Electron Microscope + Hemocytometer - (Dec/06/2013 )

First of all, you are right, I am using the cover slip which came with haemocytometer. 

My case is to count for gram +ve bacteria, which means cannot use the trypan blue as staining dye since it is not human cells. Therefore, I have create a protocol by using crystal violet, iodine and safranin to stain out the cells first before I load to the haemocytometer. 

First of all, I was dilute the sample into 10^-4 dilution factor, 1mL from the tube was transferred to 10^-5 tube (contains 8mL of water and 3 drops of crystal violet), incubate for 1 min, 3 drops of Iodine was added into the same tube and incubate for another 1 min. 1mL of sample from 10^-5 tube was transferred to 10^-6 tube (contains 7mL of water and 2mL of ethanol), incubate for 30 sec; transfer 1 mL of sample from tube 10^-6 to 10-7 (contains 8.5mL of water and 3 drops of safranin), incubate for 1 min. Small amount of sample was loaded into the haemocytometer and view under EM. 

Thank you. 

This is so far from the methodologies modified that you can't assume validity.    It's not obvious that is can be done with any accuracy but in any case you need to validate.

I also don't see how you can viability from that stain (that's what trypan blue is for), and it won't show up under the EM.  The stains you have indicated are all for light microscopy.  I really really don't see how you can be in a micro lab and not even have a single light microscope.

No wonder why you didn't see the lines... Do you know how does the EM works?

In any case, if you place the haemocytometer with the liquid sample in the EM you are going to break the EM sooner or later...

Why do you stain the cells? If you stained them with some metallic salt I would understand but safranin? crystal violet? You don't see colors on the EM and unless those compounds create precipitates (unlikely given the protocol) you won't see anything... and so many dilutions

If you want to count teh cells with the EM anyway, use Isopore filters, vacuum filter a know amount of culture suspended in a total of 20 mL PBS, saline or similar. Carefully remove filter. Freeze dry or just dry it in the oven about 60-70° C (unless you want to keep structural info of the cells). once dry, Sputter coating the filter. If you can see an even distribution of cells on the filter then is ok. You should be able to measure some areas with the EM software and counting the cells. You can backcalculate the real concentration after that...

In any case, are you working with pure cultures? Why don't you just make a growth curve base on absorbance/CFU? If you have an spectrophotometer, though. That would be way easier...