Sam:LabNotes/Human/2009-4-8: Difference between revisions

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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

Latest revision as of 08:36, 11 April 2009