Crude samples

How to measure on coarse samples and with BactoBox®

Plastic container for stool samples. We leave the sample itself for your imagination.

Crude sample taken from fermenter.

Pellicle formed at the top of the sample.

Use BactoBox® to measure on stool samples, crude fermentation samples, skin swabs, heavily aggregated bacteria, pellicle-forming bacteria, etc. You must homogenize and filter these kind of samples first. Otherwise, it is literally shit in, shit out.

See What can I measure on? and Sample preparation for details.

Overall approach

We separate bacteria from other particles in two steps:

1. Homogenize the sample

2. Filter the sample with a 5 or 10 µm nylon cell sieve

It is a fine-grained filter because BactoBox® does not count particles larger than 5 µm anyways (see Particle size).

Use live/dead fluorescence microscopy

Use a fluorescence microscope and the LIVE/DEAD™ BacLight™ staining kit to ensure that:

• Bacteria are in single-cell form (no aggregates)

• Bacteria are separate from other particles

• Bacterial cell membrane is intact

See Live/dead fluorescence microscopy.

Detailed protocol

1

Prepare your sample

Homogenize your sample as much as possible. Here are some options:

• Shake by hand

• Vortex

• Pipette boy

• Agitate with magnetic stirrer

2

Disaggregate bacteria and particles

1. Fill a flip-top tube with 9 mL buffered peptone water.

2. Transfer 1 mL or 1 g of sample to the flip-top tube.

3. Add 5 mm steel beads until the liquid level rises by 1 mL (typically 12 beads or 5 g).

4. Homogenize aggressively by bead beating (Max speed, Bead genie, 1–3 min).

   1. In some cases, vortex with glass beads (3 mm, 3 g) is sufficient.

Use live/dead fluorescence microscopy

Use a fluorescence microscope and the LIVE/DEAD™ BacLight™ staining kit to ensure that:

• Bacteria are in single-cell form (no aggregates)

• Bacteria are separate from other particles

• Bacterial cell membrane is intact

See Live/dead fluorescence microscopy.

If your sample is solid or difficult to pipette, weigh it instead.

3

Remove large particles

1. Assemble a 3 mL syringe with a 10 µm cell sieve.

2. Remove the piston from the syringe and decant 3 mL homogenized sample into the syringe.

3. Filter approximately 1–2 mL sample to an empty vial.

4

Dilute your sample

Prepare two dilution vials: Take two empty vials and add 10 mL of diluent to each.

1st dilution (total 10^-2)

Dilute 1:100: Transfer 101 μL of your sample into the first dilution vial. Vortex (15 sec., Max RPM).

2nd dilution (total 10^-4)

Dilute 1:100: Transfer 101 μL of from the first dilution vial into the second dilution vial. Vortex.

5

Measure your sample

Measure with BactoBox®. Start with the least concentrated sample and move towards higher concentrations.

First, clean your BactoBox® setup with a fresh disinfection vial.

Measure on the 10^-4-diluted sample

If there result is above the limit of detection (above 30 000 cells/mL) then you are done! Otherwise, move on.

Measure on the 10^-2-diluted sample

Is even the most-concentrated sample below the limit of detection? See Concentrate your sample for next steps.

Multiply the cells/mL concentration with the dilution factor to calculate the cells/mL concentration in your sample stock.

Multiply the cells/mL concentration in your sample stock with the density (in g/mL) to calculate cells/g of your sample stock.