Assessing Swarming of Aerobic Bacteria from Human Fecal Matter

Materials and Reagents

1. Petri dish, stackable lid, 100 mm × 15 mm, sterile, polystyrene (Fisherbrand, Fisher Scientific, catalog number: FB0875713)

2. Fecal specimen collection cup, 650 ml (Fisherbrand, Commode Specimen Collection System, catalog number: 23-038032)

3. Microcentrifuge tube, 1.5 ml (Fisherbrand, Fisher Scientific, catalog number: 05-408-130)

4. Adjustable 0.5-10 μl pipette (Eppendorf Research, catalog number: EPPR4402)

5. 10 μl pipette Tips, racked, sterile (Thomas Scientific, catalog number: 1148U48)

6. Agar, bacteriological grade (Research Products International, CAS number: 9002-18-0)

7. Sodium chloride (Fischer Chemicals, Fisher Scientific, CAS number: 7647-14-5)

8. Tryptone, microbiologically tested (Sigma-Aldrich, CAS number: 91079-40-2)

9. Yeast extract (Acumedia, Neogen Corporation, catalog number: 7184B)

10. Distilled water (Thermo Fisher Scientific, catalog number: 15230001)

11. Agar LB plates (see Recipes)

Note: All materials and reagents are stored at room temperature.

Equipment

1. Epson Expression 1600 Scanner (Epson, Expression 1600, catalog number: E1600-PRO)

2. Incubator (Thermo Scientific, NAPCO Series 8000DH Incubator, catalog number: 3541)

3. -80°C freezer (Thermo Fisher Scientific, Revco ExF, catalog number: EXF40086D)

4. Autoclave (Amsco Scientific, Eagle, catalog number: SV120)

5. Hotplate (Thermolyne, Cimarec, catalog number: HP131225)

6. Biological hood (Thermo Fisher Scientific, 1300 Series Class II, Biological Safety Cabinet, catalog number: 1353)

Software

1. ImageJ Version 1.59e (National Institutes of Health, https://imagej.nih.gov/ij/download.html)

Procedure

1. Prepare the agar LB plates with a final agar concentration of 0.4% (see Recipes).

   
   

2. Remove fecal samples from the freezer

   1. Fresh human fecal samples were obtained in 2015 (under protocols IRB# 2009-446 and 2015-4465) from healthy donors without intestinal distress and donors known to suffer from intestinal inflammation. Specimens were collected in sterile fecal specimen cups without preservatives. Specimens were then kept at 4°C for up to 15 min before transport to the laboratory at ambient temperature for processing, which consisted of aliquoting the specimen into a 100-μl microcentrifuge and storage at -80°C since 2015.

   2. Remove the fecal samples from the freezer and allow to thaw at room temperature on the bench.

   3. Under a biological hood, use a pipette to inoculate 7.5 μl healthy sample on one side of a plate (as a control) and 7.5 μl disease sample on the other half of the plate.

      Note: If the samples are not pre-homogenized, homogenization is preferable using a homogenizer for uniformity.

   4. Take care to avoid puncturing the agar with the pipette tip, as this may impede the ability to identify future swarming.

   5. Leave the cover off the Petri dish and allow to dry under a biological hood for 10 min.

      
      

3. Incubation

   1. (Optional) Once dry, take an image of the plate on the scanner, which will serve as a “before image” for later reference. See Figure 1A below.

   2. Place the plate upright (not inverted) in an incubator, set to 37°C and a humidity of 80%.

   3. Remove the plate from the incubator after 24 h and assess visually for the presence of swarming.

   4. (Optional) Scan an image of the plate and label as “after image” for record-keeping. See Figure 1B below.

   5. (Optional) Input the before and after images into the NIH’s ImageJ program to quantitatively measure the folds of expansion. Use the “Free Select” tool to highlight an area in the ‘before image’, then highlight the area of swarming in the ‘after image’. This method allows measurement of the pixels of growth and enables quantitation even when swarming occurs in irregular directions.

      
      
      Figure 1. Bacterial swarming on LB agar plates before and after incubation. A. 7.5 μl fecal samples were inoculated onto 20 ml 0.4% LB agar plates, which were then scanned. B. Plates were placed in an incubator, set to 37°C and a humidity of 80%, and were removed and scanned after 24 h.

Data analysis

An agar concentration of 0.4% was found to elicit the most swarming (compared with agar concentrations of 0.5%, 0.6%, and 0.7%), as was a sample volume of 7.5 μl (compared with volumes of 2.5 μl or 5.0 μl). At the same time, the thawing method for the samples (on ice or at room temperature) was found to have no significant impact on the presence of swarming. These conclusions were drawn from the statistical analysis of 10 independent experiments (performed in duplicate) for 6 separate patient samples. One-way ANOVA was performed in GraphPad Prism (v8) to determine significant differences between agar and inoculant volume conditions.

Recipes

1. Agar LB plates

   1 g Tryptone

   0.5 g Yeast extract

   0.5 g NaCl

   0.4 g Agar

   100 ml dH₂O

   1. Combine the water and dry ingredients while stirring on a hotplate, until the water begins to boil, then autoclave to sterilize (121°C, 15 PSI, 65 min)

   2. After autoclaving, allow the 0.4% LB agar solution to cool until warm to the touch, stirring continuously to prevent clumping or hardening

   3. Pour 20 ml 0.4% LB agar solution into sterile Petri dishes

   4. Allow LB agar plates to cool and harden on the bench at RT for at least one h

Acknowledgments

Research was conducted under a Student Research Fellowship Award from the Crohn’s and Colitis Foundation of America (CCFA) #640806 awarded to A.B, as well as #431602 awarded to S.M. The data acquisition was also partially supported by the Broad Medical Research Program (BMRP) grant #362520 at CCFA to S.M. The authors would like to thank Arpan De and Hao Li for their advice, suggestions for variables to explore, and help with formatting and editing. This protocol was modified from work completed by our group, published in March of 2021 in Gastroenterology (De et al., 2021) https://doi.org/10.1053/j.gastro.2021.03.017.

Competing interests

The authors have no competing interests or financial disclosures to report.

Ethics

All described protocols have been approved by the Albert Einstein College of Medicine Institutional Review Board (IRB# 2009-446 and 2015-4465). Informed consent was given by the human subjects from whom fecal samples were acquired.

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