In this tutorial we go through all the calculations explained in hemocytometer calculation but with a small example for both large squares (1mm wide) and small squares (0.2mm wide). We calculate the viability, the cell density, the total number of live cells and the volume to add to reach a target density.
Maria Fuentes, PhD
I did my PhD in the Department of Chemical Engineering at Imperial College London. My research focused on mathematical modeling of the cell cycle in leukemia and involved experiments with cell lines. During that time, I had to count cells with a hemocytometer so often to track growth that I got tired and decided to build an app, HemocyTap, and share my knowledge on the topic here to help as many people as possible.
in regards to the small cells…you said in the tutorial that you counted 5 small squares. Wouldn’t you multiply by the numver of cells you counted. the 0.000004 is for one of the small squares correct?
sorry, disregard previous comment. Revised…in regards to the small squares…you said in the tutorial that you counted 5 small squares. Wouldn’t you multiply by the number of small squares you counted? the 0.000004 is for one of the small squares correct?
Hi LeeAnne,
You take into account the number of squares when taking the average. So you sum the number of cells you have in total among the 5 squares (in this case, 115), you divide by the number of squares (5) and you get your average number of cells per small square. 0.000004 mL is the volume in one small square (inside the central square – see here). Since you have the average number of cells in one small square, you’re good to go!
Hope that clarifies, let me know otherwise 🙂
Hi where did you get 2 where you multiply the dilution factor?
Hi Cindy,
If you have a 1:1 dilution (considering 1 part of original sample to 1 part of dilutant), the concentration in the original sample will be doubled compared to the one in the diluted sample – that’s why you have to multiply by 2 the value of the concentration for the diluted solution.
Check out my longer reply in the Youtube comments here.
Let me know if you need more help.
Maria
when you count two cells why do you divide by 8
Hello Ritika,
That’s because you would have counted 8 of the large corner squares across the 2 chambers instead of just 4 in the 1 chamber.
Hello this is Parikshit.
I have a question for cell count in culture.
Example – I have 1000L grape juice and yeast liquid culture which 30Liters of unknown cell count. So how much cell count needs to be achieved in terms to start fermentation and how much culture volume to add in juice to start fermentation.
Please guide ahead
Hard to say, we aren’t wine experts 🙂 We can help you count, but what you do with that count is beyond us!
Hi.
Thank you so much for this tutorial, it helps me to finally understand the final volume added to get seeding density. But I have observed in different research some authors use different seeding density even for the same cell line, e.g A use 1×10^2 cfu/ml while B use 1×10^4 cfu/ml for S mutans.
So recommended seeding density is empirically determine?
Yes, the seeding density will depend on what you are doing, the types of plates being used, media, how fast you want them to grow, etc.