Team:Northwestern/Protocol/LIVE/DEAD® BacLight - Bacterial Viability Kit

'''Kit Components'''
*Component A, DMAO: 2x100 µL, 5 mM in DMSO 
*Component B, EthD-III: 2x150 µL, 2 mM in DMSO

'''Preparation of Live and Dead Bacterial Suspensions as Controls'''
#Grow 4 mL cultures of your bacteria to late log phase in nutrient broth. 
#Prepare two tubes of 1 mL of the bacteria culture in Eppendorf tubes and centrifuge at 10,000 × g for 10–15 minutes.
#Remove the supernatant and resuspend the pellet of one tube in 0.3 mL of 0.85% NaCl solution and another tube in 1 mL of 0.85% NaCl.
#Add 0.7 mL isopropyl alcohol into the tube with 0.3 mL of 0.85% NaCl and mix well (final concentration of isopropyl alcohol: 70%) for preparing dead bacteria.  
#Incubate both samples at room temperature for 1 hour, mixing every 15 minutes. 
#Pellet both samples by centrifugation at 10,000 × g for 10–15 minutes. 
#Resuspend the pellets in 1 mL of 0.85% NaCl and centrifuge again as in step 1.6. 
#Determine the optical density at 670 nm (OD670)  for a 3 mL aliquot of the bacterial suspensions in glass or acrylic absorption cuvettes (1 cm pathlength). 
#Use live and dead bacteria at your desired concentration for staining experiments shown below. 

'''Staining Bacteria in Suspension'''
#Combine one volume of Component A and two volumes of Component B in a microcentrifuge tube, mix thoroughly and add 8 volumes of 0.85% NaCl solution to derive 100X dye solution. 
#For each 100 uL of your bacteria sample and live and dead bacteria control suspensions, add 1 uL of the dye mixture. 
#Mix thoroughly and incubate at room temperature in the dark for 15 minutes. 
#Trap 5 µL of the stained bacterial suspension between a slide and an 18 mm square coverslip. 
#Observe  under a fluorescence microscope equipped with any of the filter sets as below. 

'''Selection of Optical Filters''' <br />
<u>Longpass and dual emission filters useful for simultaneous viewing of DMAO and EthD-III stains</u> <br />
Omega Filters: XF25, XF26, XF115 <br />
Chroma Filters:  11001, 41012, 71010 <br />
<u>Bandpass filters for viewing DMAO alone</u> <br />
Omega Filters: XF22, XF23 <br />
Chroma Filters:  31001, 41001 <br />
<u>Bandpass filters for viewing EthD-III alone</u> <br />
Omega Filters: XF32, XF43, XF102, XF108 <br />
Chroma Filters:  31002, 31004, 41002, 41004 <br />

 
'''Flow Cytometry''' <br />
#Adjust the E. coli suspensions (live and killed) to 1 × 108 bacteria/mL (~0.03 OD670), then dilute them 1:100 in filter-sterilized dH2O to reach a final density of 1 × 106 bacteria/mL if needed. 
#Mix 11 different proportions of E. coli in 16 × 125 mm borosilicate glass tubes according to Table 1. The volume of each of the 11 samples will be 1 mL. 
#Mix 12 µL of Component A with 24 µL of Component B in a microcentrifuge tube. Add 3 µL of the combined reagent mixture to each of the 11 samples and mix thoroughly by pipetting up and down several times. <br /> Note:  It may be desirable to prepare additional bacterial samples for staining with component A alone (stain both live and dead bacteria ) and with Component B alone (stain dead bacteria only). 
#Incubate at room temperature in the dark for 15 minutes. 
#Analyze each bacterial sample by flow cytometry using the setting for fluorescein for DMAO positive cells and propidium iodide for EtD-III positive cells. 
 
<u>'''Table 1: Volume of Live- and dead-cell suspension to mix to achieve desired ratio of live:dead cell population'''</u> <br />
{| border = "3"
|'''Ratio of Live:Dead Cells mL Live-Cell'''
|'''Suspension mL Dead-Cell'''
|'''Suspension'''
|-
|align = "center" |0:100 
|align = "center" |0 
|align = "center" |1.0 
|-
|align = "center" |10:90 
|align = "center" |0.1 
|align = "center" |0.9 
|-
|align = "center" |20:80
|align = "center" |0.2
|align = "center" |0.8
|-
|align = "center" |30:70
|align = "center" |0.3
|align = "center" |0.7
|-
|align = "center" |40:60 
|align = "center" |0.4
|align = "center" |0.6
|-
|align = "center" |50:50
|align = "center" |0.5
|align = "center" |0.5
|-
|align = "center" |60:40 
|align = "center" |0.6
|align = "center" |0.4
|-
|align = "center" |70:30 
|align = "center" |0.7 
|align = "center" |0.3
|-
|align = "center" |80:20 
|align = "center" |0.8 
|align = "center" |0.2
|-
|align = "center" |90:10 
|align = "center" |0.9 
|align = "center" |0.1 
|-
|align = "center" |100:0 
|align = "center" |1.0 
|align = "center" |0 
|-
|}


Adapted from: http://www.openwetware.org/images/b/b9/Cell_Death_Assay.pdf

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