hi, all. I'm facing a problem in counting the spore of bacillus subtilis. I had used heamocytometer to count the spore, but the size of spore are just too small , i had used 1000x dilution factor (DF), but the spore count approximately obtained was range from 300 to 900 spore count in each quadrant as i count singly of 16 box of each quadrant, and it ended up with 1000 -3000 spore count in a total 4 quadrant. Higher DF are suggested to dilute my samples, however with higher DF , it took more time for me to differentiate the spores under the microscope. I doubt with my method used, as 3 replicate i counted all show significance difference of value. Any method suggest for bacillus subtilis spore counting ?
Is easier to count if you make the dilutions and germinate spores in agar plates
Cell colonies would be obtained in the plate count right? I can't accurately count the spore number since it's very small in size ..
I agree with Dr. Olmos-Soto's suggestion. I would add that prior to spreading aliquots on the agar, you can heat shock the suspensions at 80°C for 10 min to kill off any vegetative cells that remain.
Thank you for your answer. I really appreciated for the answer. I will try this method later.
The best procedure for counting spores of Bacillus subtilis is the count on selective agar media. A good medium is Bacillus Differentiation Agar (differentiate between B. cereus and B. subtilis). Other techniques for identification can be found on www.sfam.org.uk/download.cfm?docid=A8649AF0-52D3
Totally agree with Daniel R Zeigler's opinion. Very important. To avoid false results
In order to make easier the method, you can use a contrast phase microscope. The spores looks like brilliants ovals, and vegetative cells look dark and gray.
Hello
I agree Witherspoon all of thé answers
Do not forget to perform dilution for exact country of bacterio.
Dear Ching,
You can take the help of the attached paper with my mail. The worker are also working with Bacillus group, their isolation and identification parameters.
Spores’ suspensions should be subjected to a heat shock (80°C, 10 min) and stored at 4°C, if it is necessary. Viable spore counts are done by serial decimal dilutions in distilled sterile water and 50 μL of each dilution are inoculated on a tryptone soy agar plate surface in duplicate. Plates are incubated overnight at 36°C. If you strain form a biofilm you can use a pour-plate method. I advise to use sterile glass beds at least in the first dilution agitation to avoid spores attachment and to vortex at least 1 minute after each dilution inoculation on agar plate.
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