# 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](/protocols/do-it-yourself-diy/assemble-dilution-dispenser.md) or have a [Dilution vial](/encyclopedia/item-register/vials-flasks-and-liquids/dilution-vial.md) 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](#disaggregation "mention") (optional)\ You must [Break up clumps and chains](/protocols/prepare-your-sample/break-up-clumps-and-chains.md) if your cells are prone to forming clumps or chains. 2. [#dilution](#dilution "mention")\ Most bacterial samples that are studied with BactoBox® are in the range of 1e6-1e11 cells/mL. This is outside the [Limit of detection](/encyclopedia/measurements-explained/limits-of-detection.md) 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](/protocols/prepare-your-sample/break-up-clumps-and-chains.md). Contact SBT if you are unsure. {% hint style="info" %} ## 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. {% endhint %} ## 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 limit of detection (LoD). BactoBox® outputs concentrations in these ranges: * 30 000–5 000 000 **cells/mL** (3·10⁴ to 5·10⁵ cells/mL) * 30 000–5 000 000 **total/mL** (3·10⁴ to 5·10⁵ total/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](/encyclopedia/item-register/consumables/flow-cell.md). | 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](/encyclopedia/software/error-codes.md). For more details on preparing dilutions, please see the [Hit the right concentration](/protocols/prepare-your-sample/hit-the-right-concentration.md) page. {% hint style="info" %} **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](/protocols/prepare-your-sample/hit-the-right-concentration/dilute-1-100.md) from primary sample. Sample #2 - target dilution 1:1,000: [Dilute 1:10](/protocols/prepare-your-sample/hit-the-right-concentration/dilute-1-10.md) from sample #1. Also, see [Dilute 1:1 000](/protocols/prepare-your-sample/hit-the-right-concentration/dilute-1-1-000.md) Sample #3 - target dilution 1:10,000: [Dilute 1:100](/protocols/prepare-your-sample/hit-the-right-concentration/dilute-1-100.md) from sample #1. Also, see [Dilute 1:10 000](/protocols/prepare-your-sample/hit-the-right-concentration/dilute-1-10-000.md) {% endhint %} {% hint style="success" %} ## 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. {% endhint %} ## Conductivity The [electrical conductivity](https://en.wikipedia.org/wiki/Electrical_resistivity_and_conductivity) of your [sample vial](https://help.sbtinstruments.com/encyclopedia/item-register/vials-flasks-and-liquids/sample-vial) must be within [space.vars.BB\_LIMIT\_EC](/encyclopedia/measurements-explained/what-can-i-measure-on.md#electrical-conductivity). You control the conductivity of your sample with [diluent](https://help.sbtinstruments.com/encyclopedia/item-register/vials-flasks-and-liquids/dilution-liquid-5-l). 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](/protocols/prepare-your-sample/hit-the-right-conductivity.md), some media will only work once you perform a 1:1,000 dilution. --- # Agent Instructions: Querying This Documentation If you need additional information that is not directly available in this page, you can query the documentation dynamically by asking a question. Perform an HTTP GET request on the current page URL with the `ask` query parameter: ``` GET https://help.sbtinstruments.com/measure-on-cells/measure-a-bacterial-sample.md?ask= ``` The question should be specific, self-contained, and written in natural language. The response will contain a direct answer to the question and relevant excerpts and sources from the documentation. Use this mechanism when the answer is not explicitly present in the current page, you need clarification or additional context, or you want to retrieve related documentation sections.