Measure a bacterial sample

This page explains how to measure a single bacterial sample with BactoBox® and provides and introduction on how to prepare your sample correctly. By the end, you will:

• Understand the importance of disaggregation.

• Understand the importance of dilution.

• Understand the importance of conductivity.

This guide assumes that you:

• Know how to make a DIY dilution vial or have a Dilution vial at hand.

• Know how to perform serial dilutions using pipetting.

Step-by-step

Preparing a sample to be measured with BactoBox® generally includes two steps:

1. Disaggregation (optional) You must Break up clumps and chains if your cells are prone to forming clumps or chains.

2. Dilution Most bacterial samples that are studied with BactoBox® are in the range of 1e6-1e11 cells/mL. This is outside the Limits of detection of BactoBox®, and therefore requires dilution.

Disaggregation

Disaggregation is only required if your cells form clumps or chains during the growth phase you are studying. Many strains do not aggregate during active growth, so evaluate whether it is necessary to Break up clumps and chains. Contact SBT if you are unsure.

Plate counts on bacterial clumps and chains

Bacterial aggregates are generally a pain to measure. If your bacteria form clumps or chains, neither BactoBox® nor plate counts give meaningful results.

Plate counts are measured in colony-forming units (CFUs). However, a colony does not distinguish between single cells and aggregates. What does your CFU number mean then? Answer: "It depends". Not happy with that answer? Neither are we.

Dilution

BactoBox® counts individual particles. If there are too few particles, the concentration estimate is too uncertain. If there are too many particles, we can't tell them apart. Therefore, we have limits of detection (LoD). BactoBox® outputs concentrations in these ranges:

• 30 000–5 000 000 cells/mL (3e4 to 5e6 cells/mL)

• 30 000–5 000 000 total/mL (3e4 to 5e6 cells/mL)

If you are not sure how much dilution is needed, we recommend preparing and measuring the dilution series in the table below as a starting point. Always measure the most diluted sample first to reduce the risk of clogging the Flow cell.

Sample	BB meas. ID	cells/mL	total/mL	Dilution factor
#1				1:100
#2				1:1,000
#3				1:10,000

Once you get a good feeling for how much dilution is required, you can start preparing samples with the dilution factors you know work. If the sample is outside the detection range, BactoBox® will display appropriate Error codes.

For more details on preparing dilutions, please see the Hit the right concentration page.

Preparing a dilution series with 1:100, 1:1,000 and 1:10,000 dilutions

The most convenient way of preparing three samples diluted by 1:100, 1:1,000 and 1:10,000 is listed below.

Sample #1 - target dilution 1:100: Dilute 1:100 from primary sample.

Sample #2 - target dilution 1:1,000: Dilute 1:10 from sample #1. Also, see Dilute 1:1 000

Sample #3 - target dilution 1:10,000: Dilute 1:100 from sample #1. Also, see Dilute 1:10 000

Vortex each dilution step thoroughly!

Always vortex thoroughly for at least 10 seconds. Ensure that a clear cyclone/vortex appears.

You must perform proper vortexing to ensure that the transferred bacteria are well-mixed in the prepared dilution. Without proper vortexing, you may get underestimation of concentration and variation between replicates.

Conductivity

The electrical conductivity of your prepared sample vial must be within 1500–2200 µS/cm. You control the conductivity of your sample with diluent. Keep the diluent at room temperature!

In most cases, conductivity will fall within the acceptable range after sufficient dilution since a heavy dilution also dilutes whichever salts you have in your media. In most cases, you can dilute 1:100 and measure a sample. However, as explained in the guide Hit the right conductivity, some media will only work once you perform a 1:1,000 dilution.