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.

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 .. 

yes

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.

Hi: plate count method is the best in this situation. 

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

Dr. Zeigler recommendation is ok

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. 

 I agree with Dr. Zeigler that is recommendation very well method

thanks for your suggestion ^^

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.

Thank you , Pranab and Sandra. 

The best procedures are drop plate method and Hemocytometer