# Crude samples

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](https://en.wikipedia.org/wiki/Stool_test), 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?](/encyclopedia/measurements-explained/what-can-i-measure-on.md) and [Prepare your sample](/protocols/prepare-your-sample.md) 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](/encyclopedia/item-register/consumables/cell-sieve-10-m.md) It is a fine-grained filter because BactoBox® does not count particles larger than 5 µm anyways (see [What can I measure on?](/encyclopedia/measurements-explained/what-can-i-measure-on.md#particle-size)). {% hint style="success" %} ## Use live/dead fluorescence microscopy Use a [fluorescence microscope](https://en.wikipedia.org/wiki/Fluorescence_microscope) and the [LIVE/DEAD™ *Bac*Light™](https://www.thermofisher.com/order/catalog/product/L7012?SID=srch-srp-L7012) 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 [Avoid cell lysis](/protocols/prepare-your-sample/break-up-clumps-and-chains/avoid-cell-lysis.md#live-dead-fluorescence-microscopy). {% endhint %} ## Detailed protocol
{% stepper %} {% step %} #### Prepare your sample Homogenize your sample as much as possible. Here are some options: * Shake by hand * Vortex * Pipette boy * Agitate with magnetic stirrer {% endstep %} {% step %} #### 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. {% hint style="success" %} ## Use live/dead fluorescence microscopy Use a [fluorescence microscope](https://en.wikipedia.org/wiki/Fluorescence_microscope) and the [LIVE/DEAD™ *Bac*Light™](https://www.thermofisher.com/order/catalog/product/L7012?SID=srch-srp-L7012) 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 [Avoid cell lysis](/protocols/prepare-your-sample/break-up-clumps-and-chains/avoid-cell-lysis.md#live-dead-fluorescence-microscopy). {% endhint %} {% hint style="info" %} If your sample is solid or difficult to pipette, weigh it instead. {% endhint %} {% endstep %} {% step %} #### Remove large particles 1. Assemble a 3 mL syringe with a [10 µm cell sieve](/encyclopedia/item-register/consumables/cell-sieve-10-m.md). 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](/encyclopedia/item-register/vials-flasks-and-liquids.md#empty-vial). {% endstep %} {% step %} #### Dilute your sample Prepare two [dilution vials](/encyclopedia/item-register/vials-flasks-and-liquids/dilution-vial.md): Take two [empty vials](/encyclopedia/item-register/vials-flasks-and-liquids/empty-vial.md) and add 10 mL of [diluent](/encyclopedia/item-register/vials-flasks-and-liquids/dilution-liquid-5-l.md) to each. **1st dilution (total 10****-2****)** [Dilute 1:100](/protocols/prepare-your-sample/hit-the-right-concentration/dilute-1-100.md): Transfer 101 μL of your sample into the *first* dilution vial. Vortex (15 sec., Max RPM). **2nd dilution (total 10****-4****)** [Dilute 1:100](/protocols/prepare-your-sample/hit-the-right-concentration/dilute-1-100.md): Transfer 101 μL of from the *first* dilution vial into the *second* dilution vial. Vortex. {% endstep %} {% step %} #### 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](/protocols/prepare-your-sample/hit-the-right-concentration/concentrate-your-sample.md) for next steps. {% endstep %} {% endstepper %} 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. --- # 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/meta/applications/coarse-samples.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.