LSC Application: Bacterial Detection

Laser Scanning Cytometry can be used to detect live and dead bacteria, estimate cell numbers, and calculate live/dead ratios. The methods are easier and more accurate than traditional, manual counts.

E. coli bacteria Differential staining of live and dead cells can be done using a combination of dyes. The nucleic acid dye, propidium iodide (PI), is unable to permeate the intact cell membrane of a living cell, but does label dead cells with red fluorescence. A vital dye such as SYTO^® 16 (Molecular Probes) will label the nucleic acids of living cells with green fluorescence. Shown here are e. coli bacteria captured on a membrane and stained with PI and SYTO 16.

Hematocytometer with e. coli. When analyzed on the LSC, the numbers of green and red fluorescing cells in a population can be compared to give the percentages of live and dead cells. If the cell suspension is a known volume of liquid, the concentration of cells can be estimated in the same manner as counting cells on a hemacytometer. A laser scan image of a portion of a hemacytometer loaded with a suspension of e. coli is shown on the left.

While a hemacytometer is awkward to use on the LSC, other constant volume slide types are available, such as the multi-well slide whose dot plot is shown below.

Counting bacteria in multi-well slides
Plot of a multi-well slide with increasing concentrations of bacteria added to each well, and the total cell counts per region determined by laser scanning.

The live/dead staining technique can be applied to other cell types, including mammalian cells (both adherent and in suspension) and yeast.

One practical application of the technique is detecting contaminating bacteria in apheresis platelet packs. Using DNA labeling to report the number of bacteria can lead to errors because platelet packs may contain leukocytes, which also have DNA, and these leukocytes can be live or dead (fig. A). When bacteria are seeded in a platelet pack, they stain similarly to the leukocytes (fig. B). The way around this problem is to add a low concentration of detergent to the staining mixture. The detergent will cause the leukocytes to lose their membrane integrity and appear as dead cells (fig. C). Bacteria, having a detergent-resistant cell wall, will be unaffected and continue to appear as live cells (fig. D).

Method
For 488 nm argon laser excitation, a mixture of propidium iodide at a concentration of 1 µg/ml and 5 µM SYTO 16 was used to label dead and live cells. Detergent lysing of leukocytes in platelet suspensions was done with 0.5% Triton X detergent.
(Developed by Ed Luther, CompuCyte Corp)