Chris:LabNotes/Microbiome/2010-10-17

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FISH with "Alexa 488 EBU 338 9/03/10 probe" on ETOH E. coli and ETOH/RLV MB[edit]

Background[edit]

  • Previously, we tested PU on E. coli and MB on 10/13 but only could get the universal probe to work on E. coli -> wanted to move forward to try to get probe to work on MB
  • This time, we tested a new MB template made on 10/14, and also changed the dilution from 1/2 to 1/5
  • Probe: "Alexa 488 EBU 338 9/03/10"
  • Template:
ETOH E. coli (7/23) -> new E. coli cultured sample
ETOH MB (10/14)
RLV MB (10/14)
  • Rxn's:
1=ETOH E. coli PU
2=ETOH E. coli PN
3=ETOH MB PU
4=ETOH MB PN
5=RLV MB PU
6=RLV MB PN

Procedure[edit]

  • Prepared 2M NaCl, 0.1% SDS, 2M Tris (was stored in the 1.5ml tube that was previously tested for correct pH), 0.5 EDTA, and PBS solutions (used no UV/filter PBS because not sorting)
    • Vortex each reagent before use
  • Thaw the pre-fixed bacteria E. coli/MB samples (stored in -80C)
    • Used ETOH E. coli (7/23) -> no dilution because already decreased template by half during fixation
    • Used ETOH/RLV-fixed MB (10/14/2010) -> 1/5 dilution for MB
  • Pellet the samples by 10,000xg, 5min at RT. Remove the supernatant.
  • Wash palettes with 500ul 1xPBS twice (w/ 10,000xg, 5min centrifuge after each wash)
  • After last washing, re-centrifuge the pellet again to remove the supernatant completely (10,000xg, 1min).
  • Pre-warm the incubator to 46C
Prepare H-buffer in foil wraped, clear low-binding 1.5-ml tube (didn't prepare H-buffer until every washing was done)
Because same probe, prepared H-buffer for each probe rxn's separately
                     1 rxn
---------------------------------------
H2O                  86 = used nuclease-free water
2M NaCl              90  
0.1% SDS             20  
2M Tris               2  
Probe(1000ng/uL)      2    
--------------------------------------
                    200   
repeat pipette mix 5x 
  • Resuspend the pellet (in 1.5mL tubes) with 200uL H-buffer. Make sure to break up pellet thoroughly for complete hybridization.
  • Vortex each sample for 5sec
  • Incubate at mixture at 46C for 1hr 30min (No shaking).
Prepare W-buffer in a 1.5-mL clear low-binding tube          
           1rxn      
-----------------------------
H2O        395      
0.1% SDS    50          
2M NaCl     45          
2M Tris      5          
0.5M EDTA    5          
-----------------------------
           500           


  • Adjust to incubator to 48C and pre-warm the washing buffer. (prewarmed at 48C w/ some little mixing)
  • Pellet the labeled bacteria (10,000xg, 5min at RT). Remove the supernatant carefully.
    • Pallet still substantial (barely any change in size since last PBS washing)
  • Resuspend the pellet with 500 uL washing buffer by repeat pipetting. Incubate the mixture at 48C for 20min without shaking.
    • Had less than expected amount of W-buffer so added 500ul W-buffer into one tube then <500ul into the other tube
  • Pellet the labeled bacteria (10,000xg, 5min at RT). Remove the supernatant.
  • Additional washing with 500uL 1X PBS twice (10,000xg, 8min)
    • After last wash, still substantial pallet so can still see it clearly in tube
  • Resuspend the pellet with 100uL PBS and stored in 4C fridge

Results[edit]

Microscope Images[edit]

    • ETOH MB PU (124/248)

File:10 17 2010 3 Chris.jpg

    • RLV MB PU (124/248)

File:10 17 2010 5 Chris.jpg

Results based on microscope images show that RLV-fixed mouse bacteria showed slightly better fluorescence than ETOH-fixed

Flow Cytometry Results[edit]

  • ETOH E. coli PN (used to calibrate flow cytometer PMT for ETOH E. coli)

File:002.jpg

  • ETOH E. coli PU

File:004.jpg

  • ETOH MB PN (used to calibrate flow cytometer PMT for ETOH MB)

File:003.jpg

  • ETOH MB PU

File:006.jpg

  • RLV MB PN (used to calibrate flow cytometer PMT for RLV MB)

File:10 17 2010 001.jpg

  • RLV MB PU

File:005.jpg

Overall Conclusions[edit]

  • E. coli PU doesn't seem like it's working, but still move on because successful cultured mouse gut bacteria hybridization
  • Based on both microscope and flow cytometry results, RLV-fixed MB has higher signal than ETOH-fixed MB