Break up clumps and chains

Illustration of chain-forming bacteria

Does your sample contain chain-forming or aggregating bacteria? Then you need to disaggregate 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) 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 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 or PubMed® search with your genus in question and search strings like “flow cytometry”, “disaggregation”, “single-cell”, “dispersant”, “homogenization”, etc.

Disaggregation methods

See our Disaggregate mechanically page for various options. If pure mechanical disaggregation does not work, you can try to add surfactant.

Disaggregate mechanicallyDisaggregate with surfactant

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!

When you choose a disaggregration method, monitor the effect of cell lysis. This avoids surprises down the road.

Avoid cell lysis

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 such a 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.

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).

• 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.

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.