1) What does it mean to have a countable plate? In roughly what dilution would y
ID: 56553 • Letter: 1
Question
1) What does it mean to have a countable plate? In roughly what dilution would you expect to find a countable plate?
2) You are given a sample of unknown Bacilus subtilus and need to find out the cell concentration of the original sample. Describe your experimental design. Draw out a dilution map that illustrates your experimental design. Be sure to label the amount of autoclaved water going in to each vial, volume of bacteria sample transferred, the plating volume and determine the resulting concentration that it will produce after being plated.
3) What is the original cell density of a sample that produced 117 colonies on a plate that was inoculated with a 0.1mL sample from a tube containing a 10-5 dilution?
4) What is the original cell density of a sample that produced 32 colonies on a 10-7 dilution plate?
5) If you only have three agar plates left that are suitable to grow V. fischeri and your original cell sample was 2.6x105, which three dilution vials would you inoculate in order to guarantee yourself a countable plate? Assume the inoculation volume used on each plate was 0.1mL and that each vial had a dilution factor of 10.
Explanation / Answer
1. Countable plates measure colony forming units of bacteria which are in range between 30 and 300 colonies. The dilution of sample which is used in countable plate is roughly 1 / 10 dilution of sample.
2. Suppose we have a unknown sample which produces a countable plate (100 colonies) with the inoculation of 0.01ml of sample. So, if 100 CFUs are present in the 0.01 ml inoculum, then 100 x 100 CFUs would be present in one ml; the final answer is 10,000 CFUs/ml of the sample.
Now, you can go in reverse order if you want to know make countable plate of 100 colonies.
You have 10,000 CFUs/ml.
Label 3 - 4 tubes as A ,B, C and D
Add 9.0 ml of autoclaved water in each tube
Take 1.0 ml sample and inoculate it in tube A, so we put the 10,000 CFUs into a total of 10 ml which is equivalent to 1000 CFUs per ml of the 1/10 dilution of the sample.
Take 1 ml from tube A and inoculate it in tube B, so we put the 1000 CFUs into a total of 10 ml which is equivalent to 100 CFUs per ml of the 1/10 dilution of the tube A.
(no need of tubes C and D in our case as we have got the required CFUs)
multiply dilution facor of tube A and tube B to get the requited dilution factor for producing 100 colonies on countable plate i.e. 1 / 10 (A) x 1 / 10 (B) = 1 / 100 = 10-2
So, by inoculating 1 ml of a 10–2 dilution into the plate which is subsequently counted, we are theoretically doing the equivalent of plating 10–2 ml (i.e., 0.01 ml) of the sample i.e. plating volume = 0.01 ml.
3. To calculate number of colonies / ml in original sample, if you know dilution factor and CFUs at that dilution simply go in reverse direction. No of colonies will increase in each preceding tubes by factor of 10 (inverse of dilution factor).
So, we have
10-5 = 117
10-4 = 1170
10-3 = 11700
10-2 = 117000
10-1 = 1170000
original sample = 11700000 = 117 x 105 CFUs / ml
4. Likewise original cell density produced by 32 colonies on a 10-7 dilution plate, will by 32.0 x 107 CFUs / ml
5. As, i have mentioned earlier a reliable countable plate has colonies in range of 30 - 300.
Now if we do serial dilution of we get one reasonable dilution which fits within countable plate range i.e
10-2 = 260 CFUs / 0.1 ml
To make two other dilutions, what you can do, dilute vial (having 260 CFUs / 0. 1 ml) with water in 1 : 1 ratio to get estimated cell number as 260 / 2 = 130 CFUs / 0.1 ml and similarly dilute further the vial containing 130 CFUs / 0.1 ml in the ratio of 1 : 1 to get 60 CFUs / 0.1 ml.
(You can check it with some other person also, as i have little doubt over it. Keeping the dilution of each vial in question into account, i could find only one reasonable dilution provided above)
Related Questions
Navigate
Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.