Protocol name: Live/DEAD detection
Prepared by: Monica Ponder
Date Created/Revised: 12/19/03
Purpose: Quantify the number of live
cells in high salinity media
Reference(s):
Molecular probes protocol version April 2003
Comments:
L-7012 LIVE/DEAD® BacLight. Bacterial Viability Kit *for microscopy and quantitative
assays*
Growth
1.1 Grow 70 mL cultures of Psychrobacter 273-4 to mid log phase (OD 600=0.2-0.3) in ½ TSB+5% NaCl (15gTSB,
Difco, 50gNaCl, Fisher,1000g MilliQ)
1.2 Remove 10mls for preparation of live cells control. Remove 1ml for plate counts using serial dilution. Centrifuge the
remaining cells and resuspend in 60mls of ½ TSB+2.79mNaCl. Remove 10mls in 2 hours after inoculation and 10mls the
next day. Prepare all samples as below.
Preparation for Fluorescence microscopy
1.1Concentrate 10 mL of the bacterial culture by centrifugation
at 10,000 × g for 10–15 minutes.
1.2 Filter 100mls of dd water through a 0.2 µm pore–size filter to sterilize and to remove particulate matter (Nucleopore,
125ml size) and place in clean orange cap tubes until ready to use.
1.3 Remove the supernatant and resuspend the pellet in 2 mL of
deionized water (dH2O) that has been filtered.
1.4 Add 1 mL of this suspension to each of two 30–40 mL
centrifuge tubes containing either 20 mL of filter-sterilized dH2O
(for live bacteria) or 20 mL of 70% isopropyl alcohol (for killed
bacteria).
1.5 Incubate both samples at room temperature for 1 hour,
mixing every 15 minutes.
1.6 Pellet both samples by centrifugation at 10,000 × g for
10–15 minutes.
1.7 Resuspend the pellets in 20 mL filter-sterilized dH2O and
centrifuge again as in step 1.6.
1.8 Resuspend both pellets in separate tubes with 10 mL of
filter-sterilized dH2O each.
1.9 Determine the optical density at 670 nm (OD670) of a 3 mL
aliquot of the bacterial suspensions in glass or acrylic absorption
cuvettes (1 cm pathlength).
Fluorescence Microscopy Protocols
Selection of Optical Filters
The fluorescence from both live and dead bacteria may be
viewed simultaneously with any standard fluorescein longpass
filter set Filter I3 for our microscope. Alternatively, the live (green fluorescent) (L3)and dead
(red fluorescent) cells may be viewed separately with fluorescein
and Texas Red bandpass filter sets.
Staining Bacteria in Suspension with either Kit L-7007 or
L-7012
Center for Microbial Ecology
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2.1 Combine 2.5 volumes of Component A to one volume of Component B
in a microfuge tube, mix thoroughly.
2.2 Add 3 µL of the dye mixture for each mL of the bacterial
suspension. When used at the recommended dilutions, the
reagent mixture will contribute 0.3% DMSO to the staining
solution. Higher DMSO concentrations may adversely affect
staining.
2.3 Mix thoroughly and incubate at room temperature in the dark
for 15 minutes.
2.4 Trap 5 µL of the stained bacterial suspension between a ethanol cleaned slide
and an 18 mm square coverslip.
2.5 Observe in a fluorescence microscope equipped with filter I3.
2.6 Prepare 3 slides for each sample and record 10 fields. Must count total of at least 400 bacterial cells. Count the
number of green cells and number of red cells for each field.
Center for Microbial Ecology
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