Qualitative Microscopy Analysis Of Bacterial Viability Over Time

July 26, 2016

Idival Research Institute
Microbiology
Principal Investigator

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Company:

ThermoFisher Scientific

Product Name:

LIVE/DEAD BacLight™ Bacterial Viability Kit

Catalog Number:

L7012

The kit consists of two stains, propidium iodide (PI) and SYTO9, which both stain nucleic acids in bacteria. Green fluorescing SYTO9 is able to enter all bacteria and is used for assessing total bacterial cell counts, whereas red fluorescing PI enters only bacteria with damaged cytoplasmic membranes and therefore, is often used to differentiate between active and dead bacterial cells. To learn more about bacterial survival strategies in the environment, we used this kit to study the effect of stress on the long-term survival of different bacterial species.

Experimental Design and Results Summary

Application

Fluorescence Microscopy

Starting Material

Gram-negative bacteria exposed to desiccation on glass surfaces

Protocol Overview

24-well plates with 12 mm glass coverslips. 10 μL of a bacterial suspension were directly added to each well and allowed to dry at RT. The plate was then incubated for 25 days at RT and stained with the LIVE/DEAD® BacLight. Bacterial Viability Kit according to manufacturer's instructions.

Tips

We always use 0.85-0.9% NaCl to obtain the working solution. No fixation needed. Mix very well the components, even with Vortex

Results Summary

It seems accurate to assume that membrane-compromised bacterial cells (red) can be considered dead after 25 days of stress (or different conditions). This correlates well with a decrease in CFU counts performed in parallel. The staining of elongated cells must be interpreted with caution.The staining could be dependent of the bacterial physiological conditions.

Additional Notes

Bacterial strain stained at five days (A) and 25 days (B). Scale bars 5 micrometers. Detection: Green Exc.: 488 nm Em.: 525/50; Red Exc.: 561 Em.: 595/50.

Image Gallery

Summary

The Good

Very suitable to identify dead "red" bacterial cells.

The Bad

Careful interpretation of the results with the "green" population in elongated cells

The Bottom Line

Easy differentiation between bacteria with intact and damaged cytoplasmic membranes. Great potential for rapid screening of bacterial mortality. Easy to use. This format is more flexible than the previous one because it provides separate solutions of the stains.

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