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How to Make Bacteriological Serial Dilutions


HYPOTHETICAL EXAMPLE: In the following, a bottle contains an unknown concentration of bacteria ("cfu/ml" means "colony forming units per milliliter", where each cfu equals one LIVE bacterium). One milliliter from the bottle is transferred into a tube containing 9 ml of water or saline (0.15% NaCl is called "normal saline" or "NS"). Thus the concentration of the bacteria in the tube is exactly 1/10 that in the bottle. And if 1 ml of that is transferred into a second tube of 9 ml NS, another 10-fold dilution is accomplished. It is now 1/100 the concentration in the bottle.
Hint: note that it is 10-(tube#). And you then see successive "serial" dilutions are made.

Then you see that 1 ml is transferred from each tube and put onto a agar plate. The 1 ml is then spread evenly over the surface of the plate, and allowed to dry, which leaves any bacteria that were in the liquid "high and dry" on the agar surface. Those marooned bacteria then begin to divide and do so for several hours until there are so many of them clumped together that they form a visible colony. Thus every colony that can be counted arose from a single bacterium. Hence, the number of colonies corresponded to the number of bacteria that were marooned.

Counting backwards, if there were 311 colonies, then there were 311 bacteria in the milliliter that was transferred to the plate from tube #4. Hence, there were 311 cfu/ml in tube #4. Or 3110 cfu/ml in #3, or 31100 in #2, or ... or 311x104 cfu/ml in the original bottle. And that equals 3.11x106.
("TMTC" = "too many to count.")

BUT there is a problem. This method does not work. It is hypothetical but not practicable because the whole milliliter put onto the plate would dry so slowly that the bacteria would be actively dividing, then wiggling away to form colonies of their own and giving a falsely high result. So-, what if we used less than 1 ml?



This is the most widely used plate count scheme used around the world. It differs from the hypothetical one above by only one important aspect: 0.1 ml (100 microliters = 100 μL = 100 λ) is transferred to the plate. This amount will both evaporate and soak into the agar within a minute or two to become a "dry" surface that will not allow most bacteria to wiggle away from the growing colonies. (Note: that 0.l ml from tube to plate is the crucial difference; that amounts transferred between the tubes is only critical with respect to maintaining a ratio of 1 unit added to 9 units.)

See if you can figure out the final calculation:
Flask's cfu/ml = (colonies on the plate) x 101+Tube#. Where did the "1" come from? That's right: it takes into account the need to multiply by an extra 10 because you added only 0.1 ml onto the plate from the tube.



One of the problems with the above technique - even though it is the most commonly used one - is that huge numbers of plates are needed in most experiments that entail the counting of a number of cultures.

The Walter Reed Army Institute of Research is responsible for preparing battlefield hospital facilities with the optimum procedures. All those petri plates needed above are not in the Army's best interests. Trucks must carry both medical supplies and bullets. So they had to find a way to cut down on the numbers of petri plates transported. Here is what they came up with:

Note that only one plate is needed and it does up to 5-fold duty. Each "lane" on the agar surface now acts as if it were its own whole plate. Now the formula for calculating the bacterial concentration in the bottle becomes:

Flask's cfu/ml = (colonies on the plate) x 102+Tube#

I bet that you can figure out where the "2" came from.



The MECHANICS to do this procedure

As you transfer the liquids down through the series of tubes, you MUST use a different pipet or pipet tip for each transfer. However, when transferring liquids from the tubes, you may use the same pipet or tip IF you start transferring from the most dilute tube and work your way upwards. If you need more explanation, click HERE!



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