Sam:LabNotes/Human/2009-4-8
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Mouse fecal sample collection and bacteria recovering
Objective
- Maintain mouse microflora for following tests
- FISH labeling
- Bacteria sorting
- Practice on culturable microflora before dealing with mouse unculturable microflora in the future
Procedures
- Mouse feces samples were collected directly from bedding and stored in 1.5-mL microcentrifuge tubes. Sample tubs were placed in zipped bag and taped with "biohazard" label for transfering safety.
MF#1: 0.0395 g MF#2: 0.0282 g MF#3: 0.0292 g
- Sample homonigizing: Mouse fecal samples were put in a 50-mL corning tube (mortar)with 1 mL PBS buffer. Put another clean 15-mL conrning tube (grinder)overlapped on the sample. Votex carefully to homonigize the sample in PBS buffer.
- Microflora recovering in prewarmed LB broth. Incubate at 37C overnight:
50 uL MF#1 slurry in 5 mL LB broth -> Sample 1 (37C incubater), Sample 4 (37C water bath) 50 uL MF#2 slurry in 5 mL LB broth -> Sample 2 (37C incubater), Sample 5 (37C water bath) 50 uL MF#3 slurry in 5 mL LB broth -> Sample 3 (37C incubater), Sample 6 (37C water bath)
- Plate counting: Dilute the mouse microfloria sample (only MF#1 and MF#3)based on following concenrtration:
Dilution factor Start 1/10 10^(-2) 10^(-3) 10^(-4) 10^(-5) 10^(-6) ------------------------------------------------------------------------------- Transfer(uL) 100 100 100 100 100 100 PBS(uL) 900 900 900 900 900 900
- Prewarm the LB agar plate (non-antibiotics) at RT for 20 min. Perform the bacteria plating using 50 uL diluted microflora samples
Samples x 2: MF#1, MF#3 Concentration x 3: 10^(-4) 10^(-5) 10^(-6) Replicates x 2: (1), (2)
Next Day
- Count grown CFU on the LB agar plates