We are trying to enumerate Bacillus spores by FCM. Known count of spore suspension is activated at 70 deg for 10 min followed by 45 deg cooling before staining. Counting by microbiological plate counting gives us 100% viability We used the dye TOTO 1 to stain dead cells. but found all the cells positive . we are using Canto II. Any suggestions on how to adjust the settings for bacterial enumeration using FCM?
In the attached paper, TOTO1 and CDFA have been used together. How do we dicriminate between these two dyes using CantoII?
Thanks
The information at
http://www.ncbi.nlm.nih.gov/pubmed/17200957
and
http://www.desatoya.com/PostersAndPresentations/ISAC2002BacteriaPoster.pdf
may be useful for your project.
Diether
Hello
also information here will help you
Good Luck
http://cst.ur.ac.rw/library/Food%20Science%20books/batch1/Handbook%20of%20fluorescent%20probes.pdf
http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2672.2009.04370.x/pdf
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2661013/
Article Immunofluorescence analysis of Bacillus spores and vegetativ...
Thank you for your answers.
The TOTO- 1–DNA complex has a ex of 514 nm and a em of 533 nm while carboxyfluorescein (cF) has an excitation maximum (ex) of 492 nm and an emission maximum (lem) of 517 nm.
Both the dyes are in the FITC range. How do we discriminate between these two dyes.? how to adjust the cytometer settings?
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