Dear all researchers,
Can I count the bacterial cells using hemocytometer under light microscope? Which staining dye can I use? Can I use crystal violet or trypan blue? What percentage and under what magnification of microscope? Tq.
You can use either Trypan Blue or Methylene Blue or Crystal Violet to stain the viable cells. Please refer the following link for stepwise description and procedure.....Best of luck
http://www.nexcelom.com/Applications/counting-cells-with-a-hemacytometer.php
Hi, thank you for your reply. Is this protocol apply for animal cells? Because crystal violet + citric acid is for nuclei extraction. Tq.
For animal cells (i.e. cancer cells like Hela) doesn't need any staining. You can directly take small (10uL) of cell suspension on hemocytometer and count under the microscope. The cell counting method is similar. Cheers.....
I just used a haemocyotomer to count Enterococcus cells today on a phase contrast upright microscope. I used to do so regularly for S. cerevisiae. No stain is required for either; dead yeast cells show up phase-dark actually (very convenient), and I'm trying to see if the same is true for my bacterium of interest.
But does the count of bacteria stained with methylene blue (MB) and counted with haemacytometer equivalent to the total viable cells OR it counts all cell (live and dead cell). As for yeast, we can stain with MB and count the cells that do not absorb the dye as the total viable cells.
Dear All,
By using Hemocytometer, we can calculate number of viable cells (with trypan blue staining), But can I take each individual cells as Colony forming units. I guess CFU is just to state the viable cells.
I'm not so much familiar with microbio, some one pls clarify..
Hello, do you count the bacterial cells using hemocytometer under light microscope successful? I also want to count the bacterial cells under light microscope, but I can not see my bacterial, do you have some suggestions about how to save these problems?
Sherlyn Tang
Hi all,
This website provides a detailed tutorial on how to count small cells with a hemacytometer. I count bacterial cells using the central 5x5 square and make sure to count at least 200 cells total.
I perform my calculations manually instead of using the app on that website.
https://www.hemocytometer.org/hemocytometer-calculation-tutorial/
The volume of each of the squares in that central 5x5 grid is 0.000004 mL.
https://www.hemocytometer.org/hemocytometer-square-size/
There does not seem to be a visible dye that can differentiate between live and dead bacteria, however, so you cannot determine the proportion of live cells with a direct count without using a fluorescent stain such as this product:
https://www.thermofisher.com/order/catalog/product/L7012
A viable cell count is therefore necessary at the time of an experiment that involves quantitation to determine how many CFU you used for your experiment.
https://mospace.umsystem.edu/xmlui/bitstream/handle/10355/69341/5-VPC%20Viable%20Plate%20Count.pdf?sequence=22&isAllowed=y
If someone knows of a differential, non-fluorescent viability stain that works with bacteria, please let us know. Trypan blue and methylene blue do not appear to work with bacterial cells, but if anyone has had success, please share!
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