I am growing protoplasts extracted from leaves in a liquid medium.
They've formed colonies that consist of cells without cell walls.
Now I need to count cell numbers...
So I tried the same enzyme mixtures previously used for protoplast extraction from leaves... But the cells don't seem to be separated from the colonies...
How come the cohesion between the cells from a colony can be this strong?
Is there any suggestion for separating cells from colonies (with only 10~20 cells.....)?
Dear Hyunbae Lee ,
please see the following text:
colony-forming unit (CFU or cfu) is a measure of viable bacterial or fungal cells. In direct microscopic counts (cell counting using haemocytometer) where all cells, dead and living, are counted,but CFU measures only viable cells. For convenience the results are given as CFU/mL (colony-forming units per milliliter) for liquids, and CFU/g (colony-forming units per gram) for solids. CFU can be calculated using miles and misra method, it is useful to determine the microbiological load and magnitude of infection in blood and other samples.
Example:
Calculate the number of bacteria (CFU) per milliliter or gram of sample by dividing the number of colonies by the dilution factor The number of colonies per ml reported should reflect the precision of the method and should not include more than two significant figures.
The CFU/ml can be calculated using the formula:
cfu/ml = (no. of colonies x dilution factor) / volume of culture plate
For example, suppose the plate of the 10^6 dilution yielded a count of 130 colonies. Then, the number of bacteria in 1 ml of the original sample can be calculated as follows:
Bacteria/ml = (130) x (10^6) = 1.3 × 10^8 or 130,000,000.
CFU/mL Practice Problems - CFU/mL Calculation Examples
Problem 1:
Five ml of Bacterial Culture is added to 45 ml of sterile diluent. From this suspension, two serial, 1/100 dilutions are made, and 0.1 ml is plated onto Plate Count Agar from the last dilution. After incubation, 137 colonies are counted on the plate. Calculate CFU/mL of the original Sample?
Answer:
First thing we need to know is the Dilution Factor, or how much the original sample is diluted:
here Initially 5mL in 45mL = Final Volume / Sample volume = 50/5 = 10.
Then two serial dilutions of 1/100.
Total Dilution Factor = 10 * 100 *100 = 10^5
CFU/mL = cfu/ml = (no. of colonies x dilution factor) / volume of culture plate
= (137 * 10^5)/0.1
=1.37*10^8
So Total colony forming units = 1.37*10^8 CFU/mL
Converting CFU/mL to Log value
For example,
Total colony forming units = 1.37*10^8 CFU/mL and you want to convert it into Log value,
Just take Log(CFU/mL)
Here, log (1.37*10^8) = 8.13924922.
Useful for expressing log reduction of microbes / biologic log reduction.
For illustration of the figures in this article, please use the following link:
http://technologyinscience.blogspot.co.il/2011/11/cfu-colony-forming-unit-calculation.html#.VpF-W_krLIW
Hoping this will be helpful,
Rafik
@ Mehdi Babaei
Thank you for your suggestion!
Since my samples only contain colonies consisting of countable numbers of cells, I wished to break the cohesion between cells so I could count single cells. I would probably treat bacteria in the future so I believe your suggestion would be much help then!
Protoplasts will not start to divide until new walls have formed and that usually does not happen in liquid media, but in semi-solid ones, because of the structural support needed for cell wall development.
Using the extraction solution would only make it worse.
You could try just washing and centrifuging at low acceleration (
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