High-accuracy microbial input

A practical guide to using BactoBox for measuring and validating the CFU-count of bacterial cultures prior to dosing into in vivo and in vitro experiments.

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Goal and scope

This page shows you how to use BactoBox to match the CFU input of a bacterial culture before you dose it into in vivo or in vitro experiments.

You will:

• Understand when BactoBox cells/mL and plate CFU counts agree, and when they drift apart.

• Choose between two simple workflows that keep your culture in a phase where BactoBox is a reliable proxy for CFU.

• Use BactoBox to set the inoculum and to verify the final concentration before you dose.

This guide assumes that you:

• Know how to Measure a bacterial sample with BactoBox®

• Know how to plate for CFU with your own standard method.

When BactoBox counts and CFU counts agree

You get the best match between BactoBox cells/mL and plate CFU counts when:

• The culture is in exponential or early stationary phase.

• Most cells are have intact membranes and are culturable.

• You do not have severe aggregation or clumping, or you have broken clumps up before measurement.

BactoBox cells/mL and CFU counts show a very tight linear correlation (R² close to 1.0) in this regime.

Overnight cultures of fast‑growing organisms such as E. coli, S. aureus, and P. aeruginosa will typically enter deep stationary or death phase well before 16 hours when you inoculate at densities typically observed in common lab protocols. In that phase, BactoBox counts and CFU can diverge, because stressed and viable-but-non-culturable cells still contribute to the BactoBox signal, but no longer form colonies.

You therefore need to design the overnight culture and timing so that, at the moment you use the culture, it is still in exponential or early stationary phase.

BactoBox vs CFU: how well do they agree?

When you measure cultures in exponential or early stationary phase and you avoid severe clumping, BactoBox cells/mL and plate CFU counts follow a very tight linear relationship. In our validation study on several species, the correlation had R² close to 1.0 across the tested range.

→ Link to peer-reviewed article: https://www.sciencedirect.com/science/article/pii/S0167701225002003

Overview of workflow strategies

There are two practical ways to use BactoBox to assess CFU before dosing:

1. → Same‑day subculture (strategy 1)

   • You already have an overnight culture.

   • You make a same‑day subculture in the morning.

   • You use the refreshed culture around midday while it is in exponential growth.

2. → Low‑inoculum overnight culture (strategy 2)

   • You want a culture that is ready first thing in the morning after about 16 hours.

   • You inoculate the overnight culture at a very low concentration that keeps it in late exponential or early stationary phase at that time.

Both strategies use BactoBox twice:

1. To set the inoculum for the subculture or overnight culture.

2. To verify the cells/mL in the final culture just before you dose and to calculate dosing volume.

You can choose the strategy based on your schedule:

• Culture ready at midday → Strategy 1.

• Culture ready next morning → Strategy 2.

Using BactoBox counts to calculate dosing volume

When you use an appropriate workflow strategy to ensure you have a High-quality culture at the time of dosing, you can calculate how much of that culture to add to your experimental sample in order to reach a specific CFU/mL target concentration. Try our online dosing calculator:

BactoBox® Dosing Calculatorsbtinstruments.com

This calculation uses three quantities:

1. Target cell concentration (CFU/mL) The desired concentration of bacteria in your final sample.

2. Initial volume of medium (mL) The volume of medium present in your well or tube before adding the bacterial culture.

3. Culture concentration (cells/mL) The dilution-adjusted concentration reported by BactoBox® for your culture.

With these inputs, the required volume of culture to add is calculated as:

$$V_{\text{add}} = \frac{C_{\text{target}} \times V_{\text{initial}}}{C_{\text{culture}}}$$

Where:

• $V_{add}$ is the culture volume to add (in mL)

• $C_{culture}$​ is the target cell concentration (CFU/mL)

• $V_{initial}$​ is the initial volume of medium (mL)

• $C_{culture}$​ is the culture concentration from BactoBox (cells/mL)

You can convert the result from mL to µL by multiplying by 1000.

Example

If you want 1×10⁶ CFU/mL in a 1.0 mL sample and your culture is 4×10⁸ cells/mL, then:

$$V_{\text{add}} = \frac{1\times10^{6} \times 1.0}{4\times10^{8}} = 0.0025 \text{ mL} = 2.5 \text{ µL}$$

This means you need to pipette 2.5 µL of your bacterial culture into your 1 mL sample to achieve the target concentration. The final sample volume will be slightly higher (approximately 1.0025 mL), which is acceptable for most workflows.

Pipetting low volumes can be significant source of variability. In cases where $V_{add}$ is a small volume, prepare an intermediate dilution first.

Example: If $V_{add}$ is 2.5 µL, dilute the culture, $C_{culture}$, 1:10 and then add 25 µL instead.

Protocol validation tips

It is difficult to compare BactoBox and CFU during rapid growth, because the culture changes quickly. At optimal growth conditions for E. coli in LB, a delay of about 10 minutes can already cause a large difference between the two methods.

You can reduce this problem by:

1. Sampling once Take one well‑mixed sample from the culture at the time point you care about.

2. Stopping growth immediately Place the sample in an ice bath as soon as you collect it.

3. Splitting the sample for the two methods

   • For BactoBox, prepare dilutions from this chilled sample into BactoBox diluent.

   • For CFU plating, prepare dilutions from the same chilled sample into your standard plating diluent.

This way, any difference between BactoBox cells/mL and CFU reflects the methods, not ongoing growth.

Define an acceptance range that makes sense for your application (for example, within a factor of two). Once the method falls within that range consistently, you can use BactoBox cells/mL as CFU/mL in that workflow.

Media blank controls

When you start to use a new medium formulation, you should check if the medium itself affects BactoBox measurements.

Prepare and measure two samples:

1. Media blank

   • Medium diluted 1:100 in BactoBox diluent.

2. Sterile‑filtered media blank

   • The same medium, sterile‑filtered, also diluted 1:100.

Interpretation:

• If both blanks show very low or no cells/mL, the medium does not generate a significant false cell signal.

• If the media blank shows some cells/mL but the sterile‑filtered blank does not, the signal likely comes from removable particles or microorganisms in the medium.

• If both blanks show similar cells/mL, the default cell gate (BacTotal‑v2024‑10) may classify intrinsic media particles as cells. In that case, contact SBT for guidance.

It is not uncommon to see a small apparent cell count in the non‑filtered medium, because the default gate is broad. It is very uncommon to see a significant cell count in properly sterile‑filtered medium.

Feedback or questions

If you have feedback on this workflow, or if you are unsure how to adapt it to your organism or setup, reach out to SBT Instruments and share your use case. We can help you choose inoculum settings, validation criteria, and troubleshooting steps.

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