial.
{% endstep %}
{% step %}
### Disaggregate clumps of bacteria
Vortex the sample at maximum speed for 1 minute.
Ensure that a proper vortex forms inside the tube during mixing.
{% endstep %}
{% step %}
### Set a countdown for the next measurement
Set a 30-minute countdown to remind yourself to collect the next sample for the growth curve.
{% endstep %}
{% endstepper %}
## Summary
You have now collected a sample after \~5 hours of incubation and used vortex mixing to disaggregate bacterial clumps. Proceed to [1:100 dilutions](/tutorials/measure-vitroid-tm-growth-curve/1-100-dilutions) to determine the cell concentration using BactoBox®.
# 1:100 dilutions
The lag phase, acceleration phase, and early exponential growth phase are typically monitored using 1:100 dilutions prepared in 15 mL centrifuge tubes. The procedure for preparing these dilutions is described below.
Special emphasis is placed on proper vortexing of the 15 mL tubes, as inadequate mixing is one of the most common pitfalls in sample preparation for BactoBox® analyses.
## How to make 1:100 dilutions
1:100 dilutions are done by transferring 101 µL sample to 10 mL of diluent.
Tutorials 1 explained the importance of using the correct vortexing technique for 15 mL vials. If you need a refresher, expand the section below. Skip ahead if you've already mastered the technique.
Proper vortex technique
When vortexing is done correctly, a strong vortex forms in the vial within a few seconds. Follow the recommendations in the green information box and avoid the common mistakes demonstrated in the below slow-motion video (✔ demonstrates correct. ✘ demonstrates incorrect).
{% hint style="success" %}
## Good vortexing technique
:hashnode: Choose a mixer platform that is compatible with your vial. [Some platforms](https://www.scientificindustries.com/collections/vortex-mixers-shakers-accessories/products/3-inch-platform-with-rubber-cover) can accommodate multiple vial types and sizes.
:hand-point-down: Use a gentle grip on the vial cap while vortexing the tube. Start with a firm 3-finger hold on the cap and switch to a gentle press with one finger once the motion starts.
{% endhint %}
* **The left panel demonstrates proper vortexing technique:** The cap is initially held with three fingers to start the motion, after which a single finger applies light downward pressure while allowing the tube to vortex freely.
* **The center panel demonstrates improper technique:** The vial is held too tightly using a central grip. The liquid bounces slightly, but the vortex does not form.
* **The right panel demonstrates improper technique:** A 50 mL vortex mixer attachment is used for the 15 mL tube. The vortex does not start even though the grip is correct.
## Step by step
{% stepper %}
{% step %}
### **Dilute 1:100 in BactoBox® diluent**
The below video demonstrates how to do a 1:100 dilution.
* Transfer 101 µL of your sample to 10 mL of diluent.
* Suspend ×10 to rinse the pipet tip for residual bacteria
* Vortex 10 seconds at maximum speed.
{% endstep %}
{% step %}
### **Measure the diluted sample on BactoBox®**
The below video demonstrates how to do the bacterial measurement
* Transfer the tubing kit to the 1:100 vial.
* Press Measure to start a new measurement and wait for the results.
{% endstep %}
{% step %}
### Create a table of the results
Note down your results in a table like the one below.
Measurement ID
Sampled at
Dilution
Cells/mL
Total/mL
Q17
11:30
100
89 000
130 000
{% endstep %}
{% step %}
### Clean the device
Transfer the tubing kit to the disinfection vial and press Clean .
{% endstep %}
{% step %}
### Repeat 1:100 dilutions for subsequent samples
When the 30-minute timer expires, repeat the same sampling, disaggregation, 1:100 dilution, measurement, and cleaning procedure for the subsequent samples.
Proceed to the [1:1,000 dilutions](/tutorials/measure-vitroid-tm-growth-curve/1-1-000-dilutions) when the measured cell concentration of the last sample is ≥ 1,500,000 cells/mL.
{% endstep %}
{% endstepper %}
## Summary
You measured the first time point after \~5 hours of incubation. Proceed to [1:1,000 dilutions](/tutorials/measure-vitroid-tm-growth-curve/1-1-000-dilutions) to determine cell concentration with BactoBox®.
{% hint style="info" icon="face-glasses" %}
## Workarounds to analyze dilute bacterial cultures
To keep this tutorial as simple as possible, only 1:100, 1:1,000, and 1:10,000 dilutions are used. However, custom diluents and dilution factors can also be applied.
Lower dilution factors provide higher sensitivity when measuring dilute bacterial cultures. See [Dilute 1:10](/advanced/advanced-sample-preparation/hit-the-right-concentration/dilute-1-10) for more information.
{% endhint %}
# 1:1,000 dilutions
As the bacteria multiply, the cell concentration increases, and a 1:100 dilution may eventually exceed the maximum concentration of 5,000,000 total cells/mL permitted for a BactoBox® measurement.
At this point, switch from the initial 1:100 dilution to a 1:1,000 dilution.
{% hint style="success" %}
## Rule of thumb for when to switch to 1:1,000 dilutions
To avoid the “too many particles” error during the exponential growth phase, follow this simple rule of thumb: if the previous 1:100 dilution measurement, taken approximately 30 minutes earlier, exceeded **1,500,000 cells/mL**, switch to a 1:1,000 dilution for the next measurement.
{% endhint %}
## How to make 1:1,000 dilutions
A 1:1,000 dilution is prepared by performing two sequential dilution steps. The first step is the standard 1:100 dilution, which is prepared by transferring 101 µL of sample to 10 mL of diluent.
The second step is a 1:10 dilution of the first vial. To prepare this dilution, transfer 1 mL of the 1:100 dilution to a second dilution vial. Because the dilution vials contain a standard volume of 10 mL, you must first discard 1 mL of diluent from the second vial.
## Step by step
{% hint style="success" icon="bacteria" %}
Remember to disaggregate: Vortex each culture sample for 1 min at max speed.
{% endhint %}
{% hint style="success" icon="stopwatch" %}
Set a 30 min timer after each sampling point.
{% endhint %}
{% stepper %}
{% step %}
### Dilute 1:100
Label a dilution vial 1:100 .
Vortex sample vial briefly (it has already been thoroughly vortexed when sampling).
Transfer 101 µL of your sample to the 1:100 vial. Pipette up and down ×10 in the 1:100 vial to rinse the pipette tip for residual bacteria.
Cap tightly and vortex 10 seconds at maximum speed.
{% endstep %}
{% step %}
### Dilute 1:1,000
Label a second dilution vial 1:1,000.
Aspirate and discard 1 mL diluent from the vial labeled 1:1,000
Transfer 1 mL of the 1:100 vial to the 1:1,000 vial. Pipette up and down ×10 in the 1:1,000 vial to rinse the pipette tip for residual bacteria. Cap tightly and vortex 10 seconds at maximum speed.
{% endstep %}
{% step %}
### Measure the diluted sample on BactoBox®
Transfer the tubing kit to the 1:1,000 vial.
Press Measure to start a new measurement and wait for the results.
Add your results to the measurement table.
{% endstep %}
{% step %}
### Clean the device
Transfer the tubing kit to the disinfection vial and press Clean .
{% endstep %}
{% step %}
### Repeat 1:1,000 dilutions for subsequent samples
Repeat the sampling, disaggregation, 1:1,000 dilution, measurement, and cleaning procedure for the next samples.
Proceed to the [1:10,000 dilutions](/tutorials/measure-vitroid-tm-growth-curve/1-10-000-dilutions) when the measured cell concentration of the last sample is ≥ 1,500,000 cells/mL.
{% endstep %}
{% endstepper %}
## Summary
Once the cell concentration increased it was necessary to switch from 1:100 to 1:1,000 dilutions. As the cell concentration continues to rise you will need to use [1:10,000 dilutions](/tutorials/measure-vitroid-tm-growth-curve/1-10-000-dilutions).
# 1:10,000 dilutions
When the culture reaches the deceleration and stationary growth phases you should switch to a 1:10,000 dilution.
The rule of thumb still applies when deciding whether to increase the dilution factor: switch to the next dilution scheme if the measurement obtained approximately 30 minutes earlier was ≥ 1,500,000 total cells/mL.
## How to make 1:10,000 dilutions
A 1:10,000 dilution is obtained by two sequential dilutions. First dilution is the standard 1;100 dilution done by transferring 101 µL to 10 mL of diluent. Second dilution is an additional 1:100 dilution from the first 1;100 vial.
## Step by step
{% hint style="success" icon="bacteria" %}
Remember to disaggregate: Vortex each culture sample for 1 min at max speed.
{% endhint %}
{% hint style="success" icon="stopwatch" %}
Set a 30 min timer after each sampling point.
{% endhint %}
{% stepper %}
{% step %}
### Label two dilution vials
Label first dilution vial 1:100.
Label second dilution vial 1:10,000.
{% endstep %}
{% step %}
### Dilute 1:100
Transfer 101 µL of your sample to the 1:100 vial.
Suspend ×10 to rinse the pipette tip for residual bacteria.
Vortex 10 seconds at maximum speed.
{% endstep %}
{% step %}
### Dilute 1:10,000
Transfer 101 µL of the 1:100 vial to the 1:10,000 vial.
Suspend ×10 to rinse the pipette tip for residual bacteria.
Cap tightly and vortex 10 seconds at maximum speed.
{% endstep %}
{% step %}
### Measure the diluted sample on BactoBox®
Transfer the tubing kit to the 1:10,000 vial.
Press Measure to start a new measurement and wait for the results.
Add your results to the measurement table.
{% endstep %}
{% step %}
### Clean the device
Transfer the tubing kit to the disinfection vial and press Clean .
{% endstep %}
{% step %}
### Repeat 1:10,000 dilutions for subsequent samples
Repeat the sampling, disaggregation, 1:10,000 dilution, measurement, and cleaning procedure for the next samples.
Conclude the sampling when two subsequent data points are within ±25%.
{% endstep %}
{% endstepper %}
## Summary
In deceleration and stationary phase 1;10,000 dilutions are necessary to hit the right concentration. By now you have gathered sufficient data points to plot a nice growth curve and calculate the growth rate. Proceed to [Data analysis](/tutorials/measure-vitroid-tm-growth-curve/data-analysis).
# Data analysis
Once the measurements have been completed, it is time to analyze the data. The following section explains how to plot the growth curve and determine the generation time.
We have included one of our own growth experiments as an Excel template.
{% file src="/files/XfY4WYnohyOmtMfOGR11" %}
{% hint style="info" icon="laptop-mobile" %}
## Plot data with ease in Access
To keep things simple, this tutorial demonstrates how to use BactoBox® as a stand-alone device.
Once you are familiar with the fundamentals, we recommend connecting BactoBox® to a computer and running the measurements through BactoBox® Access. This makes it easy to record sampling times and dilution factors and provides a real-time plot of your data.
See [Track growth curve](/mpd/mini-course/track-growth-curve) for a mini-course on tracking a growth curve using BactoBox® Access.
{% endhint %}
## Step by step
{% stepper %}
{% step %}
### Transfer your data to the template
Manually replace the data in the excel template with your data.
Remove any data old data entries that do not belong to your data
{% endstep %}
{% step %}
### Add information on inoculation time
Cell B14 is used for automatic calculation of incubation time. Update the cell with time stamp from your incubation time.
{% endstep %}
{% step %}
### Plot the data
The x/y plot in the template will automatically plot the data on a log-10 y-axis.
The below plot is from our run of the tutorial.
* The solid data points are used to generate an exponential fit.
* The hollow data points are excluded from the fit since they are already in deceleration/stationary stage.
{% endstep %}
{% step %}
### Identify the data belonging to exponential growth stage
Click one of the solid data points and use the mouse to select the x/y range where the data are on a straight line, i.e. the exponential growth stage. In this tutorial it will typically be from \~1×107 up to \~1×109 cells/mL.
If the exponential model provides a good fit to your data the R2 should be close to 1.
{% endstep %}
{% step %}
### Determine growth rate
The green number on the plot is the growth rate per hour calculated via the exponential fit.
{% endstep %}
{% step %}
### Calculate generation time
Change the number in cell B27 to the green number from your dataset.
This will automatically calculate the generation time in hours (cell C28) and minutes (cell C29). The SBT data set resulted in a growth rate of 1.72 h-1 and a generation time of 24.2 min.
{% endstep %}
{% endstepper %}
## Summary
Here we detailed how to plot data and calculate growth rates and generation times. To keep things simple, we used manual data entry in a classic Excel template.
On the next page, [Summary](/tutorials/measure-vitroid-tm-growth-curve/summary), we will recap the tutorial and provide inspiration for the next steps in your BactoBox® journey.
# Summary
This tutorial used an *E. coli* Vitroids™ disc to inoculate a shake flask containing LB medium. The initial cell concentration is below the limit of quantification, and approximately 5 hours of incubation are required before the culture can be reliably measured using a 1:100 dilution.
Although it may be tempting to use a lower dilution factor, this will typically result in conductivity errors when using the standard diluent because most growth media have relatively high conductivity.
As the cell concentration increases, the initial 1:100 dilution scheme is replaced by 1:1,000 and later 1:10,000 dilutions.
## Screening for suitable dilution factors
Selecting an appropriate dilution is often the most challenging aspect of BactoBox® measurements. We recommend using the following screening approach.
Prepare a dilution series up to a 1:10,000 dilution and begin by measuring the most diluted sample.
* If the 1:10,000 dilution yields a valid cell concentration, no further measurements are required.
* If the measured concentration is below 30,000 cells/mL, repeat the measurement using the 1:100 dilution instead.
The advantage of measuring the most diluted sample first is that it minimizes the risk of clogging the external filter.
{% hint style="success" %}
## Measure the lowest dilution first when screening for suitable dilution factors
For cultures with unknown bacterial concentration you often screen, try a 1:10,000 dilution first. If the cell concentration is < 30,000 try a 1:100 dilution.
{% endhint %}
## BactoBox® skills
After successfully completing this tutorial you have acquired the following skills:
:bacteria: Disaggregrate bacterial clumps by vortexing
:square-bolt: Hit the right conductivity
:bullseye-arrow: Hit the right concentration
:chart-line-up: Plot a growth curve
:calculator-simple: Calculate growth rate and generation time
## Next steps
Tutorials 1, 2, and 3 provide the basic skills needed to use BactoBox® as a stand-alone device.
After successfully completing these tutorials, you are ready to move on to more advanced studies involving your own cultures and growth media. See [Workflows](/mpd/workflows/bioprocess-workflows) for inspiration on potential use cases.
We recommend you see see the [Track growth curve](/mpd/mini-course/track-growth-curve) mini course for a brief introduction to tracking growth curves via Access. This provides a powerful way to see your growth curves in real time and skip the manual logs.
# Best practices
How to take care of your BactoBox® and get the most out of it.
## Maintenance
Proper maintenance require yearly, weekly, and daily maintenance. Here is an overview:
See the [Maintenance](/maintenance/maintenance) page for more best practices and recommendations.
### Best-practice example
Here is an example of best-practice BactoBox® use throughout a week:
## Access
BactoBox® comes with the [Access](/software/access/access) user interface, which helps you get the most out of BactoBox®. Access runs on your own computer and [connects to BactoBox® over USB](/best-practices/connect-to-access).
Among other things, you can do the following through Access:
* Download measurement files
* Customize gating
* Update your BactoBox® software
* Send device data bundles to SBT
{% content-ref url="/pages/I55cnLNxocdOs2q5EV2y" %}
[Connect to Access](/best-practices/connect-to-access)
{% endcontent-ref %}
{% content-ref url="/pages/ju8GX0Rmv7wK1yVatiFr" %}
[Update BactoBox® software](/best-practices/update-bactobox-r-software)
{% endcontent-ref %}
## BactoBox® in your inbox
Get product updates, pro tips, and workflows directly in your inbox.
{% @mailchimp/mailchimpSubscribe cta="Subscribe to our newsletter" listId="e5e5767f96" %}
# Connect to Access
## Quick clip :zap:
## Get things ready
You need these items:
* 1 × [BactoBox®](/item-register/bactobox-r/bactobox-r)
* 1 × Computer with:
* USB Type-A port
* Windows 7, 10, 11, or later
* 1 × [USB cable](/item-register/accessories/inlet-outlet-cover-1) (supplied with your BactoBox®)
{% hint style="danger" %}
### Access does not work with macOS
We do not support *any* version of macOS.
{% endhint %}
{% hint style="info" %}
### Experimental support for Linux
We do not officially support Linux but we do provide *experimental* support. It may or may not work. It may also suddenly stop to work.
{% endhint %}
## Step by step
{% stepper %}
{% step %}
#### Insert USB cable into BactoBox®
Insert the micro-B end of the USB cable into the port on the side of your BactoBox®.
{% hint style="danger" %}
**Connect only when BactoBox displays Ready**
Connect only when your BactoBox® shows the *Ready* screen. Connecting during a measurement may cause disruptions.
{% endhint %}
{% endstep %}
{% step %}
#### Insert USB cable into computer
Connect the other end to a USB Type-A port in your computer.
{% endstep %}
{% step %}
#### Open file with Access
After a short while, your computer recognizes your BactoBox® as a USB disk drive. For example, it may show up as `D:\`.
Your computer shows a window like this:
File browser that shows BactoBox® files
Open the file *SBT Access.html*. This opens Access in your browser.
{% hint style="danger" %}
### Nothing happens?
Make sure BactoBox is turned on.
Check the USB cable at both ends. Make sure that it is fully inserted.
Try to open directly in your browser.
{% endhint %}
{% endstep %}
{% step %}
#### That is it!
You now have Access open. It looks something like this:
The Software update page in Access.
{% hint style="danger" %}
### Ran into trouble?
See [Can not connect to Access](/troubleshooting/power-on-works-but.../can-not-connect-to-access) for guidance.
{% endhint %}
{% endstep %}
{% endstepper %}
# Update BactoBox® software
We continuously improve the software for your BactoBox®
This page teaches you how to download and install the latest BactoBox® software update.
{% hint style="success" %}
## Latest BactoBox® software is space.vars.SW\_LATEST\_RELEASED\_VER
Keep your BactoBox® software up to date. Follow [update BactoBox® software](/best-practices/update-bactobox-r-software).
See what [changed in space.vars.SW\_LATEST\_RELEASED\_VER](https://github.com/sbtinstruments/docsites/tree/main/software/bactobox-r-software-changelog/README.md) and the [software update FAQ](https://github.com/sbtinstruments/docsites/tree/main/software/bactobox-r-software-changelog/software-update-faq.md).
{% endhint %}
## Quick clip :zap:
From software **v2026.06**, Access fetches and installs the **latest software from the cloud** in one click. On earlier software this option is not available yet, so you install a specific **`.swu` file** instead (the steps below). That also brings you up to a version that has the cloud option.
{% columns %}
{% column %}
From v2026.06: Software update → Latest → Install latest.
{% endcolumn %}
{% column %}
Before v2026.06: install a specific .swu file, as shown in the steps below.
{% endcolumn %}
{% endcolumns %}
## Step by step
{% stepper %}
{% step %}
#### Download the software update file
Click the button below to download the latest [software update (.swu) file](/software/file-formats/swu-file) to your computer:
Download v2026.06
This software update file works for [hardware v7.5 and later](/item-register/bactobox-r/bactobox-r-hardware-changelog).
{% hint style="success" %}
## Installing an alpha or beta version? Create a backup first!
[Create a backup](https://github.com/sbtinstruments/docsites/tree/main/software/access/pages/backup-and-restore.md) before you install [pre-release software](https://github.com/sbtinstruments/docsites/tree/main/software/bactobox-r-software-changelog/pre-release-software.md). This ensures that your data is safe from any [bugs](https://en.wikipedia.org/wiki/Software_bug) in the pre-release software.
{% endhint %}
{% endstep %}
{% step %}
#### Connect to Access
Follow the instructions in [Connect to Access](/best-practices/connect-to-access).
{% endstep %}
{% step %}
#### Navigate to the *Software update* page
Click *Software update* in Access' [page navigation](/software/access/components/page-navigation).
Access with the Software update page open.
{% endstep %}
{% step %}
#### Update the software on BactoBox®
Drag-and-drop the software update file into the field.
**The software update starts immediately and takes 2–5 min.**
{% endstep %}
{% step %}
#### That is it!
When the installation is done, your BactoBox® prompts you to restart the device.
On the first start after a software update, you may see a data migration phase. This can take another 5–10 min. Once migration is done, your BactoBox® shows the *Ready* screen as usual.
{% hint style="danger" %}
### Ran into trouble?
See [Can not connect to Access](/troubleshooting/power-on-works-but.../can-not-connect-to-access) for guidance.
{% endhint %}
{% endstep %}
{% endstepper %}
# Maintenance
Our recommendations to take care of your BactoBox®
Your BactoBox® requires periodic maintenance.
Use this page as a checklist.
Beyond the periodic maintenance, there are also specific events that require further action. See the *When to X* sections below for further detail.
## Schedule
Proper maintenance require yearly, weekly, and daily maintenance. Here is an overview:
### Best-practice example
Here is an example of best-practice BactoBox® use throughout a week:
## When to do preventive maintenance
Schedule your BactoBox® for [preventive maintenance](https://github.com/sbtinstruments/docsites/tree/main/items/service-agreement/preventive-maintenance.md) in these cases:
* **At least once a year.**
* When the [Check engine light](/maintenance/check-engine-light) shows.
{% hint style="success" %}
## Preventive maintenance is part of your [service agreement](/item-register/service-agreement/service-agreement)
Your [Service agreement](/item-register/service-agreement/service-agreement) offers 24 month warranty and includes 2 × [https://github.com/sbtinstruments/docsites/tree/main/items/service-agreement/preventive-maintenance.md](https://github.com/sbtinstruments/docsites/tree/main/items/service-agreement/preventive-maintenance.md "mention").
{% endhint %}
## When to qualify your BactoBox®
[Qualify](/getting-started/qualification) your BactoBox® in these cases:
* **At least once a year.**
* When you relocate your BactoBox® setup.\ Example: You install your BactoBox® for the very first time.\ Example: You ship your BactoBox® to another physical location.\ Example: You get your BactoBox® back from service/repair at SBT.
{% hint style="info" %}
## Difference between QC test and qualification
**When you** [***qualify***](/getting-started/qualification) **your BactoBox® setup, always use an SBT dilution vial (bundled as `DV01`).**
For the regular (weekly) QC test, use the dilution vial that you intend to use going forward for your own measurements. This depends on your use case and may be either of:
* SBT dilution vial (bundled as `DV01`)
* DIY[^1] dilution vial ([made with the dilution dispenser](/item-register/do-it-yourself-diy/diy-dilution-dispenser/make-diy-dilution-vial))
Besides the choice of dilution vial, qualification and QC test are the same.
{% endhint %}
## When to QC test
Perform a [QC test](/getting-started/qc-test) in these cases:
* **At least once a week.**
* After a [deep clean](/maintenance/deep-clean-1) (of any length).
* When you change your BactoBox® setup.\ Example: You replace the flow cell.\ Example: You replace the external filter.\ Example: You replace the tubing kit.
* When you switch from the SBT dilution vial to the [DIY dilution vial](https://github.com/sbtinstruments/docsites/tree/main/items/do-it-yourself-diy/diy-dilution-dispenser/README.md).
* As part of [qualification](/getting-started/qualification).
## When to deep clean
[Deep clean](/maintenance/deep-clean-1) your BactoBox® in these cases:
* **At least once a week.** Use the 15-minute option.
* To recover a flow cell that failed the QC test.\ Try with the 15-minute option first. If the issue persists, try the 10-hour option.
See [Deep clean explained](/maintenance/deep-clean-1/deep-clean) for more information about what a deep clean does.
## When to clean
[Clean](/maintenance/clean-the-setup) your BactoBox® in these cases:
* **After each measurement.**
* After a deep clean.
* After 30+ minutes of inactivity.\ The BactoBox® software automatically prompts you to do this.
{% hint style="info" %}
### Why clean?
**Clean serves two purposes: Avoid growth inside your BactoBox® and avoid carry-over effects between measurements.**
Clean your BactoBox® to ensure that you only measure on your sample itself—not any residual particles or growth from previous measurements. E.g., to avoid [carry-over](https://en.wikipedia.org/wiki/Carryover_effect), biofilm, and the like.
Your BactoBox® has microfluidic flow paths that the liquid, bacteria, and particles pass through. It's essential to keep these paths clear of residue, microorganisms, biofilm, and other contaminants. In general, to avoid conditions that promote microbial growth.
{% endhint %}
## When to replace the syringe filter
[Replace the syringe filter](https://github.com/sbtinstruments/docsites/tree/main/items/do-it-yourself-diy/diy-dilution-dispenser/replace-syringe-filter.md) of your [dilution dispenser](https://github.com/sbtinstruments/docsites/tree/main/items/accessories/dilution-dispenser.md) in these cases:
* **At the start of each day**
* When it is difficult to dispense liquid (because the syringe filter clogs)
{% hint style="success" %}
### [**Replace the syringe filter**](/item-register/do-it-yourself-diy/diy-dilution-dispenser/replace-syringe-filter) **at the start of each day**
Even within a single day, the syringe filter clogs after repeated use. If it feels like you need to press harder to dispense, replace the syringe filter.
Each syringe filter typically clogs after 200–500 mL of liquid dispensed (20–50 dispenses of 10 mL).
{% endhint %}
{% hint style="warning" %}
### **Indications of clogged syringe filter**
A squeaking sound may indicate a clogged syringe filter. [Replace the syringe filter](/item-register/do-it-yourself-diy/diy-dilution-dispenser/replace-syringe-filter) if squeaking occurs to maintain accurate and fluent dispensing.
{% endhint %}
## When to replace the external filter
Replace the [external filter](https://github.com/sbtinstruments/docsites/tree/main/items/consumables/external-filter.md) in these cases:
* When you replace the flow cell.
See [Install tubing kit](/getting-started/installation/install-tubing-kit) for instructions on how to replace the external filter
## When to replace the flow cell
[Replace the flow cell](/getting-started/installation/install-flow-cell) in these cases:
* After 250 measurements.\ The BactoBox® software automatically prompts you to do this.
{% hint style="success" %}
[Clean](/maintenance/clean-the-setup) the flow cell before you remove it.
{% endhint %}
{% hint style="success" %}
### Replace the external filter when you replace the flow cell
It is good practice to replace the [external filter](https://github.com/sbtinstruments/docsites/tree/main/items/consumables/external-filter.md) whenever you replace the flow cell. See [Install tubing kit](https://help.sbtinstruments.com/getting-started/installation/install-tubing-kit) for details.
**Run a** [QC test](/getting-started/qc-test) **when you replace the external filter.**
{% endhint %}
## When to replace the tubing kit
It is not necessary to replace the [tubing kit](/item-register/accessories/tubing-kit).
## Found a stain on your BactoBox®?
Wipe the surface of your BactoBox® device with a clean, soft cloth and 70 % isopropanol (or another alcohol).
## Spilled liquid on your BactoBox®?
Power off the device immediately. Wipe it off with a damp, soft cloth. Wait for it to dry thoroughly.
[^1]: Do it yourself
# Clean
## Get things ready
You need these items:
* 1 × [BactoBox® setup](/item-register/bactobox-r/bactobox-r)
* 1 × [disinfection vial](/item-register/vials-flasks-and-liquids/disinfection-vial)
## Step by step
{% stepper %}
{% step %}
#### Use disinfection vial
Place a disinfection vial in the vial rack. Transfer the tubing kit to the disinfection vial.
{% hint style="success" %}
You can reuse the same disinfection vial multiple times. We recommend at most 10 uses per vial.
{% endhint %}
{% hint style="warning" %}
### The disinfection vial contains flammable liquid
The disinfection vial contains 70 % denatured alcohol. This liquid is flammable and an eye irritant.
Keep it away from heat, sparks, open flames, and hot surfaces. Avoid contact with eyes.
Refer to the Material Safety Data Sheet (MSDS) for more information.
{% endhint %}
{% endstep %}
{% step %}
#### Press the Clean button
Press the Clean button to clean the BactoBox® setup.
Press the Clean button
The display shows a countdown that tells you when the *Clean* program is done.
Countdown on the Cleaning screen.
{% endstep %}
{% step %}
#### That is it!
After about 1 min, the *Clean* program is done. You return to the *Home* screen.
{% endstep %}
{% endstepper %}
## When to clean
[Clean](/maintenance/clean-the-setup) your BactoBox® in these cases:
* **After each measurement.**
* After a deep clean.
* After 30+ minutes of inactivity.\ The BactoBox® software automatically prompts you to do this.
See [Maintenance](/maintenance/maintenance) for more information.
# Deep clean
## Get things ready
You need these items:
* 1 × [BactoBox® setup](/item-register/bactobox-r/bactobox-r)
* 1 × [deep clean vial](/item-register/vials-flasks-and-liquids/deep-clean-vial)
## Step by step
{% stepper %}
{% step %}
#### Switch to the deep clean vial
Transfer the tubing kit to the deep clean vial.
{% endstep %}
{% step %}
#### Start the *Deep clean* program
Press and hold Measure for 2 seconds to enter the *Programs* menu.
Use the navigation buttons (OK←∨∧) to select the *Deep clean* program.
Press OK to continue.
{% endstep %}
{% step %}
#### Select duration
Use the navigation buttons (OK←∨∧) to select the *Deep clean* duration.
Press OK to start the *Deep clean* program.
{% endstep %}
{% step %}
#### That is it!
The *Deep clean* program is done. **Discard the deep clean vial.** It is for *single use* only.
{% hint style="success" %}
Perform a [QC test](/getting-started/qc-test) after the *Deep clean* program is done.
{% endhint %}
{% endstep %}
{% endstepper %}
## When to deep clean
[Deep clean](/maintenance/deep-clean-1) your BactoBox® in these cases:
* **At least once a week.** Use the 15-minute option.
* To recover a flow cell that failed the QC test.\ Try with the 15-minute option first. If the issue persists, try the 10-hour option.
See [Maintenance](/maintenance/maintenance) for more information.
## Want to know more
See [Deep clean explained](/maintenance/deep-clean-1/deep-clean) for additional detail.
# Deep clean explained
## How to deep clean
See our [deep clean tutorial](/maintenance/deep-clean-1).
## When to deep clean
[Deep clean](/maintenance/deep-clean-1) your BactoBox® in these cases:
* **At least once a week.** Use the 15-minute option.
* To recover a flow cell that failed the QC test.\ Try with the 15-minute option first. If the issue persists, try the 10-hour option.
See [Maintenance](/maintenance/maintenance) for more information.
## Why deep clean is important
Your BactoBox® setup might contain hard-lived contaminants such as limescale or biofilm.
{% columns %}
{% column %}
Limescale as known from your tea kettle.
{% endcolumn %}
{% column %}
Biofilm
{% endcolumn %}
{% endcolumns %}
{% hint style="success" %}
Follow our guidelines (see [Maintenance](/maintenance/maintenance)) to avoid build-up of hard-lived contaminants such as limescale and biofilm.
{% endhint %}
Especially the microfluidic flow paths inside the flow cell are sensitive to contaminants. The smallest cross-sectional area is 10×25 µm, which is approximately one-tenth the width of a human hair. Even relatively light contamination may influence the performance of the flow cell.
## What deep clean does
The *Deep clean* program scrubs your BactoBox® setup with our [deep clean vial](/item-register/vials-flasks-and-liquids/deep-clean-vial). The deep clean vial contains a pepsin/HCl solution. In other industries, this solution is used to remove protein and limescale deposits from pH-electrodes made of glass. This solution also effectively cleans the flow cell in your BactoBox® setup.
The *Deep clean* program soaks the flow cell in the solution. It runs the pump intermittently to refresh the liquid in contact with the flow cell.
# Check engine light
Just like your car, BactoBox® has a check engine light
{% hint style="success" %}
When the [Check engine light](/maintenance/check-engine-light) appears, schedule [https://github.com/sbtinstruments/docsites/tree/main/items/service-agreement/preventive-maintenance.md](https://github.com/sbtinstruments/docsites/tree/main/items/service-agreement/preventive-maintenance.md "mention") as soon as possible.
{% endhint %}
The check engine light appears when your BactoBox® requires [Preventive maintenance](/item-register/service-agreement/preventive-maintenance).
The check engine signals that your BactoBox® setup *approaches* a failure mode. It allows you to schedule preventive maintenance before it is too late.
The check engine appears in the upper right corner in [Dash](/software/dash/user-interface).
{% hint style="success" %}
## Preventive maintenance is part of your [service agreement](/item-register/service-agreement/service-agreement)
Your [Service agreement](/item-register/service-agreement/service-agreement) offers 24 month warranty and includes 2 × [https://github.com/sbtinstruments/docsites/tree/main/items/service-agreement/preventive-maintenance.md](https://github.com/sbtinstruments/docsites/tree/main/items/service-agreement/preventive-maintenance.md "mention").
{% endhint %}
## What triggers the check engine light
The check engine light appears if any of these are true:
* It's been a year since the last [Preventive maintenance](/item-register/service-agreement/preventive-maintenance)
* You've run 2000 programs (e.g., measurements) since the last [Preventive maintenance](/item-register/service-agreement/preventive-maintenance)
* The latest QC test shows that your BactoBox® setup approaches a failure mode
## Can I use my BactoBox® while the check engine light shows?
**Yes.** The check engine light signals that your BactoBox® *approaches* a failure mode. In other words, your BactoBox® setup still works as intended even when the check engine light appears.
{% hint style="success" %}
When the [Check engine light](/maintenance/check-engine-light) appears, schedule [https://github.com/sbtinstruments/docsites/tree/main/items/service-agreement/preventive-maintenance.md](https://github.com/sbtinstruments/docsites/tree/main/items/service-agreement/preventive-maintenance.md "mention") as soon as possible.
{% endhint %}
# Shipping and storage
## Prepare BactoBox® for storage
The shutdown procedure is simple:
1. Clean the setup (See [Clean](/maintenance/clean-the-setup))
2. Power off the BactoBox® (flip the rocker switch)
Leave the tubing kit in the disinfection vial and keep the flow cell in BactoBox®.
## Prepare BactoBox® for long-term storage
Step-by-step guide:
1. Clean the setup (See [Clean](/maintenance/clean-the-setup))
2. Power off the BactoBox® (flip the rocker switch)
3. Disconnect [tubing kit](/item-register/accessories/tubing-kit) and place it in an [empty vial](/item-register/vials-flasks-and-liquids/empty-vial) for storage
4. Place the [inlet-outlet cover](/item-register/accessories/inlet-outlet-cover) (the grey protective cap) over BactoBox'® inlet and outlet
5. Preferably store the BactoBox® in the original box together with the [accessories](/item-register/accessories/accessories)
{% hint style="success" %}
It is okay (but not mandatory) to leave a flow cell clicked in place during storage.
{% endhint %}
## Prepare BactoBox® for shipping
Step-by-step guide:
1. Clean the setup (See [Clean](/maintenance/clean-the-setup))
2. Disconnect [tubing kit](/item-register/accessories/tubing-kit) and place it in an [empty vial](/item-register/vials-flasks-and-liquids/empty-vial) for storage
3. Place the [inlet-outlet cover](/item-register/accessories/inlet-outlet-cover) (the grey protective cap) over BactoBox'® inlet and outlet
4. Place the BactoBox® in the original box and then in a padded cardboard box for shipping
{% hint style="success" %}
It is okay (but not mandatory) to leave a flow cell clicked in place during shipping.
{% endhint %}
{% hint style="danger" %}
Do not ship the BactoBox® only in the original box as it does not provide sufficient protection when shipped.
{% endhint %}
## Bring BactoBox® out of storage
Whether you want to bring BactoBox® out of short-term or long-term storage, the procedure is the same: [Qualify your BactoBox® setup](/getting-started/qualification).
# Microbial process development
Use your BactoBox® in real-world bioprocess workflows. See exactly what your culture is doing over time.
Get an answer right away from our help center AI agent
Cover image
:burger:Screen growth mediaMPD-3
Use BactoBox® direct cell counts to screen growth media for their ability to support high cell concentrations.
# Why use BactoBox®
The short answer: Because BactoBox® measures real [bacterial growth](/mpd/cell-growth/growth-phases). No proxies. No guesswork.
BactoBox® helps whether you are trying to:
* [Broken mention](broken://pages/C6YcpyFfVcFZsMakakP2)
* [Broken mention](broken://pages/p621fpSADrEkfyuT5b5s)
* [Broken mention](broken://pages/Tvr51NO5VXFx6QvI0tg8)
BactoBox® fits into all key steps in your upstream [bioprocess](/mpd/encyclopedia/bioprocess): 🌱 Seed, 🌿 growth, and 🌽 harvest.
## With and without BactoBox®
Once you use BactoBox® in your workflows, it's easy to forget what life was like before. This table serves as a reminder.
| Workflow | Without BactoBox® | With BactoBox® |
| ---------------------------------------------------------------------------------------------------- | -------------------------------------------------------------------------------------------------- | ------------------------------------------------------------------------------------ |
|
Optimize starter culture ⚙️ Optimization 🌱 Seed
| TODO | One-shot setup of high quality culture (OptiSeeding) |
|
Inoculate an overnight culture ⚙️ Optimization 🌱 Seed
| | |
|
Track growth curve 💡 Insight 🌿 Growth
|
Use plate counts (CFU), which is time consuming. Use OD, which is an indirect proxy.
| Use cells/mL, which is the direct cell count. |
|
Assess growth rate in different media 💡 Insight 🌿 Growth
| | |
|
Assess carrying capacity in different media 💡 Insight 🌿 Growth
| | |
|
Optimize harvest point ⚙️ Optimization 🌽 Harvest
| TODO | Get X % better yield/titer |
|
Decide when to harvest during a cultivation ⚙️ Optimization 🌽 Harvest
| | |
|
Predict when to harvest during a production ⏲️️ Prediction 🌽 Harvest
| | Just a couple of initial measurements predict the optimal harvest time for the batch |
|
Assess onset and conclusion of sporulation 💡 Insight 🌽 Harvest
| | |
| **Consistent bacterial cell banking** | | |
| **Design seed train** | | |
| **Optimize feed profile in fed-batch** | | |
| **Assess exact number of doublings in CRISPRi workflows** | | |
# Track growth curve
MPD-7 summarized to key steps. The Access workflow, step by step.
This is the [MPD-7 workflow](/mpd/workflows/track-growth-curve) summarized to key steps.
Where you end up: a full growth curve, plotted live in Access.
***
{% stepper %}
{% step %}
### Open Access
Connect your BactoBox® to your computer and open Access in your browser. The familiar BactoBox® buttons move onto your screen.
Plug in your BactoBox® and open Access.
{% endstep %}
{% step %}
### Create a measurement group
Start a *growth curve* measurement group. Every sample lands in the same place, ready to plot.
One measurement group collects every sample.
{% endstep %}
{% step %}
### Measure and annotate each sample
Set the dilution factor and a label, then click Measure to run the measurement. Access works out the concentration in your flask. Use the same label for every sample in the series, for example `E_coli_growth`, then click that label above the chart to watch your curve build.
{% hint style="info" %}
#### The label is what ties the curve together
The label is not a free-form note. Access plots every measurement that shares a label as one growth curve, and places each point on the time axis by its measurement time. So give every sample in the series the same label. If you change the label between samples, you get scattered single points instead of one curve.
{% endhint %}
Annotate, measure, and the curve builds point by point.
{% endstep %}
{% step %}
### Watch it reach stationary
Keep sampling, with a Clean after each measurement. Continue to see the onset of stationary phase.
The curve flattens as the culture reaches stationary phase.
{% endstep %}
{% endstepper %}
***
## Do it yourself
That is the whole loop. The full guide walks you through sample prep, dilution schemes, and the medium check, step by step.
{% content-ref url="/pages/I0HcPYpXvMRDTZBltrbb" %}
[Track growth curve](/mpd/workflows/track-growth-curve)
{% endcontent-ref %}
# Screen growth media
MPD-3 summarized to key steps. Screen growth media with BactoBox®.
This is the [Screen growth media workflow](/mpd/workflows/best-medium) summarized to key steps. You measure the same strain grown in different media and pick the one that reaches the highest cell concentration.
Where you end up: the growth medium with the highest cells/mL wins.
***
{% stepper %}
{% step %}
### Grow the media you want to compare
Inoculate at least two growth media with the same starter culture, about 24 hours ahead, so every culture sits in its stable plateau at the first measurement.
Same starter culture, different media.
{% endstep %}
{% step %}
### Create a measurement group
Open Access and create a *basic* measurement group so every reading lands in the same place.
Plug in your BactoBox® and open Access.
{% endstep %}
{% step %}
### Measure each medium
Sample 1 mL, vortex 1 minute, dilute 1:100 then 1:10,000, and measure. Label each reading with the medium (for example `LB` or `TSB`) and set the dilution to 10,000. Repeat for every medium, at three time points across the plateau.
A 1:10,000 dilution lands the dense culture in range.
{% endstep %}
{% step %}
### Identify the best medium
Export the data, average the three time points per medium, and compare. The highest mean cells/mL is the best medium.
Terrific broth reaches the highest cell concentration here.
{% endstep %}
{% endstepper %}
***
## Do it yourself
That is the whole loop. The full workflow covers the sampling schedule, the dilution steps, and the data analysis, step by step.
{% content-ref url="/pages/LQH8L6gm4m1NETVVaulL" %}
[Screen growth media](/mpd/workflows/best-medium)
{% endcontent-ref %}
# Bioprocess workflows
In practical terms, a workflow helps you accomplish a goal repeatedly.
We focus on workflows in [Microbial process development](/mpd). In other words, workflows that results in a [Bioprocess](/mpd/encyclopedia/bioprocess).
Each workflow addresses a concrete problem with clear steps and reproducible outcomes. The goal is to help you reduce uncertainty, improve culture consistency, and make better decisions in your bioprocess.
## Workflow categories
We categorize workflows by:
* The workflow result: 💡 Insight, ⚙️ optimization, ⏲️ prediction
* Bioprocess step: 🌱 Seed, 🌿 growth, and 🌽 harvest
* Required BactoBox® skill level: 🎒 No skill requirement, 🎓 Intermediate, 🧩 Advanced, 🥊 Champion
* Required microbiology skill level: 🎒 No skill requirement, 🎓 Intermediate, 🧩 Advanced
* Time to complete: ⏳️ Given in hours
# Optimize your bioprocess
An optimization workflow helps you *decide* between multiple options.
In practice, an optimization workflow solves practical challenges in microbial process development such as:
* [How do I find the optimal seeding ratio for the initial cryo stock expansion?](/mpd/workflows/identify-optimal-inoculum-size)
* Which growth media should I choose?
* [When should I harvest my batch cultivation to get the highest CFU/mL?](/mpd/cell-growth/harvest-window)
# Understand your bioprocess
# Get starter culture
MPD-5
There are several ways to get a starter culture for your bioprocess. We use a specific *input* material and a defined *workflow*, to transform initially dormant cells into active cells with enhanced replication capabilities.
In the table, we summarize the pros and cons of different types of input materials. Details are given in the sections below. You can also jump straight to the step by step workflows in the subpages:
* [From cryo stock](/mpd/workflows/get-starter-cultures/from-cryo-stock)
* [From agar plate colony](/mpd/workflows/get-starter-cultures/from-agar-plate-colony)
* [From liquid culture](/mpd/workflows/get-starter-cultures/from-liquid-culture)
* [From CRM product](/mpd/workflows/get-starter-cultures/from-crm-product)
Most workflows result in starter cultures after overnight incubation, but by changing the seeding concentration you will often be able to get a same-day starter cultures.
Pros
Cons
:snowflake: Cryo stock
Locks down genetic consistency
Enables easy standardization
Simple protocol with few steps
Potential risk of contamination
Concentration of culturable cells may change during storage and re-stocking
Requires a -80 °C freezer
:bowling-ball: Agar plate colony
Simple, low-cost
Low risk of contamination
Possible to add selective growth conditions and pick colonies with desired phenotype
Does not require -80 °C freezer
Adds an additional incubation step that delays the workflow
Challenging to get accurate counts because bacteria may be partially encased in biofilm and or agar.
:flask: Liquid culture
Fast and convenient
Accurate enumeration of total cell concentration
Does not require -80 °C freezer
Risk of contamination
Risk of mutations occurring over time if subcultivation is repeated extensively
May contain mixed populations
:capsule: CRM products
Convenient, ready-to-use formats for consistency and traceability
Often comes with well-defined bacterial concentration
Higher cost
Limited shelf life
May be difficult to distinguish between live and dead cells.
## :snowflake: Cryo stock
Cryo stocks are one of the cornerstones for good manufacturing practices (GMP) in biotechnology mainly because you can effectively "reset" a culture to a consistent starting point each time. This ensures low passage numbers and thereby minimizes the risk of genetic drift. Cryo stocks are convenient, as a large bank of individual vials can be stored, with a vial being used each time a culture is required.
Using cryo stocks as input also comes with some drawbacks: The freezers are relatively expensive and culturability may drift over time which leads to inconsistent starter cultures. When the stock is depleted, re-stocking is necessary, which may lead to differences in growth dynamics. Finally, some of the individual cryo stocks may be contaminated - there is little change of spotting this contamination early because the cryo stock is added directly to the starter culture, .
## :bowling-ball: Agar plate colony
The [streak plate method](https://microbenotes.com/streak-plate-method-principle-methods-significance-limitations/) is a straightforward, cost-effective, and reliable technique for obtaining discrete colonies and verifying the purity of bacterial stocks. The primary advantage of using a colony as input material is the reduced risk of contamination.
A downside to the agar plate step is the potential for significant delays in the protocol, as bacteria need incubation time to form colonies. Additionally, accurate determination of bacterial concentration can be challenging since the cells may be partially encased in agar or biofilm-like clumps. Extensive disaggregation is needed.
## :flask: Liquid culture
The fastest way to get a starter culture is to do a subcultivation from an existing liquid culture in exponential or early-stationary growth stage. This substantially reduces the lag phase duration and may be relevant if your goal is to get an early-exponential starter culture.
It can be risky to use a liquid culture as input material due to its inherent lack of uniformity: Within the culture, both healthy and stressed cells might coexist, and genetic variability can lead to differing fitness properties. Over several passages, these differences might cause genotype drifts, resulting in inconsistent outcomes. Another drawback is that contamination may be challenging to detect: Unlike the streak plate method, real-time detection of trace contaminants in a liquid culture is challenging.
## :capsule: CRM product
Ready-to-use certified reference materials (CRMs) are available from different providers like [Microbiologics](https://www.microbiologics.com/item-type/Product/product-format/Epower), [ATCC](https://www.atcc.org/products/qc6-mini), [bioMérieux](https://biotek.com.mk/product/bioball/), [Merck](https://www.sigmaaldrich.com/DK/en/campaigns/vitroids-and-lenticule-discs?srsltid=AfmBOophpxaxYomocMiekPqjIwgOoQEeebzBEdrsLsVrRk2JCjnYVgt9), [Zeptometrix](https://www.zeptometrix.com/dk/en/analytical-reference-materials/microbiology/food-and-agriculture?product_type%5B%5D=Microbiology+Standards\&ipp=12) and [Thermo Scientific](https://documents.thermofisher.com/TFS-Assets/MBD/brochures/Culti-Loops%20Brochure.pdf).
In principle the pros and cons of the CRM products are similar to the cryo stock option, but they offer convenience and reliability with the ready-to-use, certified formats. This typically comes at a relatively high cost, especially if a large supply is needed.
# From cryo stock
| Skill level | Time to complete | Hands-on time | Requirements |
| -------------------------------------- | ------------------------------------------------------------ | ----------------------------------------------- | ----------------------------------------------------------------------------------------------------------------------------------------------------------------- |
| :school\_satchel: No skill requirement | :hourglass: 16 hours (*E. coli*) | :stopwatch: 3 hours | :hard-drive: [7.6a](/item-register/bactobox-r/bactobox-r-hardware-changelog) :floppy-disk: ≥ v2026.02a |
To reactivate bacteria from cryo stocks, first add a precise volume of the cryo stock to a shake flask. Incubate the flask to revive the dormant bacteria and allow them to multiply, thereby creating a starter culture of active cells.
{% hint style="info" %}
#### Choose between overnight and same-day reactivation of cells in cryo stocks
:square-1: Overnight incubation helps activate cells and prepare starter cultures. We recommend this approach.
:square-2: Same-day starter culture is also possible if a higher seeding ratio is used.
{% endhint %}
## :square-1: Overnight starter cultures from cryo stocks
[Get starter culture](/mpd/workflows/get-starter-cultures) provides a powerful approach to tune the seeding ratio for a [High-quality culture](/mpd/cell-growth/high-quality-culture) when preparing an overnight culture. With this approach it is possible to get extremely consistent starter cultures.
## :square-2: Same-day starter culture from cryo stock
Usually it is possible to get a starter culture within the same day if you use a relatively high seeding ratio. The step by step guide is detailed in the subsequent pages.
1. [Get things ready](/mpd/workflows/get-starter-cultures/from-cryo-stock/get-things-ready)
2. [Inoculate culture](/mpd/workflows/get-starter-cultures/from-cryo-stock/inoculate-culture)
3. [Determine initial concentration](/mpd/workflows/get-starter-cultures/from-cryo-stock/determine-initial-concentration)
4. [Determine concentration after incubation](/mpd/workflows/get-starter-cultures/from-cryo-stock/determine-concentration-after-incubation)
Proceed to [Get things ready](/mpd/workflows/get-starter-cultures/from-cryo-stock/get-things-ready) for initializing the same-day starter culture from cryo stock protocol.
# Get things ready
You will need these items for getting a same-day starter culture from a cryo stock.
## Hardware
* [BactoBox® setup](https://help.sbtinstruments.com/items/bactobox-r#bactobox-r-setup) including a [flow cell](https://help.sbtinstruments.com/items/consumables/flow-cell).
* HW: [7.6a](/item-register/bactobox-r/bactobox-r-hardware-changelog)
* SW: ≥ v2026.02a
* Vortex mixer.
* Incubator shaker with clamps for 250 mL shake flask
* Rack for holding 15 mL centrifuge vials.
* P20 and P200 pipette. Alternatively, P10 and P100 pipettes may also be used.
* Sterile pipette tips.
* Permanent marker.
* Sterile, serological pipettes. Preferably 10 mL version.
* Serological pipet controller.
* Sterile 5 mL centrifuge vials (or similar) for collecting and disaggregating samples.
## Reagents
* 1 × cryo stock of your bacterial species.
* 1 × shake flask with growth medium. E.g. 250 mL flask with 50 mL growth medium. Preferably filtered through a 0.2 µm filter to [minimize contribution of background particulates from growth medium](https://help.sbtinstruments.com/custom/advanced-sample-preparation/focus-on-target-objects).
* [Dilution vials](https://help.sbtinstruments.com/items/vials-flasks-and-liquids/dilution-vial) either ready-to-use or freshly prepared using the [dilution dispenser](https://help.sbtinstruments.com/items/accessories/dilution-dispenser).
* [Disinfection vials](https://help.sbtinstruments.com/items/vials-flasks-and-liquids/disinfection-vial).
## Summary
With these things ready, proceed to [Inoculate culture](/mpd/workflows/get-starter-cultures/from-cryo-stock/inoculate-culture).
# Inoculate culture
First step to get a starter culture from a cryo stock is to inoculate the growth medium.
## Step by step
{% stepper %}
{% step %}
**Thaw a cryo stock.**
Allow the cryo vial to thaw at room temperature.
{% endstep %}
{% step %}
**Inoculate**
Once thawed, add 1 mL cryo stock to 50 mL growth medium in a shake flask for a \~2% (v/v) seeding.
{% endstep %}
{% step %}
**Swirl**
Swirl the bottle for 10 seconds to disperse the cryo stock in the cultivation medium.
{% endstep %}
{% endstepper %}
## Summary
Once the growth medium is inoculated, proceed to [Determine initial concentration](/mpd/workflows/get-starter-cultures/from-cryo-stock/determine-initial-concentration).
# Determine initial concentration
We determine the concentration immediately after inoculation, T0, to know the starting concentration. This number will help compare post-incubation results to determine if cell concentration increases. Use a 1:100 dilution before performing a BactoBox® measurement, as the cryo stock is already diluted in the growth medium.
## Step by step
{% stepper %}
{% step %}
**Pull a sample at T0**
Use a sterile serological pipette to collect 1 mL sample immediately after inoculation.
{% endstep %}
{% step %}
**Disaggregate cell clumps**
Vortex the sample 1 min at max speed to disaggregate cell clumps.
{% endstep %}
{% step %}
**Dilute 1:100**
Transfer 101 µL of T0 culture to 10 mL of diluent. Vortex 10 sec. at max speed.
{% endstep %}
{% step %}
**Measure**
Transfer tubing kit to the diluted sample. Press Measure and wait for the result to appear on the BactoBox® screen.
{% endstep %}
{% step %}
**Calculate concentration**
Multiply the cells/mL result with the dilution factor, in this case 100, to get the cell concentration in the culture at T0.
{% endstep %}
{% step %}
**Incubate**
Place the shake flask at desired growth conditions.
{% endstep %}
{% endstepper %}
## Summary
After determining the concentration at T0, proceed to [Determine concentration after incubation](/mpd/workflows/get-starter-cultures/from-cryo-stock/determine-concentration-after-incubation).
# Determine concentration after incubation
For every e.g. 2 hours, pull a sample to check if the cell concentration has increased. The starter culture is ready when the cell concentration is at least ten-fold higher.
{% hint style="info" icon="arrow-up-right-dots" %}
#### Extend incubation until cell concentration is at least ten-fold higher compared to the T0 result
* A cryo stock contains a complex mix of culturable, viable-but-non-culturable, and dead cells. The initial BactoBox® cell concentration right after inoculation detects all these variants and does therefore not only reflect the concentration of culturable cells.
* Incubate the culture until the cell concentration is at least a 10× higher. At this point at least 90% of the detected cells are culturable.
{% endhint %}
## Step by step
{% stepper %}
{% step %}
**Retrieve a sample after 2 hours**
After 2 hours of incubation, use a serological pipette to pull 1 mL sample. Transfer to e.g. a 5 mL centrifuge vial.
{% endstep %}
{% step %}
**Disaggregate cell clumps**
Vortex the sample 1 min at max speed to disaggregate cell clumps.
{% endstep %}
{% step %}
**Dilute 1:100**
Transfer 101 µL of T0 culture to 10 mL of diluent. Vortex 10 sec. at max speed.
{% endstep %}
{% step %}
**Measure**
Transfer tubing kit to the diluted sample. Press Measure and wait for the result to appear on the BactoBox® screen.
{% endstep %}
{% step %}
**Calculate concentration**
Multiply the cells/mL result with the dilution factor, in this case 100, to get the cell concentration in the culture at T0.
{% endstep %}
{% step %}
**Stop or repeat**
Your starter culture is ready when the cell concentration is at least ten-fold higher than at T0,
If the concentration is still too low, extend the incubation by two hours and repeat steps 1 to 5 until ten-fold increase is obtained.
{% endstep %}
{% endstepper %}
## Summary
The starter culture is ready once the cell concentration has increased by at least 10-fold. You can now use the culture for experiments or to continue with the next step in your seed train.
# From agar plate colony
Get an active culture using a fresh colony from an agar plate.
| Skill level | Time to complete | Hands-on time | Requirements |
| -------------------------------------- | --------------------------------------------------------------- | ----------------------------------------------- | ----------------------------------------------------------------------------------------------------------------------------------------------------------------- |
| :school\_satchel: No skill requirement | :hourglass: 16-24 hours (*E. coli*) | :stopwatch: 3 hours | :hard-drive: [7.6a](/item-register/bactobox-r/bactobox-r-hardware-changelog) :floppy-disk: ≥ v2026.02a |
A fresh colony from an agar plate is a good starting point to ensure a single-species (axenic) culture. The standard workflow uses overnight incubation of the liquid culture, but it is often possible to get a same-day starter culture from an agar plate (see hint box).
Illustration of workflow. A fresh colony is collected and transferrred to a capture tube. Cell clumps are disaggregated using bead-beater or vortex mixer. Finally, a sample of capture tube is diluted and subjected to BactoBox® measurement.
{% hint style="info" icon="sneaker-running" %}
## Get a same-day starter culture
* If you want a same-day starter culture, try using higher seeding concentration, e.g. 5× 107 cells/mL.
* Check the liquid culture frequently for increase in cell concentration, e.g. for each 2 hours.
* We recommend that - prior to use - the active starter culture should have cell concentration is at least tenfold higher than at T0.
{% endhint %}
The protocol assumes that you have already used the [streak plate method](https://microbenotes.com/streak-plate-method-principle-methods-significance-limitations/) to get fresh colonies on an agar plate. The steps are outlined below.
1. [Get things ready](/mpd/workflows/get-starter-cultures/from-agar-plate-colony/get-things-ready)
2. [Collect and disperse bacteria from agar plate](/mpd/workflows/get-starter-cultures/from-agar-plate-colony/collect-and-disperse)
3. [Determine cells/mL in harvest tube](/mpd/workflows/get-starter-cultures/from-agar-plate-colony/determine-concentration)
4. [Inoculate liquid culture](/mpd/workflows/get-starter-cultures/from-agar-plate-colony/inoculate-liquid-culture)
5. [Determine concentration of overnight culture](/mpd/workflows/get-starter-cultures/from-agar-plate-colony/determine-concentration-after-incubation)
Next, the [get things ready](/mpd/workflows/get-starter-cultures/from-agar-plate-colony/get-things-ready) section will provide you with a check-list of necessary items.
# Get things ready
You will need these items.
## Hardware
* [BactoBox® setup](https://help.sbtinstruments.com/items/bactobox-r#bactobox-r-setup) including a [flow cell](https://help.sbtinstruments.com/items/consumables/flow-cell).
* HW: [7.6a](/item-register/bactobox-r/bactobox-r-hardware-changelog)
* SW: ≥ v2026.02a
* Vortex mixer or alternatively a bead beater like [bead genie](https://www.scientificindustries.com/products/bead-genie%E2%84%A2?srsltid=AfmBOopRmRHhvdvEtPAo7snGZNBxssDgbZpMhqlaAFGGuBxu2WSzUJrF).
* Rack for holding 5 and 15 mL centrifuge vials.
* Sterile 1 µL Inoculation loops. Preferably made of polystyrene (PS). See below hint.
* P200 or P100 pipette.
* Sterile pipette tips.
* Serological pipettes. Preferably 10 and 2 mL versions.
* Serological pipet controller.
{% hint style="success" %}
#### Use polystyrene (PS) inoculation loops
After picking a suitable colony, you will snap the inoculation loop in the capture vial. Choose [polystyrene (PS)](https://www.sarstedt.com/en/products/laboratory/microbiology/accessories/product/86.1567.050/) because they snap easier than more "bendable" materials like polypropylene.
{% endhint %}
## Reagents
* 1 × agar plate with fresh colonies. Preferably same agar type as the liquid growth medium.
* 1 × shake flask with growth medium. E.g. 250 mL flask with max 50 mL growth medium.
* 1 × capture vial: [5 mL centrifuge vial with screw cap](https://www.eppendorf.com/dk-en/Products/Lab-Consumables/Lab-Tubes/EppendorfTubes-50mL-p-PF-156668) containing 4 mL cultivation medium and 1 mL 3 mm glass beads. See below hint box for more information.
* [Dilution vials](https://help.sbtinstruments.com/items/vials-flasks-and-liquids/dilution-vial) either ready-to-use or freshly prepared using the [dilution dispenser](https://help.sbtinstruments.com/items/accessories/dilution-dispenser).
* [Disinfection vials](https://help.sbtinstruments.com/items/vials-flasks-and-liquids/disinfection-vial).
{% hint style="info" icon="vial-vertical" %}
#### How to make capture vials
Prepare capture vials with the optimal growth medium for the bacterium of interest. Remember to add [3 mm glass beads](https://help.sbtinstruments.com/items/consumables/glass-beads-3-mm-50-g) as these greatly aid in disaggregation of cell clumps.
1. Prepare relevant growth medium as recommended by the manufacturer. Often, heating and magnetic stirring is needed to dissolve the powder completely.
2. Let medium cool to room temperature before moving to next step.
3. Filter by 0.2 µm syringe or vacuum filter to [remove medium particulates](https://help.sbtinstruments.com/custom/advanced-sample-preparation/focus-on-target-objects).
4. Place [5 mL centrifuge vial with screw cap](https://www.eppendorf.com/dk-en/Products/Lab-Consumables/Lab-Tubes/EppendorfTubes-50mL-p-PF-156668) in an autoclavable rack.
5. Transfer 4 mL growth medium to each 5 mL vial.
6. Add [3 mm glass beads](https://help.sbtinstruments.com/items/consumables/glass-beads-3-mm-50-g) to each vial until the liquid level has risen to the 5 mL mark.
7. Cap the vials loosely and apply a small piece of autoclave tape to keep the cap in place.
8. Autoclave as recommended by the growth medium supplier.
9. Once vials have reached room temperature, cap the vials tightly.
{% endhint %}
With these things in hand, you are ready to start the [Collect and disperse](/mpd/workflows/get-starter-cultures/from-agar-plate-colony/collect-and-disperse) step.
# Collect and disperse
A fresh colony is collected using an inoculation loop and the cells are dispersed in a 5 mL centrifuge vial. We refer to this as the capture vial.
{% hint style="success" icon="dice-one" %}
#### Collect a single, well-isolated, discrete colony
To ensure a single genetic origin (monoclonal), it is best to collect a well-isolated, discrete colony. Avoid fused colonies, as they may contain different clones. For very small colonies, it may be necessary to gather several to achieve a BactoBox® result within the desired range.
{% endhint %}
The video shows all the collect-and-disperse steps.
{% stepper %}
{% step %}
**Collect a colony**
Use a 1 µm inoculation loop to collect a single, fresh colony. See the above info field for details
{% endstep %}
{% step %}
**Transfer colony to capture vial**
Insert the tip of the inoculation loop into the capture vial containing 3 mm glass beads. Press the screw cap down on the rod while bending the loop up and down until the rod snaps. Tighten the cap securely.
{% endstep %}
{% step %}
**Disperse cells**
Use [bead-genie](https://www.scientificindustries.com/products/bead-genie%E2%84%A2?srsltid=AfmBOopeCmTHw8Fo4wj9wkggx4sjCx21NFo6YwXjXQwrP5Ks5Rf3_702) for 1 minute at max speed to disperse cells. If you don't have a bead-beater beater, vortex the capture vial 1 min at max speed.
{% endstep %}
{% endstepper %}
## Summary
A suitable colony is collected, and the dispersed cells in the capture vial are now ready for BactoBox® analysis. Next step is to [determine the cell concentration](/mpd/workflows/get-starter-cultures/from-agar-plate-colony/determine-concentration).
# Determine concentration
The dispersed cells in the capture vial will be used to inoculate a liquid culture at a well-defined seeding concentration. To know which volume to add, first determine the cell concentration and then use the subcultivation calculator to determine the volume. The steps are demonstrated below.
{% stepper %}
{% step %}
**Prepare a 1:201 dilution**
Transfer 50 µL of your sample to 10 mL of diluent. Vortex 10 seconds at maximum speed.
{% endstep %}
{% step %}
**Measure on the diluted sample**
Transfer the tubing kit to the diluted sample.\
Press Measure and wait for the result to appear on the BactoBox® screen.
{% endstep %}
{% step %}
**Calculate cell concentration in capture vial**
Multiply the cells/mL with the dilution factor. In this case, it is 201
{% endstep %}
{% endstepper %}
## Summary
Now you know the cell concentration in the capture vial. Proceed to [Inoculate liquid culture](/mpd/workflows/get-starter-cultures/from-agar-plate-colony/inoculate-liquid-culture) to calculate the volume of capture vial suspension needed to inoculate growth medium in the shake flask.
# Inoculate liquid culture
Use the [subcultivation calculator tool](https://www2026.sbtinstruments.com/wp-content/uploads/2025/12/202512-BactoBox-sub-cultivation-calculator.html) to determine the volume needed to seed the growth medium. A typical seeding concentration for an overnight culture is 1 x 106 cells/mL.
{% hint style="success" %}
#### Use Optiseeding workflow to determine suitable seeding concentration
The seeding concentration may need adjustment to get a [high-quality culture](/mpd/cell-growth/high-quality-culture). Do a screening experiment with a broad 1:10 dilution series to find a suitable seeding concentration. See [Optimize starter culture](/mpd/workflows/identify-optimal-inoculum-size) for more details.
{% endhint %}
{% stepper %}
{% step %}
**Open the subcultivation calculator**
Open the [subcultivation calculator tool](https://www2026.sbtinstruments.com/wp-content/uploads/2025/12/202512-BactoBox-sub-cultivation-calculator.html) in a browser window.
{% endstep %}
{% step %}
**Calculate inoculation volume**
* Fill in cells/mL of the capture vial in the first field.
* Add volume information in the second field. In this case it is 40 mL
* Set the desired inoculation concentration in the final field. In this case 1 × 106 cells/mL
* Click Calculate V\_add to get the result.
{% endstep %}
{% step %}
**Add starter culture to growth curve flask**
Transfer starter culture to growth curve flask and swirl aggressively to disperse the inoculum.
Record the inoculation time. Record it on the autoclave tape as a backup, ensuring you have a reference if you forget to document it elsewhere.
Inoculation of shake flask at well-defined concentration. In this video the 50 mL vial with the red cap represents the capture vial.
{% endstep %}
{% endstepper %}
## Summary
The overnight culture is inoculated at a well-defined starting concentration of 1 x 106 cells/mL. Next step is [Determine concentration after incubation](/mpd/workflows/get-starter-cultures/from-agar-plate-colony/determine-concentration-after-incubation).
# Determine concentration after incubation
Now, you will determine the concentration after overnight incubation. Cell concentration is typically in a ball-park range of 1 × 1010 cells/mL, and therefore a dilution of 1:10,000 is usually suitable. This dilution is obtained by two sequential 1:100 dilutions. Below we demonstrate this step by step using the Access interface. You can also use BactoBox® as a standalone device and do the calculations manually.
{% stepper %}
{% step %}
**Retrieve and disaggregate a sample of the overnight culture**
Use a sterile serological pipette to transfer 10 mL of the overnight culture to a sterile vial.
Vortex thoroughly to disperse any cell clumps. For an *E. coli* culture it is usually sufficient to vortex 30 seconds at max speed.
{% hint style="warning" %}
## Proper vortexing is important for reliable results!
Ensure that you see a proper vortex forming. Poor vortexing can cause inadequate disaggregation, leading to low precision in replicates and non-linearity in dilution series.
Different pedestals exists for different sizes of vials. In the video, we show a multipurpose pedestal that works for many types of vials. Ensure that your pedestal is compatible with the given vial.
{% endhint %}
{% endstep %}
{% step %}
**Dilute 1:100**
Transfer 101 µL of your sample to 10 mL of diluent. Vortex 10 seconds at maximum speed.
{% endstep %}
{% step %}
**Dilute 1:10 000**
Transfer 101 µL of the diluted sample to 10 mL of diluent. Vortex 10 seconds at maximum speed.
{% endstep %}
{% step %}
**Measure the diluted sample on BactoBox®**
Transfer the tubing kit to the 1:10,000 vial.
Add the metadata:
* Dilution: 10 000
* Label: Overnight\_culture
Then press Measure in Access to start a new measurement.
After completing the measurement, Access will automatically calculate the bacterial concentration in the shake flask. To view the full table, drag the panel sideways.
{% endstep %}
{% endstepper %}
## Summary
Now you know the bacterial concentration of your overnight culture. With cells from an agar plate as input material, you now have a liquid starter culture with a well-defined cell concentration.
# From liquid culture
| Skill level | Time to complete | Hands-on time | Requirements |
| -------------------------------------- | -------------------------------------------------------------- | ----------------------------------------------- | ----------------------------------------------------------------------------------------------------------------------------------------------------------------- |
| :school\_satchel: No skill requirement | :hourglass: 8-24 hours (*E. coli*) | :stopwatch: 3 hours | :hard-drive: [7.6a](/item-register/bactobox-r/bactobox-r-hardware-changelog) :floppy-disk: ≥ v2026.02a |
Subcultivation from an existing liquid culture is a fast approach to get a same-day starter culture. Because the cells are often in exponential or stationary stage, the adaptation period (lag phase) is short and it won't be long before the new culture is in exponential stage.
Note that subcultivation from a liquid culture carries certain risks. Liquid cultures often contain genetically diverse populations, and there may also be contaminants present.
Here we provide a step by step guide for how to subcultivate from a liquid culture.
1. [Get things ready](/mpd/workflows/get-starter-cultures/from-liquid-culture/get-things-ready)
2. [Determine concentration of inoculum](/mpd/workflows/get-starter-cultures/from-liquid-culture/determine-concentration-of-inoculum)
3. [Inoculate culture](/mpd/workflows/get-starter-cultures/from-liquid-culture/inoculate-culture)
4. [Determine initial concentration](/mpd/workflows/get-starter-cultures/from-liquid-culture/determine-initial-concentration)
5. [Determine concentration after incubation](/mpd/workflows/get-starter-cultures/from-liquid-culture/determine-concentration-after-incubation)
Proceed to [Get things ready](/mpd/workflows/get-starter-cultures/from-liquid-culture/get-things-ready) for initializing the protocol for subcultivation from a liquid culture.
# Get things ready
You will need these items for the protocol.
## Hardware
* [BactoBox® setup](https://help.sbtinstruments.com/items/bactobox-r#bactobox-r-setup) including a [flow cell](https://help.sbtinstruments.com/items/consumables/flow-cell).
* HW: [7.6a](/item-register/bactobox-r/bactobox-r-hardware-changelog)
* SW: ≥ v2026.02a
* Vortex mixer.
* Incubator shaker with clamps for 250 mL shake flask
* Rack for holding 15 mL centrifuge vials.
* P20 and P200 pipette. Alternatively P10 and P100 is also ok.
* Sterile pipette tips.
* Permanent marker.
* Sterile, serological pipettes. Preferably 10 mL version.
* Serological pipet controller (not shown).
* Sterile 5 mL centrifuge vials (or similar) for collecting and disaggregating samples.
## Reagents
* 1 × liquid culture of your bacterial species.
* 1 × shake flask with growth medium. E.g. 250 mL flask with 50 mL growth medium. Preferably filtered through a 0.2 µm filter to [minimize contribution of background particulates from growth medium](https://help.sbtinstruments.com/custom/advanced-sample-preparation/focus-on-target-objects).
* [Dilution vials](https://help.sbtinstruments.com/items/vials-flasks-and-liquids/dilution-vial) either ready-to-use or freshly prepared using the [dilution dispenser](https://help.sbtinstruments.com/items/accessories/dilution-dispenser).
* [Disinfection vials](https://help.sbtinstruments.com/items/vials-flasks-and-liquids/disinfection-vial).
## Summary
With these things ready, proceed to [Determine concentration of inoculum](/mpd/workflows/get-starter-cultures/from-liquid-culture/determine-concentration-of-inoculum).
# Determine concentration of inoculum
We want to inoculate using a well-defined seeding concentration of cells. For this you need to know the cell concentration of the liquid culture used to seed the growth medium. A 1:10 000 dilution is usually suitable for the BactoBox® measurement. See [Hit the right concentration](https://help.sbtinstruments.com/custom/advanced-sample-preparation/hit-the-right-concentration) if the result is not within the working range.
## Step by step
{% stepper %}
{% step %}
**Retrieve a sample of the inoculum**
Swirl the inoculum well to suspend cells and use a sterile serological pipette to collect 1 mL sample. Transfer to a sterile 5 mL centrifuge tube
{% endstep %}
{% step %}
**Disaggregate cell clumps**
Vortex the sample 1 min at max speed to disaggregate cell clumps.
{% endstep %}
{% step %}
**Dilute 1:100**
Transfer 101 µL of T0 culture to 10 mL of diluent. Vortex 10 sec. at max speed.
{% endstep %}
{% step %}
**Dilute 1:10 000**
Transfer 101 µL of the 1:100 dilution to 10 mL of diluent. Vortex 10 sec. at max speed.
{% endstep %}
{% step %}
**Measure**
Transfer tubing kit to the diluted sample. Press Measure and wait for the result to appear on the BactoBox® screen.
{% endstep %}
{% step %}
**Calculate concentration**
Multiply the cells/mL result with the dilution factor, in this case 10 000, to get the cell concentration of the inoculum
{% endstep %}
{% endstepper %}
## Summary
Once you know the cell concentration of the inoculum, proceed to [Inoculate culture](/mpd/workflows/get-starter-cultures/from-liquid-culture/inoculate-culture).
# Inoculate culture
Once you know the cell concentration of the inoculum you can calculate the volume necessary to hit a target seeding concentration (here 1 × 107 cells/mL). Use the [subcultivation calculator tool](https://www2026.sbtinstruments.com/wp-content/uploads/2025/12/202512-BactoBox-sub-cultivation-calculator.html) for the calculations.
{% hint style="success" %}
## Use Optiseeding workflow to determine suitable seeding concentration
The seeding concentration may need adjustment to get a [high-quality culture](/mpd/cell-growth/high-quality-culture). Do a screening experiment with a broad 1:10 dilution series to find a suitable seeding concentration. See [Optimize starter culture](/mpd/workflows/identify-optimal-inoculum-size) for more details.
{% endhint %}
{% stepper %}
{% step %}
**Open the subcultivation calculator**
Open the [subcultivation calculator tool](https://www2026.sbtinstruments.com/wp-content/uploads/2025/12/202512-BactoBox-sub-cultivation-calculator.html) in a browser.
{% endstep %}
{% step %}
**Calculate inoculation volume**
* Fill in cells/mL of the inoculum in the first field.
* Add volume information in the second field. In this case it is 50 mL
* Set the desired inoculation concentration in the final field. In this case 1 × 107 cells/mL
* Click Calculate V\_add to get the result.
{% endstep %}
{% step %}
**Add inoculum culture to growth medium**
Transfer the calculated volume of inoculum to growth curve flask and swirl aggressively to disperse the inoculum.
Record the inoculation time. Note it down on the autoclave tape as a backup, ensuring you have a reference if you forget to document it elsewhere.
Inoculation of shake flask at well-defined concentration. In this video the 50 mL vial with the red cap represents the inoculum.
{% endstep %}
{% endstepper %}
## Summary
Once the growth medium is inoculated, proceed to [Determine initial concentration](/mpd/workflows/get-starter-cultures/from-liquid-culture/determine-initial-concentration).
# Determine initial concentration
We determine the concentration immediately after inoculation, T0, to know the starting concentration. This number will help compare post-incubation results to determine if cell concentration has increased.
The target seeding concentration is 1 × 107 cells/mL and therefore a 1:100 dilution will usually be suitable to hit the BactoBox® working range.
## Step by step
{% stepper %}
{% step %}
**Pull a sample at T0**
Use a sterile serological pipette to collect 1 mL sample immediately after inoculation and swirling. Transfer to a 5 mL centrifuge vial.
{% endstep %}
{% step %}
**Disaggregate cell clumps**
Vortex the sample 1 min at max speed to disaggregate cell clumps.
{% endstep %}
{% step %}
**Dilute 1:100**
Transfer 101 µL of T0 culture to 10 mL of diluent. Vortex 10 sec. at max speed.
{% endstep %}
{% step %}
**Measure**
Transfer tubing kit to the diluted sample. Press Measure and wait for the result to appear on the BactoBox® screen.
{% endstep %}
{% step %}
**Calculate concentration**
Multiply the cells/mL result with the dilution factor, in this case 100, to get the cell concentration in the culture at T0.
{% endstep %}
{% step %}
**Incubate**
Place the shake flask at desired growth conditions.
{% endstep %}
{% endstepper %}
## Summary
Now that concentration at T0 is known, proceed to [Determine concentration after incubation](/mpd/workflows/get-starter-cultures/from-liquid-culture/determine-concentration-after-incubation).
# Determine concentration after incubation
For every e.g. 2 hours, pull a sample to check if the cell concentration has increased. The starter culture is ready when the cell concentration has increased by at least ten-fold.
{% hint style="info" icon="arrow-up-right-dots" %}
#### Incubate until cell concentration has increased by at least ten-fold
* The inoculum used to seed the growth medium may contain a complex mix of culturable, viable-but-non-culturable, and dead cells. The initial BactoBox® cell concentration right after inoculation detects all these variants and does therefore not only reflect the concentration of culturable cells.
* We recommend that the culture is incubated until the cell concentration is at least 10× higher. At this point at least 90% of the detected cells are culturable.
{% endhint %}
## Step by step
{% stepper %}
{% step %}
**Retrieve a sample after 2 hours**
After 2 hours of incubation, use a serological pipette to pull 1 mL sample. Transfer to e.g. a 5 mL centrifuge vial.
{% endstep %}
{% step %}
**Disaggregate cell clumps**
Vortex the sample 1 min at max speed to disaggregate cell clumps.
{% endstep %}
{% step %}
**Dilute 1:100**
Transfer 101 µL of T0 culture to 10 mL of diluent. Vortex 10 sec. at max speed.
{% endstep %}
{% step %}
**Measure**
Transfer tubing kit to the diluted sample. Press Measure and wait for the result to appear on the BactoBox® screen.
{% endstep %}
{% step %}
**Calculate concentration**
Multiply the cells/mL result with the dilution factor, in this case 100, to get the cell concentration in the culture at T0.
{% endstep %}
{% step %}
**Stop or repeat**
Your starter culture is ready if the cell concentration is at least ten-fold higher than at T0,
If the concentration is still too low, extend the incubation by two hours and repeat steps 1 to 5 until ten-fold increase is obtained.
{% endstep %}
{% endstepper %}
## Summary
The culture is ready once the cell concentration are at least 10-fold higher. You can now use the starter culture for experiments or to continue with the next step in your seed train.
# From CRM product
| Skill level | Time to complete | Hands-on time | Requirements |
| -------------------------------------- | ---------------------------------------------------------- | ----------------------------------------------- | ----------------------------------------------------------------------------------------------------------------------------------------------------------------- |
| :school\_satchel: No skill requirement | :hourglass: 2 days (*E. coli*) | :stopwatch: 4 hours | :hard-drive: [7.6a](/item-register/bactobox-r/bactobox-r-hardware-changelog) :floppy-disk: ≥ v2026.02a |
Certified reference materials are available from e.g. [Microbiologics](https://www.microbiologics.com/item-type/Product/product-format/Epower), [ATCC](https://www.atcc.org/products/qc6-mini), [bioMérieux](https://biotek.com.mk/product/bioball/), [Merck](https://www.sigmaaldrich.com/DK/en/campaigns/vitroids-and-lenticule-discs?srsltid=AfmBOophpxaxYomocMiekPqjIwgOoQEeebzBEdrsLsVrRk2JCjnYVgt9), [Zeptometrix](https://www.zeptometrix.com/dk/en/analytical-reference-materials/microbiology/food-and-agriculture?product_type%5B%5D=Microbiology+Standards\&ipp=12), and [Thermo Scientific](https://documents.thermofisher.com/TFS-Assets/MBD/brochures/Culti-Loops%20Brochure.pdf). Some formats require different preparations:
* **Agar stabs:** Need disaggregation.
* **Disk-types:** Require rehydration.
* **Glycerol stocks:** Must be thawed.
In this step by step guide we provide an example of how to work with the [Microbiologics](https://www.microbiologics.com/item-type/Product/product-format/Epower) [lyfo diskTM](https://www.microbiologics.com/item-type/Product/product-format/LYFO-DISK). In principle the disks are compatible with the workflow [From agar plate colony](/mpd/workflows/get-starter-cultures/from-agar-plate-colony) and are also compatible with the workflow [From liquid culture](/mpd/workflows/get-starter-cultures/from-liquid-culture).
Here we use the workflow [From agar plate colony](/mpd/workflows/get-starter-cultures/from-agar-plate-colony) in combination with the [lyfo diskTM](https://www.microbiologics.com/item-type/Product/product-format/LYFO-DISK). First the disk is rehydrated and the cell-containing rehydrate is used to streak an agar plate. Once single, fresh colonies are available they are used to inoculate growth medium at a well-defined concentration.
The step by step guide is divided in the following sections.
1. [Get things ready](/mpd/workflows/get-starter-cultures/from-crm-product/get-things-ready)
2. [Rehydrate and streak](/mpd/workflows/get-starter-cultures/from-crm-product/rehydrate-and-streak)
3. [Collect and disperse](/mpd/workflows/get-starter-cultures/from-crm-product/collect-and-disperse)
4. [Determine concentration](/mpd/workflows/get-starter-cultures/from-crm-product/determine-concentration)
5. [Inoculate liquid culture](/mpd/workflows/get-starter-cultures/from-crm-product/inoculate-liquid-culture)
6. [Determine concentration after incubation](/mpd/workflows/get-starter-cultures/from-crm-product/determine-concentration-after-incubation)
{% hint style="info" %}
#### See [Microbiologics](https://www.microbiologics.com/item-type/Product/product-format/Epower) [lyfo diskTM](https://www.microbiologics.com/item-type/Product/product-format/LYFO-DISK) documentation for full details
We use an adapted version of the [lyfo diskTM](https://www.microbiologics.com/item-type/Product/product-format/LYFO-DISK) rehydration protocol. For full description please inspect the original [supporting material](https://www.microbiologics.com/item-type/Product/product-format/LYFO-DISK).
{% endhint %}
Proceed to [Get things ready](/mpd/workflows/get-starter-cultures/from-crm-product/get-things-ready) for a check list of items necessary for the workflow.
# Get things ready
You will need these items for the protocol.
## Hardware
* [BactoBox® setup](https://help.sbtinstruments.com/items/bactobox-r#bactobox-r-setup) including a [flow cell](https://help.sbtinstruments.com/items/consumables/flow-cell).
* HW: [7.6a](/item-register/bactobox-r/bactobox-r-hardware-changelog)
* SW: ≥ v2026.02a
* Vortex mixer or alternatively a bead beater like [bead genie](https://www.scientificindustries.com/products/bead-genie%E2%84%A2?srsltid=AfmBOopRmRHhvdvEtPAo7snGZNBxssDgbZpMhqlaAFGGuBxu2WSzUJrF).
* Rack for holding 5 and 15 mL centrifuge vials.
* Sterile 1 µL Inoculation loops. Preferably made of polystyrene (PS). See below hint.
* P200 or P100 pipette.
* Sterile pipette tips.
{% hint style="success" %}
#### Use polystyrene (PS) inoculation loops
After picking a suitable colony, you will snap the inoculation loop in the capture vial. Choose [polystyrene (PS)](https://www.sarstedt.com/en/products/laboratory/microbiology/accessories/product/86.1567.050/) because they snap easier than more "bendable" materials like polypropylene.
{% endhint %}
## Reagents
* 1 × [Lyfo diskTM](https://www.microbiologics.com/item-type/Product/product-format/LYFO-DISK).
* 1 × agar plate. Preferably same agar type as the liquid growth medium.
* 1 × shake flask with growth medium. E.g. 250 mL flask with max 50 mL growth medium.
* 2 × capture vials: [5 mL centrifuge vial with screw cap](https://www.eppendorf.com/dk-en/Products/Lab-Consumables/Lab-Tubes/EppendorfTubes-50mL-p-PF-156668) containing 4 mL cultivation medium and 1 mL 3 mm glass beads. See below hint box for more information.
* [Dilution vials](https://help.sbtinstruments.com/items/vials-flasks-and-liquids/dilution-vial) either ready-to-use or freshly prepared using the [dilution dispenser](https://help.sbtinstruments.com/items/accessories/dilution-dispenser).
* [Disinfection vials](https://help.sbtinstruments.com/items/vials-flasks-and-liquids/disinfection-vial).
With these things in hand, you are ready to [Rehydrate and streak](/mpd/workflows/get-starter-cultures/from-crm-product/rehydrate-and-streak).
# Rehydrate and streak
The first goal to obtain a streaked agar plate of single, well-isolated colonies. The [lyfo diskTM](https://www.microbiologics.com/item-type/Product/product-format/LYFO-DISK) is used as a starting material.
{% hint style="info" %}
#### See [Microbiologics](https://www.microbiologics.com/item-type/Product/product-format/Epower) [lyfo diskTM](https://www.microbiologics.com/item-type/Product/product-format/LYFO-DISK) documentation for full details
We use an adapted version of the [lyfo diskTM](https://www.microbiologics.com/item-type/Product/product-format/LYFO-DISK) rehydration protocol. For full description please inspect the [supporting material provided by Microbiologics](https://www.microbiologics.com/item-type/Product/product-format/LYFO-DISK).
{% endhint %}
## Step by step
{% stepper %}
{% step %}
**Transfer disk to capture tube**
Use aseptic technique to transfer a LYFO DISK to a preheated (37 °C) 5 mL capture tube containing glass beads.
{% endstep %}
{% step %}
**Dissolve disk and disperse cells**
Rehydrate the disk by bead-beating, manual shaking or vortexing for 1 min at max speed (or until the suspension is homogenous)
{% endstep %}
{% step %}
**Streak agar plate**
Dip a sterile 1 µL inoculate loop into the suspension and [streak](https://microbenotes.com/streak-plate-method-principle-methods-significance-limitations/) to obtain single, well-resolved colonies.
{% endstep %}
{% step %}
**Incubate**
Incubate at optimal growth conditions until colonies appear (typically overnight). Avoid over-incubation as this may lead to presence of dead cells.
{% endstep %}
{% endstepper %}
## Summary
The [Lyfo diskTM](https://www.microbiologics.com/item-type/Product/product-format/LYFO-DISK) is rehydrated and an agar plate is streaked to get single colonies. Next step is to [Collect and disperse](/mpd/workflows/get-starter-cultures/from-crm-product/collect-and-disperse).
# Collect and disperse
A fresh colony is collected using an inoculation loop and the cells are dispersed in a 5 mL centrifuge vial. We refer to this as the capture vial.
{% hint style="success" icon="dice-one" %}
#### Collect a single, well-isolated, discrete colony
To ensure a monoclonal origin, it is best to collect a well-isolated, discrete colony. Avoid fused colonies, as they may contain different clones. For very small colonies, it may be necessary to gather several to achieve a BactoBox® result within the desired range.
{% endhint %}
The video shows the collect-and-disperse steps.
{% stepper %}
{% step %}
**Collect a colony**
Use a 1 µm inoculation loop to collect a single, fresh colony. See the above info field for details
{% endstep %}
{% step %}
**Transfer colony to capture vial**
Insert the tip of the inoculation loop into the capture vial containing 3 mm glass beads. Press the screw cap down on the rod while bending the loop up and down until the rod snaps. Tighten the cap securely.
{% endstep %}
{% step %}
**Disperse cells**
Use [bead-genie](https://www.scientificindustries.com/products/bead-genie%E2%84%A2?srsltid=AfmBOopeCmTHw8Fo4wj9wkggx4sjCx21NFo6YwXjXQwrP5Ks5Rf3_702) for 1 minute at max speed to disperse cells. If you don't have a bead-beater beater, vortex the capture vial 1 min at max speed.
{% endstep %}
{% endstepper %}
## Summary
A suitable colony has been collected, and the dispersed cells in the capture vial are now ready for BactoBox® analysis. Next step is to [Determine concentration](/mpd/workflows/get-starter-cultures/from-crm-product/determine-concentration).
# Determine concentration
The dispersed cells in the capture vial will be used to inoculate a liquid culture at a well-defined seeding concentration. To know which volume to add, first determine the cell concentration and then use the subcultivation calculator to determine the volume. The steps are demonstrated below.
{% stepper %}
{% step %}
**Prepare a 1:201 dilution**
Transfer 50 µL of your sample to 10 mL of diluent. Vortex 10 seconds at maximum speed.
{% endstep %}
{% step %}
**Measure on the diluted sample**
Transfer the tubing kit to the diluted sample.\
Press Measure and wait for the result to appear on the BactoBox® screen.
{% endstep %}
{% step %}
**Calculate cell concentration in capture vial**
Multiply the cells/mL with the dilution factor. In this case, it is 201
{% endstep %}
{% endstepper %}
## Summary
Now you know the cell concentration in the capture vial. Proceed to [Inoculate liquid culture](/mpd/workflows/get-starter-cultures/from-crm-product/inoculate-liquid-culture) to calculate the volume of capture vial suspension needed to inoculate growth medium in the shake flask.
# Inoculate liquid culture
Use the [subcultivation calculator tool](https://www2026.sbtinstruments.com/wp-content/uploads/2025/12/202512-BactoBox-sub-cultivation-calculator.html) to determine the volume needed to seed the growth medium. A typical overnight seeding concentration is 1 x 106 cells/mL.
{% hint style="success" %}
#### Use Optiseeding workflow to determine suitable seeding concentration
The seeding concentration may need adjustment to get a [high-quality culture](/mpd/cell-growth/high-quality-culture). Do a screening experiment with a broad 1:10 dilution series to find a suitable seeding concentration. See [Optimize starter culture](/mpd/workflows/identify-optimal-inoculum-size) for more details.
{% endhint %}
{% stepper %}
{% step %}
**Open the subcultivation calculator**
Open the [subcultivation calculator tool](https://www2026.sbtinstruments.com/wp-content/uploads/2025/12/202512-BactoBox-sub-cultivation-calculator.html) in a browser.
{% endstep %}
{% step %}
**Calculate inoculation volume**
* Fill in cells/mL of the capture vial in the first field.
* Add volume information in the second field. In this case it is 40 mL
* Set the desired inoculation concentration in the final field. In this case 1 × 106 cells/mL
* Click Calculate V\_add to get the result.
{% endstep %}
{% step %}
**Add starter culture to growth curve flask**
Transfer starter culture to growth curve flask and swirl aggressively to disperse the inoculum.
Record the inoculation time. Record it on the autoclave tape as a backup, ensuring you have a reference if you forget to document it elsewhere.
Inoculation of shake flask at well-defined concentration. In this video the 50 mL vial with the red cap represents the capture vial.
{% endstep %}
{% endstepper %}
## Summary
You have now inoculated the overnight culture at a well-defined starting concentration of 1 x 106 cells/mL. Next step is [Determine concentration after incubation](/mpd/workflows/get-starter-cultures/from-crm-product/determine-concentration-after-incubation).
# Determine concentration after incubation
Here, you will determine the concentration after overnight incubation. Cell concentration is typically in a ball-park range of 1 × 1010 cells/mL, and therefore a dilution of 1:10,000 is usually suitable. This dilution is obtained by two sequential 1:100 dilutions. Below we demonstrate this step by step using the Access interface. You can also use BactoBox® as a standalone device and do the calculations manually.
{% stepper %}
{% step %}
**Retrieve and disaggregate a sample of the overnight culture**
Use a sterile serological pipette to transfer 10 mL of the overnight culture to a sterile vial.
Vortex thoroughly to disperse any cell clumps. For an *E. coli* culture it is usually sufficient to vortex 30 seconds at max speed.
{% hint style="warning" %}
## Proper vortexing is important for reliable results!
Ensure that you see a proper vortex forming. Poor vortexing can cause inadequate disaggregation, leading to low precision in replicates and non-linearity in dilution series.
Different pedestals exists for different sizes of vials. In the video, we show a multipurpose pedestal that works for many types of vials. Ensure that your pedestal is compatible with the given vial.
{% endhint %}
{% endstep %}
{% step %}
**Dilute 1:100**
Transfer 101 µL of your sample to 10 mL of diluent. Vortex 10 seconds at maximum speed.
{% endstep %}
{% step %}
**Dilute 1:10 000**
Transfer 101 µL of the diluted sample to 10 mL of diluent. Vortex 10 seconds at maximum speed.
{% endstep %}
{% step %}
**Measure the diluted sample on BactoBox®**
Transfer the tubing kit to the 1:10,000 vial.
Add the metadata:
* Dilution: 10 000
* Label: Overnight\_culture
Then press Measure in Access to start a new measurement.
After completing the measurement, Access will automatically calculate the bacterial concentration in the shake flask. To view the full table, drag the panel sideways.
{% endstep %}
{% endstepper %}
## Summary
This concludes the workflow on how to get an active starter culture from a CRM product - in this case the [Lyfo diskTM](https://www.microbiologics.com/item-type/Product/product-format/LYFO-DISK). The final starter culture is ready for experiments or to initialize your bioprocess seed train.
# Optimize starter culture
MPD-6. Get a high-quality overnight culture after the desired incubation time.
| Skill level | Time to complete (E. coli) | Hands-on time | Requirements |
| -------------------------------------- | ------------------------------------------------ | ----------------------------------------------- | ----------------------------------------------------------------------------------------------------------------------------------------------------------------- |
| :school\_satchel: No skill requirement | :hourglass: 16 hours | :stopwatch: 2 hours | :hard-drive: [7.6a](/item-register/bactobox-r/bactobox-r-hardware-changelog) :floppy-disk: ≥ v2026.02a |
## **Get a high-quality starter culture**
Most bioprocesses are initiated from a cryo stock. The cryo stock is thawed at room temperature and a volume is added to growth medium in a shake flask for overnight incubation. This starter culture will later be used to inoculate a bioreactor. The ratio between volume of cryo stock to volume of growth medium is called inoculation ratio (or seeding ratio).
A typical seed train for a bioprocesses. The initial step is the reactivation and expansion of bacteria from a cryo stock.
A common pitfall at this stage is overseeding. Too much salt ruins your food; overseeding ruins your culture. In practice high inoculation ratio may cause the starter culture to enter death phase prematurely. As a result, the subsequent expansion suffers from a prolonged lag phase, requiring extended incubation times before the culture is ready for transfer to the next bioreactor. To achieve a [high-quality culture](/mpd/cell-growth/high-quality-culture), it is crucial to establish the correct inoculation ratio.
## Introducing OptiSeeding
OptiSeeding is a step-by-step workflow designed to help you select the optimal seeding ratio for the initial cryo stock expansion. The workflow is essentially a screening experiment with 1:10 dilution series prepared directly in shake flasks.
High-level view of OptiSeeding: Dilution series are prepared directly in shake flasks and then incubated overnight.
OptiSeeding ensures efficient culture expansion, and thereby expedites process completion. The explainer [Overnight cultures](/mpd/cell-growth/overnight2) presents proof of principle for the OptiSeeding approach with investigation of culturability using different initial inoculation ratios.
## Step by step overview
In this how to example, we use *E. coli* in a shake flask with tryptic soy broth (TSB) as the growth medium. We illustrate the procedure using [Access](https://help.sbtinstruments.com/software/access) for automated dilution factor calculations. You can also use the BactoBox® buttons for measurements and perform the calculations manually.
The general workflow follows the below steps
1. [Get things ready](/mpd/workflows/identify-optimal-inoculum-size/get-things-ready) for the experiment.
2. [Prepare flasks and media](/mpd/workflows/identify-optimal-inoculum-size/prepare-flasks-and-media) for the overnight cultures.
3. [Create measurement group](/mpd/workflows/identify-optimal-inoculum-size/create-measurement-group) to link the measurements.
4. [Measure on cryo stock](/mpd/workflows/identify-optimal-inoculum-size/measure-on-cryo-stock) to determine initial total cell concentration.
5. [Prepare dilution series](/mpd/workflows/identify-optimal-inoculum-size/prepare-dilution-series) directly in shake flasks and incubate flasks overnight.
6. [Select relevant cultures](/mpd/workflows/identify-optimal-inoculum-size/select-relevant-cultures) by visual inspection.
7. [Check lowest culture](/mpd/workflows/identify-optimal-inoculum-size/check-lowest-culture) by 1:1 000 dilution and BactoBox® measurement.
8. [Check remaining cultures](/mpd/workflows/identify-optimal-inoculum-size/check-remaining-cultures) by 1:10 000 dilution and BactoBox® measurement.
9. [Choose best seeding ratio](/mpd/workflows/identify-optimal-inoculum-size/choose-best-seeding-ratio) for future overnight cultures.
## Summary
You are now ready to start your journey to get a [high-quality culture](/mpd/cell-growth/high-quality-culture). Let's jump into it. First we will [get things ready](/mpd/workflows/identify-optimal-inoculum-size/get-things-ready).
# Get things ready
You will need these items for the experiment.
Items required for the experiment: Along with these, ensure to have sterile serological pipettes and a pipette controller available.
## Hardware
* [BactoBox® setup](https://help.sbtinstruments.com/items/bactobox-r#bactobox-r-setup) including a [flow cell](https://help.sbtinstruments.com/items/consumables/flow-cell) with at least 10 measurements.
* HW: [7.6a](/item-register/bactobox-r/bactobox-r-hardware-changelog)
* SW: ≥ v2026.02a
* Windows 10/11 computer.
* 10 × shake flasks with vented caps. Preferably [50/125 mL capacity](https://www.dwk.com/na/kimble-kimax-baffled-shake-flask-125-ml-25630-125). Alternative: 2 × [6-deepwell microplates](https://www.enzyscreen.com/product/6-deepwell-plate/).
* Incubator shaker that can hold 10 flasks (not shown).
* Permanent marker.
* Vortex mixer.
* Pipettes and sterile pipette tips: P10,000, P1000, P200. Use extended pipette tips to reduce contamination risks.
* Sterile, serological pipettes. Preferably 10 mL version.
* Serological pipet controller (not shown).
* Sterile 15 mL centrifuge vial (or similar) for disaggregating overnight culture.
* Rack for 15 mL centrifuge tubes.
## Reagents
* 1 × cryo stock of your bacterial species.
* Growth medium. Preferably filtered through a 0.2 µm filter to [minimize contribution of background particulates from growth medium](https://help.sbtinstruments.com/custom/advanced-sample-preparation/focus-on-target-objects).
* [Dilution vials](https://help.sbtinstruments.com/items/vials-flasks-and-liquids/dilution-vial) either ready-to-use or freshly prepared using the [dilution dispenser](https://help.sbtinstruments.com/items/accessories/dilution-dispenser).
* [Disinfection vials](https://help.sbtinstruments.com/items/vials-flasks-and-liquids/disinfection-vial).
{% hint style="info" icon="flask" %}
#### Fill level for different vessels.
* 50/125 mL shake flasks or [6-deepwell microplates](https://www.enzyscreen.com/product/6-deepwell-plate/): Add 9 mL cultivation medium to each shake flask. Dilution series is prepared by stepwise transfer of 1 mL from flask to flask
* 250 mL shake flasks: Add 18 mL to each shake flask. Dilution series is prepared by stepwise transfer of 2 mL from flask to flask.
{% endhint %}
## Summary
Once you have located the items needed for the experiment, proceed to [Prepare flasks and media](/mpd/workflows/identify-optimal-inoculum-size/prepare-flasks-and-media).
# Prepare flasks and media
{% stepper %}
{% step %}
**Prepare growth medium**
Dissolve 500 mL culture medium in an autoclavable bluecap flask as recommended by the manufacturer.
{% endstep %}
{% step %}
**Get flasks ready**
Loosen the lids on 10 shake flasks slightly to allow steam entry.
Place a piece of autoclave tape between the flask and the cap.
Label each flask clearly with a permanent marker on the autoclave tape.
* Label the first flask 10-1.
* Proceed with the remaining flasks until reaching 10-10.
{% endstep %}
{% step %}
**Sterilize growth medium and flasks**
Sterilize the bluecap and shake flasks according to the medium manufacturer's autoclaving instructions: usually at 121 °C and \~15 psi for 15 minutes.
{% endstep %}
{% step %}
**Transfer growth medium to each flask**
Once the flasks and cultivation medium have cooled, transfer 9 mL growth medium to each flask using proper aseptic technique.
{% endstep %}
{% endstepper %}
{% hint style="info" icon="cloud-fog" %}
#### Liquid evaporates during the autoclave process
For a reliable dilution series, we need the volume of growth medium in each flask to be accurate.
To ensure consistent liquid levels and reliable dilutions, we autoclave the growth medium separately before transferring it to shake flasks. Autoclaving directly in flasks can cause unpredictable evaporation.
{% endhint %}
## Summary
Shake flasks and growth medium is ready for the experiment.\
Next step: [Create measurement group](/mpd/workflows/identify-optimal-inoculum-size/create-measurement-group).
# Create measurement group
{% stepper %}
{% step %}
#### Connect computer to BactoBox®
1. Connect your BactoBox® to a computer using the provided USB cable.
2. When the external BactoBox drive appears, double-click the Access file. This will open a browser window. The preferred browsers are Chrome and Firefox.
3. Activate full screen mode, press F11 (or Fn+F11 depending on keyboard configuration).
{% hint style="danger" %}
#### No connection to Access?
For troubleshooting of connectivity issues, please see [Can not connect to Access](/troubleshooting/power-on-works-but.../can-not-connect-to-access)
{% endhint %}
{% endstep %}
{% step %}
**Create a basic measurement group**
1. Click the *Measurement group* menu item.
2. Click *Create measurement group.*
3. Provide the experiment details in the input fields. You can use either the pointer device or the tab key to move between input fields.
4. Choose the *basic* measurement group type.
5. Click *Create* to complete the procedure.
6. Finally, click the *open* icon to enter the measurement group you have just created.
{% endstep %}
{% endstepper %}
## Summary
You created and opened a *basic* measurement group for the OptiSeeding experiment.\
Next step is [Measure on cryo stock](/mpd/workflows/identify-optimal-inoculum-size/measure-on-cryo-stock).
# Measure on cryo stock
The cryo stock is the starting point for preparing the dilution series. It will likely contain a mix of culturable, viable-but-non-culturable (VBNC), and dead cells. Knowing the cell concentration helps determine how far the dilution can go before cells are virtually absent in the growth medium. We will use a 1:1 000 dilution in two sequential dilution steps: An initial 1:100 dilution followed by a additional 1:10 dilution.
{% stepper %}
{% step %}
**Disaggregate cell clumps**
Allow the cryo stock to thaw at room temperature.
Vortex the cryo stock 1 minute at maximum speed to disaggregate cell clumps.
{% endstep %}
{% step %}
**Dilute 1:100**
Transfer 101 µL of your sample to 10 mL of diluent. Vortex 10 seconds at maximum speed.
{% endstep %}
{% step %}
**Dilute 1:1000**
Remove 1 mL diluent from a 10 mL diluent vial for a residual volume of 9 mL.
Transfer 1 mL of the 1:100 dilution to the vial containing 9 mL of diluent. Vortex 10 seconds at maximum speed.
{% endstep %}
{% step %}
**Measure on the diluted sample**
Transfer the tubing kit to the 1:1 000 vial.
Add the metadata:
* Dilution: 1 000
* Label: E\_coli\_cryo
Then press Measure in Access to start a new measurement.
After completing the measurement, Access will automatically calculate the bacterial concentration in the cryo stock in the field Cells/mL × Dil.
{% endstep %}
{% endstepper %}
## Summary
Now you know the bacterial concentration of your cryo stock.\
Next step: [Prepare dilution series](/mpd/workflows/identify-optimal-inoculum-size/prepare-dilution-series).
# Prepare dilution series
## Pick the right number of flasks
{% hint style="success" %}
#### Choose the right number of flasks
If the concentration in the cryo stock is lower than 1 × 108, using 10 flasks is excessive. As a general guideline, add 2 to the exponent. In this example, the number of flasks would be 8 + 2 = 10.
{% endhint %}
In the present example, the concentration in the cryo vial is 9.6 × 108 cells/mL. As shown in the table, diluting beyond the 10-10 flask is unnecessary, as higher dilutions likely contain fewer than one cell. Thus, the growth medium will likely remain sterile after overnight incubation. This is why we choose 10 flasks for the dilution series.
| Flask | Cells per mL | Cells per 10 mL |
| :--------------: | :-------------------: | :-------------: |
| 10-1 | 9.6 × 107 | 960 000 000 |
| 10-2 | 9.6 × 106 | 96 000 000 |
| 10-3 | 9.6 × 105 | 9 600 000 |
| 10-4 | 9.6 × 104 | 960 000 |
| 10-5 | 9.6 × 103 | 96 000 |
| 10-6 | 9.6 × 102 | 9 600 |
| 10-7 | 9.6 × 101 | 960 |
| 10-8 | 9.6 × 100 | 96 |
| 10-9 | 9.6 × 10-1 | \~10 |
| 10-10 | 9.6 × 10-2 | \~1 |
## Introduction to workflow
The dilution series is prepared as a 1:10 dilution series directly in shake flasks.
High-level view of OptiSeeding: Dilution series are prepared directly in shake flasks and then incubated overnight.
For each shake flask 1 mL of the previous dilution (or cryo stock) is transferred to 9 mL of growth medium. The flask is capped and the bacteria are dispersed by figure 8 movements.
## Step by step
{% stepper %}
{% step %}
**Vortex the cryo stock for a single-cell suspension**
Briefly vortex the cryo stock for 10 seconds at maximum RPM. This assumes that the cryo stock was already thoroughly vortexed in the previous step [Measure on cryo stock](/mpd/workflows/identify-optimal-inoculum-size/measure-on-cryo-stock).
{% endstep %}
{% step %}
**Prepare first 1:10 dilution**
Transfer 1 mL cryo stock to 9 mL growth medium.
Use long, sterile pipette tips to avoid contamination from the non-sterile pipette.
Cap flask and make figure 8 motion to distribute the cells evenly.
{% endstep %}
{% step %}
**Continue dilution series**
Repeat step 2 for the subsequent 10-2 to 10-10 flasks.
{% endstep %}
{% step %}
**Incubate overnight**
Incubate overnight in shaking incubator at the desired growth conditions. For this *E. coli* example we used 37 °C, 200 RPM.
{% endstep %}
{% endstepper %}
{% hint style="info" icon="turtle" %}
#### Do your bacteria have slow generation time?
The workflow presented here works for bacteria with a generation time of ≤ 30 min. Adjustments may be necessary when working for slow-growing bacteria:
* Increase incubation time.
* Use 1:5 dilutions when preparing the shake flask dilution series.
{% endhint %}
## Summary
A tenfold dilution series has been prepared from the cryo stock for overnight incubation.\
Next step: [Select relevant cultures](/mpd/workflows/identify-optimal-inoculum-size/select-relevant-cultures).
# Select relevant cultures
After overnight incubation the flasks seeded at high concentrations will likely be in stationary or death stage. The goal is to identify the flask that has just reached stationary phase as this is where you will find the highest concentration of culturable cells. To save time, initially inspect and discard flasks that seem irrelevant before measuring concentrations in the remaining ones.
In this example we will only keep vials -4 to -8. Below we will explain why.
{% stepper %}
{% step %}
**Transfer culture to centrifuge vial**
Use a sterile serological pipette to transfer 5 mL of each culture to a sterile 15 mL vial.
{% endstep %}
{% step %}
**Remove vials with low turbidity**
Remove the vials with very low turbidity because they likely have very low bacterial concentrations. In this example, we remove vials -9 and -10.
{% endstep %}
{% step %}
**Remove vials with high initial seeding concentration**
Remove vials with high initial seeding concentration because these will likely contain a significant number of non-culturable cells.
* In this example, vial -1 to -6 have similar turbidity.
* We remove vials -1, -2, and -3 because these cultures are likely already deep in stationary or death stage.
{% endstep %}
{% endstepper %}
{% hint style="info" icon="hourglass" %}
#### Choose relevant incubation time
* The incubation time will strongly affect the cell concentration. Choose the same incubation time as you expect to use for your process.
* Here we incubated the *E. coli* culture for 11 hours. Typically an overnight culture is started in the afternoon and analyzed in the morning, i.e. a normal incubation time is approximately 16 hours.
{% endhint %}
## Summary
We have chosen the relevant cultures from the shake flask dilution series. In the present example we removed half of the vials leaving only vials -4 to -8.
In the next step, [Check lowest culture](/mpd/workflows/identify-optimal-inoculum-size/check-lowest-culture), we will measure the cell concentration in these cultures.
# Check lowest culture
After removing irrelevant cultures, it's time to measure the concentration of the remaining overnight cultures. Begin with the culture having the lowest turbidity, which in this case is the vial -8 . We will use a 1:1 000 dilution in two sequential dilution steps: An initial 1:100 dilution followed by a additional 1:10 dilution.
{% stepper %}
{% step %}
**Disaggregate cell clumps**
Vortex the vial 1 minute at maximum speed to disaggregate cell clumps. See [Break up clumps and chains](https://help.sbtinstruments.com/custom/advanced-sample-preparation/break-up-clumps-and-chains) if your bacterium needs more aggressive disaggregation.
{% endstep %}
{% step %}
**Dilute 1:100**
Transfer 101 µL of your sample to 10 mL of diluent. Vortex 10 seconds at maximum speed.
{% endstep %}
{% step %}
**Dilute 1:1 000**
Remove 1 mL diluent from a 10 mL diluent vial for a residual volume of 9 mL.
Transfer 1 mL of the 1:100 dilution to the this vial. Vortex 10 seconds at maximum speed.
{% endstep %}
{% step %}
**Measure on the diluted sample**
Transfer the tubing kit to the 1:1 000 vial.
Add the metadata:
* Dilution: 1 000
* Label: overnight\_-8
Then press Measure in Access to start a new measurement.
{% endstep %}
{% endstepper %}
## Summary
Of the remaining vials, the culture with the lowest turbidity has a concentration of 1.4 × 109 cells/mL. Next, we will [check remaining cultures](/mpd/workflows/identify-optimal-inoculum-size/check-remaining-cultures) to identify the culture that has just reached onset of stationary phase.
# Check remaining cultures
A 1:1 000 dilution worked well for the -8 culture. The remaining cultures from -7 to -4 were seeded with more cells and therefore a dilution factor of 1:10 000 will be used for these overnight cultures. This is achieved through two sequential 1:100 dilutions.
{% stepper %}
{% step %}
**Disaggregate cell clumps**
Vortex the vial 1 minute at maximum speed to disaggregate cell clumps. See [Break up clumps and chains](https://help.sbtinstruments.com/custom/advanced-sample-preparation/break-up-clumps-and-chains) if your bacterium needs more aggressive disaggregation.
{% endstep %}
{% step %}
**Dilute 1:100**
Transfer 101 µL of your sample to 10 mL of diluent. Vortex 10 seconds at maximum speed.
{% endstep %}
{% step %}
**Dilute 1:10 000**
Transfer 101 µL of the diluted sample to 10 mL of diluent. Vortex 10 seconds at maximum speed.
{% endstep %}
{% step %}
**Measure the diluted sample on BactoBox®**
Transfer the tubing kit to the 1:10 000 vial.
Add the metadata:
* Dilution: 10 000
* Label: overnight\_-7 , overnight\_-6 , overnight\_-5 or overnight\_-4 ,
Then press Measure in Access to start a new measurement.
After completing the measurement, Access will automatically calculate the bacterial concentration in the cryo stock in the field Cells/mL × Dil.
{% endstep %}
{% step %}
**Check remaining cultures**
Repeat above steps with 1:10 000 dilutions of the remaining cultures, in this case vial -6, -5, and -4.
{% endstep %}
{% endstepper %}
## Summary
You have now determined the cell concentration in the relevant cultures.\
Proceed to the next step: [Choose best seeding ratio](/mpd/workflows/identify-optimal-inoculum-size/choose-best-seeding-ratio).
# Choose best seeding ratio
The measurements are now completed and it is time to choose the inoculation ratio that results in the best [High-quality culture](/mpd/cell-growth/high-quality-culture). In this 11-hour *E. coli* example, the 10-5 cryo stock dilution produces a culture that has likely just arrived at stationary stage. The 10-4 cryo stock dilution has a similar cell concentration but is less suitable due to being in the stationary phase longer. It therefore likely contains a higher proportion of cells that have lost culturability.
{% hint style="warning" %}
#### The inoculation ratio is only valid for the given cryo stock and growth parameters.
Conduct a new optimization experiment if there are any changes to cryo stocks, vessel type, broth, or incubation time.
{% endhint %}
## How to prepare high dilutions of the cryo stock
In the present example, a 10-5 dilution resulted in the best [High-quality culture](/mpd/cell-growth/high-quality-culture). This ratio is equivalent to a 1:100,000 dilution of the cryo stock, i.e substantially higher dilution than the typical 1:100 volumetric seeding ratio.
For future 11-hour overnight cultures, this would correspond to adding just 1 µL cryo stock to 100 mL (100,000 µL) growth medium. Because it it tricky to accurately pipette 1 µL we recommend that you first prepare a 1:100 dilution and then dilute this 1:1 000 for the final 1:100,000 dilution:
1. Prepare 1:100 dilution: Transfer 0.1 mL cryo stock to 9.9 mL sterile growth medium
2. Prepare 1:100,000 dilution: Transfer 0.1 mL 1:100 dilution to \~100 mL sterile growth medium in shake flask
## Summary and next steps
We hope that you are enjoyed the insights from this step by step guide. We are confident that your new and shiny [High-quality culture](/mpd/cell-growth/high-quality-culture) will improve your future bioprocesses.
Now that you have the conditions for a great starter culture try to [Track growth curve](/mpd/workflows/track-growth-curve) for it. For example, you could compare the [High-quality culture](/mpd/cell-growth/high-quality-culture) head-to-head with your previous starter culture protocol. You will likely observe a shorter lag phase duration for the [High-quality culture](/mpd/cell-growth/high-quality-culture).
# Track growth curve
MPD-7. Visualize growth curves in real-time with BactoBox®.
| Skill level | Time to complete (E. coli) | Hands-on time | Requirements |
| -------------------------------------- | ----------------------------------------------- | ----------------------------------------------- | ----------------------------------------------------------------------------------------------------------------------------------------------------------------- |
| :school\_satchel: No skill requirement | :hourglass: 8 hours | :stopwatch: 4 hours | :hard-drive: [7.6a](/item-register/bactobox-r/bactobox-r-hardware-changelog) :floppy-disk: ≥ v2026.02a |
Access lets you instantly add metadata about your sample. With the computer as input device, you can interact with familiar BactoBox® buttons on your screen. Growth curves are generated with ease when you add information on dilution factors and sampling time.
In this step-by-step example we will demonstrate how to track growth curves. We'll use a straightforward example with *E. coli* in a shake flask. At 37 °C, *E. coli* has a generation time, g, of \~20 min in LB medium. We will sample at g x 1.5, that is a sample for every 30 min.
Notice that you will collect samples every 30 minutes for \~8 hours. This requires that you set aside a full lab day for the experiment if you are working with *E. coli*. Understanding the lag phase duration, growth rates, and carrying capacity can significantly enhance your fermentation process design. Trust us; it will be worth it!
Here’s how the results will appear after measurements are completed.
The overall steps are shown below. Click the links to jump to the relevant section.
1. [Get things ready](/mpd/workflows/track-growth-curve/get-things-ready)
2. [Create measurement group](/mpd/workflows/track-growth-curve/create-measurement-group)
3. [Check growth medium](/mpd/workflows/track-growth-curve/check-growth-medium)
4. [Measure starter culture](/mpd/workflows/track-growth-curve/measure-starter-culture)
5. [Inoculate](/mpd/workflows/track-growth-curve/inoculate)
6. [1:100 dilution after inoculation](/mpd/workflows/track-growth-curve/1-100-dilution-post-inoculation)
7. [1:100 dilutions](/mpd/workflows/track-growth-curve/1-100-dilutions)
8. [1:10 000 dilutions](/mpd/workflows/track-growth-curve/1-10-000-dilutions)
9. [Conclude growth curve](/mpd/workflows/track-growth-curve/conclude-growth-curve)
# Get things ready
You will need these items for the growth curve experiment.
## Hardware
* [BactoBox® setup](https://help.sbtinstruments.com/items/bactobox-r#bactobox-r-setup) including a [flow cell](https://help.sbtinstruments.com/items/consumables/flow-cell) with at least 50 measurements.
* HW: [7.6a](/item-register/bactobox-r/bactobox-r-hardware-changelog)
* SW: ≥ v2026.02a
* Windows 10/11 computer.
* Incubator shaker.
* Vortex mixer.
* Pipettes and sterile pipette tips: P1000, P200, P20.
* Serological pipettes. Preferably 10 and 2 mL versions.
* Serological pipet controller.
* Sterile 50 mL centrifuge vial (or similar) for disaggregating overnight culture.
* 2 mL eppendorf vials (or similar) for individual sampling points.
## Reagents
* 1 x [high-quality culture](/mpd/cell-growth/high-quality-culture).
* 1 x Erlenmeyer flask with cultivation medium.
* [Dilution vials](https://help.sbtinstruments.com/items/vials-flasks-and-liquids/dilution-vial) either ready-to-use or freshly prepared using the [dilution dispenser](https://help.sbtinstruments.com/items/accessories/dilution-dispenser).
* [Disinfection vials](https://help.sbtinstruments.com/items/vials-flasks-and-liquids/disinfection-vial).
{% hint style="success" %}
#### Use proper aseptic technique
In the example videos, we use a simple lab bench. Always use proper aseptic techniques, including sterile pipette tips, laminar air flow (LAF), and disinfection of the work environment.
{% endhint %}
Next, [create a measurement group](/mpd/workflows/track-growth-curve/create-measurement-group).
# Create measurement group
{% stepper %}
{% step %}
**Connect computer to BactoBox®**
1. Connect your BactoBox® to a computer using the provided USB cable.
2. When the external BactoBox drive appears, double-click the Access file. This will open a browser window. The preferred browsers are Chrome and Firefox.
3. Activate full screen mode, press F11 (or Fn+F11 depending on keyboard configuration).
{% hint style="danger" %}
#### No connection to Access?
For troubleshooting of connectivity issues, please see [Can not connect to Access](https://help.sbtinstruments.com/power-on-works-but.../can-not-connect-to-access).
{% endhint %}
{% endstep %}
{% step %}
**Create a growth curve measurement group**
1. Click the *Measurement group* menu item.
2. Click *Create measurement group.*
3. Provide the experiment details in the input fields. You can use either the pointer device or the tab key to move between input fields. Choose the type *growth curve*
4. *Click Create to complete the procedure*
5. Finally, click the *open* icon to enter the measurement group you have just created.
{% endstep %}
{% endstepper %}
## Summary
You created and opened a measurement group for the growth curve experiment. Next, check the contribution of background particulates from your growth medium.
# Check growth medium
Certain growth media have unexpectedly high particle concentrations. This can lead to confusing results, particularly during the lag phase when bacterial concentration is still low.
Here, you will measure a 1:100 dilution of the growth medium **prior** to inoculation. If the cells/mL concentration is below 30,000 cells/mL the growth medium contribution is negligible and you can skip ahead to the next section. If the cells/mL exceeds 30,000 cells/mL a suitable workaround must be chosen. See the info box at the bottom of this page for more information.
{% stepper %}
{% step %}
**Prepare the sample for BactoBox® measurement**
1. Use a serological pipette to collect a small sample from the shake flask.
2. Return the shake flask to the incubator shaker.
3. Vortex 30 seconds at max speed to ensure an even distribution of particles
4. Dilute 1:100 in diluent by adding 101 µL of sample to 10 mL of diluent. Vortex 10 sec., max speed.
5. Transfer the tubing kit to the diluted sample.
{% endstep %}
{% step %}
**Measure the diluted sample on BactoBox®**
7. Add metadata:
1. Dilution: 100.
2. Label: medium\_ctrl.
8. Click the Measure button in Access.
{% hint style="danger" %}
## Do you get the error *conductivity too high*?
Some cultivation media have very high conductivity and a 1:100 dilution in standard diluent is not sufficient to bring the diluted sample within the accepted conductivity limits.
You can typically solve this issue by using a 1:201 dilution: Simply add 50 µL of sample to 10 mL of diluent. For more information, please see the section [Hit the right conductivity](/advanced/advanced-sample-preparation/hit-the-right-conductivity).
{% endhint %}
{% endstep %}
{% step %}
**Clean the device**
9. Transfer the tubing kit to the disinfection vial.
10. Click Clean.
{% endstep %}
{% endstepper %}
## Summary
Now you know the particle contribution from the growth medium. Next, measure the bacterial concentration in your starter culture.
## Mitigate background particulates in growth medium
{% hint style="info" %}
## Does the cells/mL exceed 30,000 cells/mL at 1:100 dilution?
Several strategies are possible if the [particle concentration in the medium is substantial](/advanced/advanced-sample-preparation/focus-on-target-objects/mitigate-high-background). Consult [Mitigate high background](/advanced/advanced-sample-preparation/focus-on-target-objects/mitigate-high-background) for more information.
{% endhint %}
# Measure starter culture
To track a full growth curve, you need a starter culture with a known concentration. From this, we calculate the volume required to reach a final concentration of 1 × 10⁷ cells/mL in the flask.
Here we have already prepared the overnight culture based on the guidance provided in [Optimize starter culture](/mpd/workflows/identify-optimal-inoculum-size).
{% stepper %}
{% step %}
**Retrieve a sample of the starter culture**
1. Use a serological pipette to aseptically transfer 10 mL of the overnight culture to a sterile vial.
2. Vortex thoroughly to disperse any cell clumps. For an *E. coli* culture it is usually sufficient to vortex 30 seconds at max speed.
{% hint style="warning" %}
## Proper vortexing is important for reliable results!
Ensure that you see a proper vortex forming. Poor vortexing can cause inadequate disaggregation, leading to low precision in replicates and non-linearity in dilution series.
Different pedestals exists for different sizes of vials. In the video, we show a multipurpose pedestal that works for many types of vials. Ensure that your pedestal is compatible with the given vial.
{% endhint %}
{% endstep %}
{% step %}
**Prepare the sample for BactoBox® measurement**
1. Prepare a 1:10 000 dilution using two sequential 1:100 dilution steps.
1. First make a 1:100 dilution:
1. Transfer 101 µL of your sample to 10 mL of diluent.
2. Vortex 10 seconds at maximum speed.
2. Then make the 1:10 000 dilution:
1. Transfer 101 µL of the 1:100 dilution to 10 mL diluent.
2. Vortex 10 seconds at maximum speed.
2. Transfer the tubing kit to the 1:10 000 vial.
{% endstep %}
{% step %}
**Measure the diluted sample on BactoBox®**
1. Transfer the tubing kit to the 1:10,000 vial.
2. Add metadata:
1. Dilution: 10 000
2. Label: Overnight\_culture
3. Click Measure to start a new measurement. Notice that there is a group icon on the button because you are within a measurement group.
4. After completing the measurement, Access will automatically calculate the bacterial concentration in the shake flask. To view the full table, drag the panel sideways.
{% endstep %}
{% step %}
**Clean the device**
1. Transfer the tubing kit to the disinfection vial.
2. Click Clean. The button in Access works the same way as the one on BactoBox®.
{% endstep %}
{% endstepper %}
## Summary
Now you know the bacterial concentration of your starter culture. Proceed to the next step to calculate the volume of starter culture needed to inoculate the growth curve flask.
# Inoculate
Use the [subcultivation calculator tool](https://www2026.sbtinstruments.com/wp-content/uploads/2025/12/202512-BactoBox-sub-cultivation-calculator.html) to determine the volume of starter culture needed for the growth curve experiment. Here we are targeting an inoculation concentration of 1 x 107 cells/mL.
{% stepper %}
{% step %}
**Calculate inoculation volume**
1. In Access, triple-click the cell containing Cells/mL x Dil. Press ctrl+c to copy the value.
2. Open a new window and use the [subcultivation calculator tool](https://www2026.sbtinstruments.com/wp-content/uploads/2025/12/202512-BactoBox-sub-cultivation-calculator.html) to determine which volume to add (Tip: To exit full screen mode, press F11 (or Fn+F11 depending on keyboard configuration):
1. Press ctrl+v in the first field to input the concentration from the starter culture shake flask.
2. Add the volume information in the second field.
3. Decide on the target inoculation concentration in the last field.
4. Click Calculate V\_add to get the result.
{% endstep %}
{% step %}
**Add starter culture to growth curve flask**
1. Use proper aseptic technique to transfer starter culture to growth curve flask.
2. Swirl aggressively to disperse the inoculum.
3. Remember to record the inoculation time. Record it on the autoclave tape as a backup, ensuring you have a reference if you forget to document it elsewhere.
{% endstep %}
{% endstepper %}
## Summary
You have now inoculated the growth medium at a well-defined starting concentration of 1 x 107 cells/mL. Next step is to determine the bacterial concentration at T0.
# 1:100 dilution after inoculation
Once the shake flask is inoculated, immediately collect a sample to obtain a measurement as close to T0 as possible.
{% stepper %}
{% step %}
**Prepare the sample for BactoBox® measurement**
1. Use a serological pipette to transfer a sample from the shake flask to a vial.
2. Use a permanent marker to label the vial cap with the sampling time.
3. Return the shake flask to the incubator shaker.
4. Start a 30-minute countdown to remind you to collect the next sample.
5. Vortex 30 seconds at max speed to disaggregate bacteria.
6. Dilute 1:100 in diluent by adding 101 µL of sample to 10 mL of diluent. Vortex 10 sec., max speed.
7. Transfer the tubing kit to the diluted sample.
{% hint style="success" %}
#### Put the flask back in the incubator shaker immediately after collecting the sample.
Collect the sample quickly to ensure prompt return. Minimize the time the flask is outside the incubator shaker to avoid drops in temperature and dissolved oxygen levels.
{% endhint %}
{% endstep %}
{% step %}
**Measure the diluted sample on BactoBox®**
1. Add metadata:
1. Dilution: 100.
2. Label: E\_coli\_growth.
2. Click the Measure button in Access.
3. Once measurement is done, click the label E\_coli\_growth above the chart to visualize the growth curve data.
{% hint style="danger" %}
## Do you get the error *conductivity too high*?
Some cultivation media have very high conductivity and a 1:100 dilution in standard diluent is not sufficient to bring the diluted sample within the accepted conductivity limits.
You can typically solve this issue by using a 1:201 dilution: Simply add 50 µL of sample to 10 mL of diluent. For more information, please see the section [Hit the right conductivity](/advanced/advanced-sample-preparation/hit-the-right-conductivity).
{% endhint %}
{% endstep %}
{% step %}
**Clean the device**
1. Transfer the tubing kit to the disinfection vial.
2. Click Clean.
{% endstep %}
{% endstepper %}
## Summary
You now know the bacterial concentration at T0. Next we will collect a sample for every 30 min and keep repeating the 1:100 dilution scheme for lag and early exponential growth stage.
# 1:100 dilutions
For every 30 minutes, collect a fresh sample and repeat the procedure from the 1:100 dilution.
{% hint style="success" %}
#### :spray-can: Do a *Clean* program after each measurement.
{% endhint %}
{% hint style="success" %}
#### :hourglass: Start a 30-minute countdown to remind you to collect the next sample.
{% endhint %}
{% stepper %}
{% step %}
**Prepare the sample for BactoBox® measurement**
1. Use a serological pipette to transfer a sample from the shake flask to a vial.
2. Use a permanent marker to label the vial cap with the sampling time.
3. Start a 30-minute countdown to remind you to collect the next sample.
4. Vortex 30 seconds at max speed to disaggregate bacteria.
5. Make a 1:100 dilution:
1. Transfer 101 µL of your sample to 10 mL of diluent.
2. Vortex 10 seconds at max speed.
6. Transfer the tubing kit to the diluted sample.
{% endstep %}
{% step %}
**Measure the diluted sample on BactoBox®**
1. Add metadata:
1. Dilution: 100.
2. Label: E\_coli\_growth.
2. Click the Measure button in Access.
{% endstep %}
{% step %}
**Clean the device**
1. Transfer the tubing kit to the disinfection vial.
2. Click Clean.
{% endstep %}
{% endstepper %}
## Summary
The 1:100 dilution scheme works well for the measurements during lag and early-exponential growth stage. As the concentration increases, you'll need to dilute more. We will explain this in detail in the next section.
# 1:1 000 dilutions
As the bacteria multiply, the concentration increases, and a 1:100 dilution is no longer suitable. The maximum concentration in the diluted sample is 5 000 000 total/mL. If this limit is exceeded, an error will display instead of a concentration. In such cases, clean the setup and measure a more diluted sample.
{% hint style="info" %}
#### Avoid getting *too many particles.*
To prevent the "too many particles" error, follow this simple rule of thumb during the exponential growth stage: If the 1:100 diluted sample was above 1 500 000 at the last measurement \~30 minutes ago, it is time to start using a 1;1 000 dilution.
This rule works for all bacteria as long as the sampling frequency is set to 1.5 x generation time.
{% endhint %}
{% hint style="success" %}
#### :spray-can: Do a *Clean* program after each measurement.
{% endhint %}
{% hint style="success" %}
#### :hourglass: Start a 30-minute countdown to remind you to collect the next sample.
{% endhint %}
{% stepper %}
{% step %}
**Prepare the sample for BactoBox® measurement**
1. Use a serological pipette to transfer a sample from the shake flask to a vial.
2. Use a permanent marker to label the vial cap with the sampling time.
3. Return the shake flask to the incubator shaker.
4. Start a 30-minute countdown to remind you to collect the next sample.
5. Vortex 30 seconds at max speed to disaggregate bacteria.
6. Make a 1:1 000 dilution
1. First make a 1:100 dilution:
1. Transfer 101 µL of your sample to 10 mL of diluent.
2. Vortex 10 seconds at max speed.
2. Then make the 1:1 000 dilution:
1. Remove 1 mL diluent from a 10 mL diluent vial for a residual volume of 9 mL.
2. Transfer 1 mL of the 1:100 dilution to the vial containing 9 mL of diluent.
3. Vortex 10 seconds at max speed.
7. Transfer the tubing kit to the 1:1 000 vial.
{% hint style="info" %}
## Pro tip: 1-step 1:1 001 dilution.
The described 2-step procedure for a 1:1,000 dilution is reliable with high precision. However, it demands a longer sample preparation time. If you have good pipetting skills, you can save time, sample and diluent by doing a 1-step dilution. Simply transfer 10 µL of sample to 10 mL of diluent to [get a 1:1 001 dilution](/advanced/advanced-sample-preparation/hit-the-right-concentration).
{% endhint %}
{% endstep %}
{% step %}
**Measure the diluted sample on BactoBox®**
1. Add metadata:
1. Dilution: 1 000.
2. Label: E\_coli\_growth.
2. Click the Measure button in Access.
{% endstep %}
{% step %}
**Clean the device**
1. Transfer the tubing kit to the disinfection vial.
2. Click Clean.
{% endstep %}
{% endstepper %}
## Summary
The 1:1 000 dilution scheme works well for mid- to late-exponential stage.
Monitor when the diluted sample's concentration surpasses 1,500,000. When this happens, jump to the next section where we will detail how to dilute the sample at a ratio of 1:10 000.
# 1:10 000 dilutions
Use a 1:10,000 dilution if the 1:1,000 dilution results in more than 1,500,000 total/ml, as observed in the sample analyzed approximately 30 minutes ago.
{% hint style="success" %}
#### :spray-can: Do a *Clean* program after each measurement.
{% endhint %}
{% hint style="success" %}
#### :hourglass: Start a 30-minute countdown to remind you to collect the next sample.
{% endhint %}
{% stepper %}
{% step %}
**Prepare the sample for BactoBox® measurement**
1. Use a serological pipette to transfer a sample from the shake flask to a vial.
2. Use a permanent marker to label the vial cap with the sampling time.
3. Return the shake flask to the incubator shaker.
4. Start a 30-minute countdown to remind you to collect the next sample.
5. Vortex 30 seconds at max speed to disaggregate bacteria.
6. Prepare a 1:10 000 dilution using two sequential 1:100 dilution steps.
1. First make a 1:100 dilution:
1. Transfer 101 µL of your sample to 10 mL of diluent.
2. Vortex 10 seconds at maximum speed.
2. Then make the 1:10 000 dilution:
1. Transfer 101 µL of the 1:100 dilution to 10 mL diluent.
2. Vortex 10 seconds at maximum speed.
7. Transfer the tubing kit to the 1:10 000 vial.
{% endstep %}
{% step %}
**Measure the diluted sample on BactoBox®**
8. Add metadata:
1. Dilution: 10 000.
2. Label: E\_coli\_growth.
9. Click the Measure button in Access.
{% endstep %}
{% step %}
**Clean the device**
1. Transfer the tubing kit to the disinfection vial.
2. Click Clean.
{% endstep %}
{% endstepper %}
## Summary
You have now tracked the concentration through lag, exponential, deceleration and stationary stage. The final step is to determine when to conclude the growth curve experiment.
# Conclude growth curve
Continue with 1:10 000 dilutions until the concentration stabilizes. This occurs when two consecutive data points are approximately within ±25%.
{% hint style="success" %}
#### :spray-can: Do a Clean program after each measurement.
{% endhint %}
{% hint style="success" %}
#### :hourglass: Start a 30-minute countdown to remind you to collect the next sample.
{% endhint %}
{% stepper %}
{% step %}
**Prepare the sample for BactoBox® measurement**
1. Use a serological pipette to transfer a sample from the shake flask to a vial.
2. Use a permanent marker to label the vial cap with the sampling time.
3. Return the shake flask to the incubator shaker.
4. Start a 30-minute countdown to remind you to collect the next sample.
5. Vortex 30 seconds at max speed to disaggregate bacteria.
6. Prepare a 1:10 000 dilution using two sequential 1:100 dilution steps.
1. First make a 1:100 dilution:
1. Transfer 101 µL of your sample to 10 mL of diluent.
2. Vortex 10 seconds at maximum speed.
2. Then make the 1:10 000 dilution:
1. Transfer 101 µL of the 1:100 dilution to 10 mL diluent.
2. Vortex 10 seconds at maximum speed.
7. Transfer the tubing kit to the 1:10 000 vial.
{% endstep %}
{% step %}
**Measure the diluted sample on BactoBox®**
1. Add metadata:
1. Dilution: 10 000.
2. Label: E\_coli\_growth.
2. Click the Measure button in Access.
{% endstep %}
{% step %}
**Clean the device**
1. Transfer the tubing kit to the disinfection vial.
2. Click Clean.
{% endstep %}
{% endstepper %}
## Summary
You have now completed your growth curve experiment!
With these data you can calculate growth rate (µ), lag phase duration (ƛ), and carrying capacity (κ). These metrics will help you design of efficient seed trains.
[Contact SBT](https://help.sbtinstruments.com/troubleshooting/contact-our-support-team) if you need assistance extracting information from the growth curve experiment.
# Screen growth media
MPD-3. Use BactoBox® direct cell counts to screen growth media for their ability to support high cell concentrations.
| BactoBox® skill level | Time to complete (E. coli) | Hands-on time | Requirements |
| --------------------- | ---------------------------------- | ------------------- | ----------------------------------------------------------------------------------------------------------------------------------------------------------------- |
| :footprints: Beginner | :hourglass\_flowing\_sand: 8 hours | :stopwatch: 2 hours | :hard-drive: [7.6a](/item-register/bactobox-r/bactobox-r-hardware-changelog) :floppy-disk: ≥ v2026.02a |
Maximizing CFU/mL is crucial for producing bacterial vaccines, probiotics, soil bio-stimulants, and live biotherapeutics. The growth medium significantly influences the maximum achievable cell concentrations.
## BactoBox® cells as proxy for max CFU/mL
BactoBox® cells/mL serve as an effective proxy for determining maximum CFU/mL in a given growth medium. Obviously, BactoBox® offers greater speed and precision than traditional plate counts. But crucially, BactoBox® maintains a stable cell concentration after reaching the [stationary phase](/mpd/cell-growth/growth-phases) (see lavender curve below).
Contrary to this, plate counts (yellow curve below), usually drops during decline phase and may take different times to plateau across media types. This makes BactoBox® advantageous, allowing for more relaxed sampling without compromising result accuracy. Overall the rationale is that - if present in the growth medium - the cell must have been alive at one point in time.
The explainer section [Carrying capacity, Κ](/mpd/cell-growth/carrying-capacity-k) provides more insights on the benefits of using BactoBox® for determining max cell concentration.
K. aerogenes shake flask growth curve. The data points represent BactoBox® (lavender) and plate counts (yellow). The grey box highlights the stationary and decline phases.
## A simple experiment on a bacterial sample
This workflow gives you a simple step by step guide to screen which growth medium results in the highest cell concentrations. You will get a feel for how to analyze a concentrated bacterial sample by serial dilutions and BactoBox® measurements. Subsequently, you can try other workflows like [Track growth curve](/mpd/workflows/track-growth-curve) to determine growth rate and [Identify harvest time](/mpd/workflows/mpd1) to determine optimal harvest time.
We show an example with four different growth media inoculated with the same *E. coli* starter culture. We suggest that you compare at least two different growth media. The workflow assumes that the cultures have been prepared in advance. The section [Get starter culture](/mpd/workflows/get-starter-cultures) gives information on how to prepare the cultures for the experiments, but you are also welcome to use your own standard operating protocols.
The workflow is illustrated below: A culture is inoculated 24 hours before the experiment, to be well within the stable plateau by the first BactoBox® measurement in the morning. Subsequent measurements occur around noon, 4 hours later. The final BactoBox® measurement takes place in the afternoon, approximately 4 hours afterward.
Example of inoculation time and sampling time. The goal is to get three measurement points within the stable plateau where cell concentrations are maximal. Note that the illustration is for demonstration only; the workflow does not lead to a full growth curve.
## Overview
The overall steps in the workflow are given below.
1. [Get things ready](/mpd/workflows/best-medium/get-things-ready)
2. [Create measurement group](/mpd/workflows/best-medium/create-measurement-group)
3. [Morning measurement](/mpd/workflows/best-medium/check-remaining-cultures)
4. [Noon measurement](/mpd/workflows/best-medium/check-remaining-cultures-1)
5. [Afternoon measurement](/mpd/workflows/best-medium/check-remaining-cultures-2)
6. [Identify best medium](/mpd/workflows/best-medium/identify-best-medium)
7. [Summary](/mpd/workflows/best-medium/summary)
## Summary
You are now ready to use BactoBox® to find out which growth medium results in highest cell concentrations. Let's jump into it. First step is [Get things ready](/mpd/workflows/best-medium/get-things-ready).
# Get things ready
You will need these items for the experiment.
## Hardware
* [BactoBox® setup](https://help.sbtinstruments.com/items/bactobox-r#bactobox-r-setup) including a [flow cell](https://help.sbtinstruments.com/items/consumables/flow-cell) with at least 10 remaining measurements.
* HW: [7.6a](/item-register/bactobox-r/bactobox-r-hardware-changelog)
* SW: ≥ v2026.02a
* Vortex mixer.
* Rack for holding 5 and 15 mL centrifuge vials.
* P1000 and P200 pipette.
* Sterile pipette tips.
* Serological pipettes. Preferably 2 mL version.
* Serological pipet controller.
* Marker
## Reagents
* Cultures of the same species in different growth media
* For example *E. coli* in 50 mL growth medium in a shake flask with 250 mL total volume.
* The cultures should be in stationary or decline [growth phase](/mpd/cell-growth/growth-phases).
* Different growth media should be used. Follow the suppliers recommendations on how to prepare the growth medium.
* [Dilution vials](https://help.sbtinstruments.com/items/vials-flasks-and-liquids/dilution-vial) either ready-to-use or freshly prepared using the [dilution dispenser](https://help.sbtinstruments.com/items/accessories/dilution-dispenser).
* [Disinfection vials](https://help.sbtinstruments.com/items/vials-flasks-and-liquids/disinfection-vial).
## Summary
With these things in hand, you are ready to [Create measurement group](/mpd/workflows/best-medium/create-measurement-group).
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