Be sure your haematocytometer has been commissioned and qualified (IOQ or Installation Operating Qualification).  Follow ICH Q2.  I am rather dubious this method can be validated since the results depends on RI, density, spore size...

The haematocytometer is non specific as ALL spores are detected (like TOC and organic molecules).  This drives all other parameters.

For linearity use an ATCC culture (like a pure strain of Bacillus subtillus).  LOQ and LOD are derived statistically from the linearity curve.

 Thank you Bruce. I have also performed molecular analysis by PCR and sequencing to confirm the belonging of the active substance of the product to the fungal species.

I don't know if a pure culture of the fungal agent is available.

I have time to change my mind!  Current USP section only requires verification or a demonstration that this instrument is appropriate for your samples in your hands.

Validation per USP (sections ?) indicates that you have a fundamental misunderstanding of microbiology.  You can validate the haemocytometer for an ATCC strain but what about the sporeformers in your samples?  If you are going to do this, there must a procedure in your method so that no vegetative cells are counted like pretreating conditions that will form spores (dehydration, cold, vacuum..).  

In addition your acceptance criteria must be wide (CRITICAL!!). For chemicals the linearity correlation coefficient is 0.990 but you will be lucky if you get 0.70 - 1.30.  There are other parameters that may affect your results like cell shape, size...

 Good luck!  You will need it.

There are sections in the USP that you can use.  They are and .

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Hi Barbara Amato

I am agree with Dr.Bruce Neagle, also, You can take 1 ml of the spore suspension and do culture then account the colonies