For your isolate project, you want to isolate a microbe from, and determine the

Question

For your isolate project, you want to isolate a microbe from, and determine the total number of cells in, a sample of hamburger that you found at the back of your refrigerator and that looks kind of nasty. You take 0.5 gram of the hamburger and mix it with 5 mL of sterile phosphate buffered saline (PBS) solution, and homogenize the sample to mix the meat and cells as thoroughly as possible with the PBS. You then make 10-fold serial dilutions of this mixture (up to 10-6), and plate out 50 microliters of each dilution onto a beef extract agar plate. After incubation, you observe 983 colonies on the plate onto which the 10-3 dilution was plated, 135 colonies on the plate onto which the 10-4 dilution was plated, and 9 colonies on the plate onto which the 10-5 dilution was plated. No colonies were observed on the 10-7 dilution plate, and a lawn (to many colonies to count) were observed on the 10-1 and 10-2 dilutions.

Using this data, estimate the number of microbes per gram in your original sample of hamburger that you were able to cultivate, and comment on whether or not you think that this is the total number of microbe present in your sample

Explanation / Answer

First of all using the serial dilution and plating technique we cannot cultivate each organism present in a particular sample. We can only estimate the no. of culturable microbes which can fom colonies on agar plate. Thus we use this term, CFU or colony forming unit.

Now suppose 0.5gm sample contains x CFU. so when mixed, 5 ml PBS will contain x CFU. 1 gm will contain 2x CFU.This tube containing 0.5gm sample with saline can be marked as 10-1. from there 1ml is transferred to 9ml of PBSand mixed, this will be marked as 10-2. from this we can make serial dilutions 10-3, 10-4,10-5,10-6,10-7.

50microlitre from each tube was used for plating. After incubation, colonies on all plates were counted.

For estimation of total CFU in a sample, we should choose a plate where no. of colonies is between 30-300. In the given question, only one plate fulfil this criteria, i.e. 10-2. It contains 135 colonies.

50 microliters contain 135 CFU, then 1000microliters or 1ml contains = 2700 CFU.

so we can saysuspension from 10-4 dilution contains 2700 CFU/ml.

x= no. of colonies * dilution factor. * amount of PBS used to homogenize the hamburger

x =2700* 104 *5

=135*106 CFU

so 1gm of hamburger contains 2x=2*135*106

=27*107CFU

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