# Break up clumps and chains

Does your sample contain **chain-forming** or **aggregating** bacteria? Then you need to disaggregate[^1] them into *single* cells before you measure on them with BactoBox®. This page guides you through the process. ## Which disaggregation method should I use? It depends. There is no one-size-fits-all protocol. However, a simple starting protocol is outlined below: 1. Collect primary sample. 2. Add [glass-beads-3-mm-50-g](https://help.sbtinstruments.com/encyclopedia/item-register/consumables/glass-beads-3-mm-50-g "mention") to your primary until the liquid level rises 1 mL. 3. Vortex your sample vial (1 min, max RPM), or better yet, use a bead beater. We recommend validating the disaggregation with microscopy if clumping or chain-forming is a large concern. We have many [disaggregation methods](#disaggregation-methods) in our toolbox but it's impossible to say which method works best for *your* specific bacteria. Take inspiration from the list of disaggregation methods and apply your own due diligence on top. You are always welcome to reach out to us for guidance. Try a [Google Scholar](https://scholar.google.com/) or [PubMed®](https://pubmed.ncbi.nlm.nih.gov/) search with your genus in question and search strings like “flow cytometry”, “disaggregation”, “single-cell”, “dispersant”, “homogenization”, etc. ## Disaggregation methods See our [disaggregate-mechanically](https://help.sbtinstruments.com/protocols/prepare-your-sample/break-up-clumps-and-chains/disaggregate-mechanically "mention") page for various options. If pure mechanical disaggregation does not work, you can try to add surfactant. {% content-ref url="break-up-clumps-and-chains/disaggregate-mechanically" %} [disaggregate-mechanically](https://help.sbtinstruments.com/protocols/prepare-your-sample/break-up-clumps-and-chains/disaggregate-mechanically) {% endcontent-ref %} {% content-ref url="break-up-clumps-and-chains/disaggregate-with-surfactant" %} [disaggregate-with-surfactant](https://help.sbtinstruments.com/protocols/prepare-your-sample/break-up-clumps-and-chains/disaggregate-with-surfactant) {% endcontent-ref %} {% hint style="warning" %} ## Disaggregation methods may be dangerous Disaggregation methods may result in, e.g., hearing damage (ultrasound) and production of aerosol droplets (rotor/stator and ultrasound probes). Always check the safety instructions of the equipment and procedures that you use! {% endhint %} When you choose a disaggregration method, monitor the effect of cell [lysis](https://en.wikipedia.org/wiki/Lysis). This avoids surprises down the road. {% content-ref url="break-up-clumps-and-chains/avoid-cell-lysis" %} [avoid-cell-lysis](https://help.sbtinstruments.com/protocols/prepare-your-sample/break-up-clumps-and-chains/avoid-cell-lysis) {% endcontent-ref %} ## Extreme example: Actinobacteria

Scanning electron micrograph of *Actinomyces israelii* (false colour).
By Graham Beards at English Wikipedia, CC BY 3.0,
https://commons.wikimedia.org/w/index.php?curid=4197579

*Streptomyces griseus*. Scale bar (lower right corner) is 5 µm.

An extreme case is [actinobacteria](https://en.wikipedia.org/wiki/Actinomycetota) such a [*Streptomyces*](https://en.wikipedia.org/wiki/Streptomyces). Actinobacteria make up huge filamentous yarn balls often more than 100 µm in diameter. Actinobacteria are likely to clog the microfluidic flow paths in the BactoBox® setup. We did extensive testing to disaggregate this bacterium but have not yet found a way to get a single-cell suspension without unacceptable membrane [lysis](https://en.wikipedia.org/wiki/Lysis). Presently, we do *not* recommend BactoBox® for analysis of actinobacterial filaments. On the other hand, BactoBox® works well for the single-cell actinobacterial exospores. ## Motivation Bacteria clumps and chains pose two challenges: * The clumps and chains may be too large to measure ([outside the 0.5–5 µm limits](https://help.sbtinstruments.com/encyclopedia/measurements-explained/what-can-i-measure-on#particle-size)). * The clumps and chains may be miscounted as a *single* bacterium (leading to underestimation). * The clumps and chains may clog the BactoBox® setup To avoid the above, break up the clumps and chains before you measure your sample with BactoBox®. The image below show a side view of the microfluidic flow paths inside BactoBox®. Note how some of the bacteria chains clog some of the flow paths. Over time this can cause a lot of trouble.

Microscope image of the external filter.
Shows *Streptomyces griseus* (in green) on top of the filter mesh (in black).
The filter mesh size is 40 µm.
Note how this filamentous bacteria is too large to pass though the filter mesh.

{% 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 %} [^1]: break apart into individual cells --- # 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/protocols/prepare-your-sample/break-up-clumps-and-chains.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.