The use of flow cytometry to study the germination of Bacillus cereus endospores.
BACKGROUND
At present the study of endospore germination is conducted using microbiological methods which are slow and yield data based on the means of large heterogeneous populations. Flow cytometry (FCM) offers the potential to rapidly quantify and identify germination and outgrowth events for large numbers of individual endospores.


METHODS
Standard methods were employed to arrest the germination of Bacillus cereus endospores at defined stages. Endospores were then stained with SYTO 9 alone or carboxyfluorescein diacetate (CFDA) together with Hoechst 33342 and analysed using FCM. Comparisons were made between FCM as a method to measure germination rate and standard microbiological techniques.


RESULTS
Germinating endospores displayed increases in permeability to SYTO 9 and hydrolysis of CFDA compared with controls. Statistically significant correlations were found between the standard plate count method and both FCM methods for measuring the percentage of germinating and outgrowing endospores up to 75 min after addition of germinant.


CONCLUSIONS
Using FCM, the percentage of germinating or outgrowing endospores at various time points during germination and/or outgrowth can be quantified. FCM with CFDA/Hoechst 33342 staining may be used to estimate overall germination rate, whereas FCM with SYTO 9 staining may be used to quantify ungerminated, germinating and outgrowing endospores.
Cronin
Ultan P
UP
Department of Life Sciences, University of Limerick, Castletroy, Co. Limerick, Ireland.
Wilkinson
Martin G
MG
D001409:Bacillus cereus / Q000254:growth & development* / Q000378:metabolism; D001562:Benzimidazoles / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods*; D005452:Fluoresceins / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009930:Organic Chemicals / Q000378:metabolism; D013171:Spores, Bacterial / Q000254:growth & development
D001562:Benzimidazoles; D005452:Fluoresceins; D005456:Fluorescent Dyes; D009930:Organic Chemicals; C103389:SYTO 9; C084577:5-carboxyfluorescein diacetate; C017807:bisbenzimide ethoxide trihydrochloride