Question

1. You have performed a serial dilution of a sample with an unknown concentration of microorganisms. You counted 55 CFU on a countable plate inoculated with 0.1 mL of a 10^-3 dilution. What was the population density of the original sample per milliliter?
2. Many of the quantitative methods discussed are popular because they enable the microbiology researcher to selectively count live cells only. Why do you think this might be an important or desirable feature in a medical or environmental research situation? Provide a scenario in which it might not be important to differentiate between live and dead cells when counting cell numbers.
3. What are some potential sources of error in the serial dilution/direct plate counting method?

Answer 1

Ans) The formula used is CFU/ml = (Number of colonies x Dilution factor) / Volume of culture plated

Given : Number of colonies = 55 ,

Dilution factor = 10^-3

Volume of culture plated = 0.1ml

Substituting in the formula we get,

CFU/ml = 55 x 10^-3 / 0.1

CFU/ml = 55 x 0.001 / 0.1

CFU/ml = 0.55 is the population density of the original sample per ml.

This is important in medical or environmental research in order to know the bacterial load or count in the infected sample. For example, when the patient suffers from UTI (Urinary tract infection), Doctor prescribe to do Aerobe culture urine test to observe the growth of pathogenic organsims from the urine sample on the culture plate and then it becomes easier to suggest the correct antibiotic to treat the urinary tract infection (Without knowing the bacterial strain which is infecting,no antibiotics can be given.If incorrect antibiotics are given,multidrug resistant bacteria can be produced which is very dangerous to treat later) In the environmental research area also it has various advantages.For example, during isolation of antibiotics resistant organisms from the soil sample,etc we streak the soil sample (diluted) on the culture plate followed by the addition of antibiotics and if the antibiotic resistant organisms are present in the soil sample, we can observe the zone of inhibition on the culture plate. In this scenario, it is not important to differentiate between live and dead cells, we just have to isolate antibiotic resistant colonies from the culture plate.

Counting becomes important during RBC,WBC concentration determination from the blood sample using hemocytometer , calculating the number of live cells grown in animal tissue culture lab by trypan blue assay etc.

Potential sources of error are the sensitivity of the plate counter (It always has some zero error), Pipette should be well calibrated or else inaccurate volume can also alter the results, Accurate transfer is required with each serial dilution as with each dilution,the quantity becomes less and less to be transferred.