Sam:LabNotes/Human/2009-4-8

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Revision as of 17:51, 10 April 2009 by >Sam Chiang
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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