Dilution Theory – Page 2:More Dilution Plating 

A quick review of highlights from the previous page ("Dilution Theory–Page 1"):
On this page:

When we inoculate plates already containing medium (the usual case in our lab courses), we find that it would take too long for a one ml inoculum to soak into the medium. So, we generally plate 0.1 ml from each dilution made. For each plate, you can readily see that we are then inoculating onetenth the number of CFUs there would have been in a one ml inoculum. In the following diagram, we have built on the last illustrated example given on Page 1 by adding inoculations of 0.1 ml from each of the dilutions into respective plates. (The numbers of colonies in parentheses are either "too many" or "too few" for counting – remembering our "30300 rule" above.) Instead of the letters we labeled our plates with previously, we have labeled each plate with the dilution it represents – as if one ml had been inoculated from that dilution. For example, a plate inoculated with one ml of a 10^{–2} dilution would have the same label (10^{–2}) as a plate inoculated with 0.1 ml of a 10^{–1} dilution, as they are equivalent plates. This value (10^{–2}) has been traditionally called the "plated dilution." (A more fitting term we have come up with – and may officially substitute some day – is "virtual dilution"!) Remembering our discussion on Page 1, you can see that the value of the "plated (virtual) dilution" is equivalent to the actual amount (in ml or g) of undiluted sample that is being plated out. For example, a plate labeled "10^{–2}" represents 10^{–2} ml or gram of sample being inoculated onto the plate. A quick example problem: Suppose you inoculate a plate with 0.1 ml of a 10^{–1} dilution of a sample of milk. After incubation, you find that 80 colonies have arisen on the plate. How may CFUs were there per ml of the milk?
Often it is handy to utilize formulas to work out dilution problems. In Bacteriology 102, we show continuously that the following set of formulas always work (if they are used properly). We could use one "universal" formula, but we have traditionally used these: the first (already used above) to find what portion of our sample is being analyzed (expressed as our socalled plated or virtual dilution) and the second to inflate our colony count proportionately, resulting in the number of CFUs that were in one gram or ml of the original, undiluted sample. (Don't just take our word for it. Spend a little time here and see how ultimately we can always come up with the number of CFUs per one ml or one gram of the undiluted sample.)



You can also look at the problem this way: If 220 colonies arose from plating (the equivalent of) 10^{–4} ml of the culture, then (proportionally) there would have been 220 X 10^{4} or 2.2 X 10^{6} CFUs per one ml of the culture. This is the reasoning behind the second of the two dilution formulas. 

A 1/10 dilution is achieved when 5 ml of sample are added to 45 ml of diluent. Remember that a 1/10 dilution can be made in a variety of ways – as long as there is one part of sample added to 9 parts of diluent. Even if we had a dilution we could not so reduce – e.g., something like 3 grams of hamburger added to 80 ml of diluent which would result in a 3/83 dilution – we can still simply "plug it into" the formula and we could wind up with the answer. And remember that the formulas will always give the answer as no. of CFUs per one ml (or one gram) no matter what amount we start with. Wouldn't this problem have the same answer if we had put 1 ml of milk into 9 ml of diluent? 



As for the above problem, you can look at this one as follows: If 180 colonies arose from plating (the equivalent of) 10^{–3} ml of the milk, then (proportionally) there would have been 180 X 10^{3} or 1.8 X 10^{5} CFUs per one ml of the original, undiluted milk sample. 
dilutions made 
X  amount inoculated 
=  "plated dilution" 
1  X  5  =  5 


dilution factor  X  # colonies  =  # CFUs/ml 
1/5  X  50  =  10 
Here's a treat for the general public: Click here for some more practice problems from the Bacteriology 102 Lab Manual with the solutions given on a separate page.
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" Index to the Dilution Theory pages. " Site Outline of related pages. 
These general microbiology pages have copyright by John Lindquist 