SERIAL DILUTIONS (Quantitative Techniques in Microbiology)
In quantitative microbiology, we are concerned with determining the concentration of colony-forming units
(CFUs) in our sample i.e., the number of CFUs per ml or per gram of the sample. For example, if we were
to plate out one ml of a lake water sample and then after incubation of the plates find that 100 colonies
have arisen, we would then conclude that there were 100 CFUs per ml of the lake water.
More realistically (as with most of our area lakes), the concentration of CFUs in the water could have been
considerably greater. Counting the colonies on a plate inoculated with one ml of water may be impossible.
We would like to have "countable" plates containing between 30 and 300 colonies. If fewer than 30, we run
into greater statistical inaccuracy. If greater than 300, the colonies would be tedious to count and also would
tend to run together.
Then you see that 1 ml is transferred from each tube and put onto an 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.
We may try to plate out smaller and smaller amounts, as in the example shown at right. With this new lake
water sample, a countable plate (Plate C: 100 colonies) is achieved with the inoculation of 0.01 ml of sample.
Figuring out the number of CFUs per ml of the sample would go like this: Whatever the number of CFUs in
the inoculum of Plate C (one-hundreth of a ml of the sample), there would be one hundred times as many
CFUs in one ml of the sample. So, if 100 CFUs are determined to be 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.
Two drawbacks to this procedure: It is difficult with our equipment to dispense amounts smaller than 0.1 ml.
Also, the smaller the amount tested, the less representative it is of the sample.
So we now get into "dilution theory" to accomplish the equivalent of plating out succeedingly smaller
amounts of sample. Making serial decimal dilutions (i.e., successive 1/10 dilutions, each made by adding
one part of inoculum to 9 parts of diluent) and inoculating one ml into each of the plates, we can construct a
plating procedure (shown at right) that is equivalent to the above.
Illustrating further how the concentration of CFUs decreases according to how these cell suspensions are
diluted: We determined above that the sample contains 10,000 CFUs per ml. Taking out one ml and
inoculating it into a 9 ml dilution blank (the second tube) would 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. The density of CFUs
continues to decrease ten-fold with each subsequent dilution.
DEFINITIONS:
Aliquot: a measured sub-volume of original sample.
Diluent: material with which the sample is diluted.
Dilution factor (DF): ratio of final volume/aliquot volume (final volume=aliquot + diluent)
Concentration factor (CF): ratio of aliquot volume divided by the final volume (inverse of the dilution
factor)
To calculate a dilution factor:
Remember that the dilution factor is the final volume/aliquot volume.
EXAMPLE: What is the dilution factor if you add 0.1 mL aliquot of a specimen to 9.9 mL of diluent?
1. The final volume is equal the the aliquot volume plus the diluent volume: 0.1 mL + 9.9 mL = 10 mL
2. The dilution factor is equal to the final volume divided by the aliquot volume: 10 mL/0.1 mL = 1:100
1
dilution (10 2)
-2
The Concentration Factor for this problem = aliquot volume/final volume = 0.1/(0.1 + 9.9) = 0.01 or 10
concentration
To prepare a desired volume of solution of a given dilution:
1. Calculate the volume of the aliquot: it is equal either to
the final volume/dilution factor or
the concentration factor x final volume
2. Calculate the volume of the diluent: which is equal to (the final volume - aliquot volume)
3. Measure out the correct volume of diluent, add the correct volume of aliquot to it, mix.
EXAMPLE: How would you prepare 20 mL of a 1:50 dilution?
1. Determine required aliquot by dividing final volume by dilution factor: 20 mL/50 = 0.4 mL sample
2. Subtract the aliquot volume from the final volume: 20 mL - 0.4 mL = 19.6 mL diluent
3. Measure out 19.4 mL diluent, add 0.4 mL sample to it, mix thoroughly
SAMPLE PROBLEMS: (Note that these are in a different order than originally given out in class)
1. How much sample (what sized aliquot) is required to prepare 10 mL of a 1 to 10 dilution, and how much
diluent would you need? 1 mL sample + 9.0 mL diluent
2. What is the dilution factor when 0.2 mL is added to 3.8 mL diluent? What is the concentration factor? DF =
20, CF = 0.05
3. You are to prepare 5 mL of a 102 dilution. What should the aliquot and diluent volumes be? aliquot = 0.05
mL, diluent = 4.95 mL
4. How would you prepare 20 mL of a 1:400 dilution? 0.05 mL sample, 19.95 mL diluent
5. What is the dilution factor when you add 2 mL sample to 8 mL diluent? DF = 5
6. You add a pint of STP gas treatment to a 12 gallon fuel tank, and fill it up with gas. What is the dilution
factor? (8 pints/gallon) F.V. = 12 gallons x 8 pints/gallon = 96 pints. Therefore 96pints/1 pint = D.F. =96
7. You want 1 liter of 0.1 M NaCl, and you have 4 M stock solution. How much of the 4 M solution and how
much dH2O will you measure out for this dilution? 25 mL 4.0 M stock solution + 975 mL dH2O
For problems like the following, you need to know the ratio of the diluent to the aliquot. For instance,
if you are making a 1:20 dilution, the ratio of diluent to aliquot will be1 less than the dilution factor, or
19 parts diluent, 1 part aliquot:
8. You have 0.6 mL of sample, and want to dilute it all to a fiftieth of its present concentration. How much
diluent will you add, and what will the final volume be? 29.4 mL diluent, final volume = 30 mL
9. You diluted a bacterial culture 10 6, plated out 0.2 mL and got 45 colonies on the plate. How many
bacteria/mL were in the original undiluted culture? 2.25 x 108
A harder one which requires a little algebra:
10.You have 100.00 mL of dH2O. How much glycerine would you have to add in order to make a 2.000 %
v/v dilution? (Hint, let the volume of glycerine = X, set up the standard equation for a dilution factor using
X, and solve for X.) 2.04 mL glycerine
11. The same dilution can be obtained in each of the following situations:
a. The addition of 1 ml of a sample to 9 ml of sterile diluent.
b. The addition of ml of the same sample to 27 ml of diluent.
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c. The addition of 11 ml of the same sample to ml of diluent.
12.One should expect the same number of CFUs in one ml of an undiluted sample as in ml of a
1/10 dilution of the same sample.
CFU AND DILUTION THEORY
Counting 100 colonies in Plate C, note how we can work back to a concentration of 10,000 CFUs per ml of
the sample. 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 102 ml (i.e., 0.01 ml) of the lake water sample.
IF 100 colonies arise from plating one ml of a 1/100 dilution of the lake water,
THEN there were 10,000 CFUs (from 100 X 100) per one ml of the undiluted water sample.
Compare the solution just obtained with the previous solution where Plate C was inoculated with 0.01 ml of
sample:
IF 100 colonies arise from plating 0.01 ml of the water sample,
THEN there were 10,000 CFUs per one ml of the water sample.
1. For Bacteriology 102 students, the above setup is applicable to Experiment 1, Period 2 where we
plated one ml of a 102 (i.e., 1/100) dilution of lake water. In Period 3 after incubation of the plates
if we were to find 75 colonies on our plate, we could determine the CFUs per ml of the sample (at
the time of Period 2) as follows:
IF 75 colonies arise from plating one ml of a 1/100 dilution of the lake water,
THEN there were 7,500 CFUs (from 75 X 100) per one ml of the undiluted water sample.
2. As we used a 104 (i.e., 1/10,000) dilution of soil in the same experiment, we could figure out the
CFUs per gram of the soil the same way, remembering that we consider milliliters and grams to
be equivalent:
IF 75 colonies arise from plating one ml of a 1/10,000 dilution of the soil,
THEN there were 750,000 CFUs (from 75 X 10,000) per one gram of the undiluted soil sample.
3. Consider this problem, the likes of which we often give in quizzes and on problem sets: Five
ml (not one!) of an undiluted spring water sample were added to a petri dish to which 15 ml of melted
Plate Count Agar were then added. After mixing, the plate was allowed to solidify and then was
incubated appropriately. After incubation, 50 colonies were counted. How many CFUs were present
per one ml of the original, undiluted spring water sample?
Here's the solution (and note the careful use of correct terminology): If 50 colonies arise from
plating 5 ml of the sample, then there were 10 CFUs per one ml of the sample.
4. The following is a sample problem from Bacteriology 102 worked out with the formulas: One
ml of a bacterial culture was pipetted into a 9 ml dilution blank. One-tenth ml of this dilution was
pipetted into a 9.9 ml dilution blank. From this dilution, one-tenth ml was plated with 25 ml of culture
medium. 220 colonies arose after incubation. How many colony-forming units were present per ml of
the original culture? (Does the amount of medium in the plate matter in the calculations?)
dilutions amount
X = "plated dilution"
made inoculated
1/10 X 1/100 X 1/10 = 1/10,000 or 104
dilution factor X # colonies = # CFUs/ml
104 X 220 = 2.2 X 106
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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 104 or 2.2 X 106 CFUs per one ml of the
culture. This is the reasoning behind the second of the two dilution formulas.
When checking your answer, which you can always do as follows for such problems: Start with the
sample which you have determined to contain 2.2 X 106 CFUs per ml. Then, see if you wind up with the
stated number of colonies on the plate, after making the specified dilutions in which the number of CFUs per
ml is sequentially reduced.
a) The first, 1/10 dilution contains 2.2 X 105 CFUs per ml.
b) The second, 1/100 dilution contains 2.2 X 103 CFUs per ml.
c) As 0.1 ml of the second dilution was inoculated into the plate, we would expect the number of CFUs in
the inoculum to then be 2.2 X 102 which is 220, the number of colonies we counted on the plate.
5. Here is a problem where we start out with something other than 1 ml or 1 gram of sample being
diluted: Five ml of milk were pipetted into 45 ml of diluent. One ml of this dilution was pipetted into 9 ml of
diluent. From this dilution, 0.1 ml was plated. After incubation, 180 colonies were counted. Determine the
number of colony-forming units per ml of the original milk sample.
dilutions amount
X = "plated dilution"
made inoculated
1/10 X 1/10 X 1/10 = 1/1000 or 103
dilution factor X # colonies = # CFUs/ml
103 X 180 = 1.8 X 105
As for the above problem, you can look at this one as follows: If 180 colonies arose from plating (the
equivalent of) 103 ml of the milk, then (proportionally) there would have been 180 X 103 or 1.8 X 105 CFUs
per one ml of the original, undiluted milk sample.
6. For a problem presented on the previous page (as no. III) in which no dilutions were made, we can
still work it out with the formulas: Five ml of an undiluted spring water sample were added to a petri dish
to which 15 ml of melted Plate Count Agar were then added. Fifty colonies were counted after incubation.
How many CFUs were present per ml of the original, undiluted spring water sample?
dilutions amount
X = "plated dilution"
made inoculated
1 X 5 = 5
dilution factor X # colonies = # CFUs/ml
1/5 X 50 = 10
Note that when there are no dilutions, we indicate "1" not zero! for the dilutions made. As always, the
"dilution factor" is the inverse of the so-called "plated dilution" (according to how we defined our terms), and
the "plated dilution" always represents the amount of sample being plated.
7. One ml of a bacterial culture was pipetted into a 9 ml dilution blank. One-tenth ml of this dilution was
pipetted into a 9.9 ml dilution blank. From this dilution, one-tenth ml was plated with 25 ml of culture
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medium. 220 colonies arose after incubation. How many colony-forming units were present per ml of the
original culture?
dilutions made X amount inoculated = "plated dilution"
1/10 X 1/100 X 1/10 = 1/10,000 or 104
dilution factor X no. colonies = no. CFUs/ml
104 X 220 = 2.2 X 106
8. Ten ml of spring water were added to a petri dish to which 40 ml of melted Plate Count Agar were added.
After incubation, 35 colonies arose on the plate. What was the count of CFUs per ml of the spring water?
As 35 colonies arose from the 10 ml inoculum, then there had been 3.5 CFUs per ml of the spring water.
Note that no formulas are needed for this problem, but if they were to be used, the dilution factor would be
101. Why? Remember that the "plated dilution" represents the actual amount of sample being tested
which in this case is 10 ml. (In the usual dilution problem but not in this case this amount is a small
fraction of a gram or ml.) The inverse of the plated dilution which is the dilution factor would therefore
be 101.
9. You are given eight petri dishes of Nutrient Agar and an abundance of pipettes and 9.0 ml dilution blanks,
and you need to plate out a sample of milk such that plated dilutions of 10 1, 102, 103 and 104 are
achieved in duplicate.
a. Clearly diagram how this may be done with the materials at hand. (As it may take quite a while for large
inocula to soak into plates, do not plate any amount larger than 0.2 ml.)
b. On the plates containing the 10 4 plated dilution, you count 48 colonies on one plate and 54 colonies on
the other after incubation. Calculate the number of colony-forming units (CFUs) per one ml of the original
milk sample.
dilution factor X no. colonies = no. CFUs/ml
104 X 51 (the average) = 5.1 X 105
10. Consider the following dilution scheme:
a. Report the total number of CFUs in the entire 100 ml amount of the original lake water sample. (TNTC=too
numerous to count.)
dilutions made X amount inoculated = "plated dilution"
1/10 X 1/10 X 1/10 X 1/10 = 1/104 or 104
dilution factor X no. colonies = no. CFUs/ml
4
10 X 58 = 5.8 X 105
5
Multiply CFUs/ml by 100 to get CFUs/100ml. Answer = 5.8 X 10 7.
b. Would you expect any change in the answer of the above problem if the first dilution was made by adding
one ml of sample to 9 ml of diluent? Why or why not?
No. In each case you're making a 1/10 dilution. So the concentration of CFUs per ml of that dilution will be
the same.
c. Would you expect the number of viable (living) cells in the original sample to be greater than, less than or
the same as the number of CFUs in the sample?
Greater! As a CFU can be one or more cells, there is no one-to-one relationship between the number of
colonies seen and the number of original cells. Also, you must consider that there may be viable cells which
will not grow under the conditions given (medium, temperature, etc.). So our plate count always gives us a
number lower than the real number.
Be sure you know the definitions of cell, CFU and colony as given in the introduction to Experiment 1!
Realize that CFU is not strictly a quantitative term. (The cells don't know if their colonies are going to be
counted or not!)
d. What dilution is achieved by adding 2 ml of inoculum to 19 ml of diluent? (No, it wasn't a misprint in the
diagram.)
It's simply a 2/21 dilution! We get that from 2/(19+2). There is no rule that dilutions have to be 1/10 or 1/100
or something else convenient. As long as we keep track of the math, we can plug any number into our
dilution formulas and grind out the number of CFUs per ml or gram.
11. You have obtained a pond water sample and wish to determine the concentration of bacteria which are
gram-negative and lactose-fermenting. After making two 1/100 dilutions, you plate 0.1 ml of the second
dilution onto each of two plates of MacConkey Agar. After appropriate incubation, you find that one plate
contains 155 red colonies and 45 white colonies, and the other plate contains 160 red colonies and 35
white colonies. What was the concentration of gram-negative, lactose-fermenting CFUs per ml of the
pond water sample?
As we see in Experiment 4 and Appendix D and throughout the course, only gram-negative bacteria are
expected to grow (form colonies) on MacConkey Agar. Of these colonies, the red ones are lactose-
fermenters. In this problem, the average number of colonies of lactose-fermenting gram-negative bacteria is
157.5.
dilutions made X amount inoculated = "plated dilution"
1/100 X 1/100 X 1/10 = 1/105 or 105
dilution factor X no. colonies = no. CFUs/ml
1.6 X 107
105 X 157.5 =
(rounded off)
12. One should expect the same number of CFUs in one ml of an undiluted sample as in 10 ml of a
1/10 dilution of the same sample.
If you dilute something so it's 1/10 the concentration, then you will need 10 times as much to get the same
number of CFUs. Consider this analogy: To get 10 dollars, you can work 1 hour for 10 dollars an hour or 10
hours for 1 dollar an hour.
13. You are given a flask containing a 1/10 dilution of sauerkraut juice. Without making any further dilutions,
how much of this dilution of juice can be plated in order to achieve each of the following plated dilutions?
(As these will be pour-plates as opposed to the surface-inoculated plates in problem 1 you can
inoculate more than 0.1 ml to your plates.)
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plated dilution amount of 1/10 dilution of juice
100 10 ml
101 1 ml
102 0.1 ml
14.One gram of yogurt was added to 99 ml of sterile diluent. Decimal (1/10) dilutions were then made,
and one-tenth ml was plated in duplicate on Plate Count Agar. After incubation, the following
colony counts were made:
5f
dilution colony count
initial dilution (1/100) TNTC, TNTC
first 1/10 dilution TNTC, TNTC
second 1/10 dilution 412, 422
third 1/10 dilution 54, 56
fourth 1/10 dilution 6, 4
Using the above data, calculate the number of CFUs per gram of the yogurt.
dilutions made X amount inoculated = "plated dilution"
1/100 X 1/10 X 1/10 X 1/10 X 1/10 = 1/106 or 106
dilution factor X no. colonies = no. CFUs/ml
106 X 55 = 5.5 X 107
15. One gram of hamburger was added to a 99 ml dilution blank. Two 1/10 dilutions were then made. From
the last dilution made, 0.2 ml was plated onto an all-purpose medium. After incubation, 60 colonies were
counted.
Each of the 60 colonies was inoculated onto a slant of Heart Infusion Agar (to grow cultures for gram
staining) and into a tube of Glucose Fermentation Broth. Exactly one half of the cultures were determined to
be gram-negative. Of these gram-negative cultures, twelve produced acid and gas in the broth and the rest
produced only acid.
As indication of acid (with or without gas) indicates fermentation, therefore all of the gram-negative colonies
in this problem ferment glucose.
Determine the number of gram-negative, glucose-fermenting CFUs per gram of the meat.
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dilutions made X amount inoculated = "plated dilution"
1/100 X 1/10 X 1/10 X 2/10 = 2/105 or 2 X 105
(easier to keep it as the fraction)
dilution factor X no. colonies = no. CFUs/g
105/2
(simple inverse X 30 = 15 X 105 or 1.5 X 106
of 2/105)
16. You added 10 ml of a lake water sample to 90 ml of sterile diluent; this is the "first dilution" indicated in
the table below. After mixing thoroughly, one ml of this dilution was added to 99 ml of sterile diluent to make
the second dilution. To make the third dilution, one ml of the second dilution was added to 99 ml of sterile
diluent. From each of these three dilutions, 1 ml or 0.1 ml amounts were inoculated into pour-plates of Plate
Count Agar (PCA) and tubes of Lactose Lauryl Tryptose Broth (LLTB) as shown in the table below.
After appropriate incubation, colonies were counted and the tubes were checked for positive results, and the
data are summarized below. Subsequent inoculations into BGLB and EC Broth were appropriately made and
incubated; their results are also shown below.
first dilution second dilution third dilution
dilution of lake water
=101 =103 =105
Amount inoculated into each of two plates of
1 ml 0.1 ml 1 ml 0.1 ml 1 ml 0.1 ml
PCA and each of three tubes of LLTB:
Plated dilutions: (dilution of sample 101 102 103 104 105 106
multiplied by the amount inoculated)
Dilution factors: (inverse of the plated 10 102 103 104 105 106
dilution)
205 18 0 0
Colony count on PCA plates: TNTC TNTC
215 22 2 0
No. of positive LLTB tubes: 3 2 1 0 0 0
3 2 0 0 0 0
No. of positive BGLB tubes:
1 1 0 0 0 0
No. of positive EC Broth tubes:
a. What was the "total aerobic plate count" (in CFUs per ml) of the original, undiluted sample of water?
dilutions made X amount inoculated = "plated dilution"
1/10 X 1/100 X 1 = 1/103 = 103
dilution factor X (ave.) no. colonies = no. CFUs/ml
103 X 210 = 2.1 X 105
17. What was the confirmed, most probable number of fecal coliforms per ml of the original sample of water.
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Use the EC Broth results, choosing the results of the 1st, 2nd and 3rd set of MPN tubes, as their results
are 1, 1 and 0. (1,1,0 tells us more about how the coliforms are being diluted to extinction than do any
other set of results, such as 1,0,0 or 0,0,0.) What plates you count have no bearing on which MPN tubes
you choose or vice-versa.
Note in our example of the MPN method on page 96 in the manual that no plates were inoculated at all.
(The only thing we should really worry about is if the coliform count winds up higher than the "total" count!
18. A sample of yogurt prepared with the usual organisms (i.e., Streptococcus thermophilus and
Lactobacillus bulgaricus) was bacteriologically tested as follows: One tablespoon of yogurt was mixed in a
sterile flask with four tablespoons of sterile milk. One ml of this mixture was added to 99 ml of saline.
One-tenth ml of this last dilution was plated onto a medium known to support the growth of yogurt
organisms.
After appropriate incubation, 250 colonies were counted on the plate. Fifty colonies were then picked at
random (a "random sample"), and half of these colonies were found to be composed of cocci in chains.
As half of the randomly-tested colonies were determined to be S. thermophilus, we could then say there
were (probably) 125 S. thermophilus colonies on the plate.
What was the number of Streptococcus thermophilus CFUs per gram of the yogurt?
dilutions made X amount inoculated = "plated dilution"
1/5 X 1/100 X 1/10 = 1/5000 = 1/(5 X 103)
= 2 X 104
dilution factor X no. colonies = no. CFUs/ml
6.25 X 105
5 X 103 X 125 =
better rounded to 6.3 X
105
During the grading of this problem set, one student suggested this sequential solution, keeping in mind that
the so-called "plated dilution" (2 X 10 4 in this problem) really represents the amount of undiluted sample
being tested (as we show here):
4
125 CFUs / 2 X 10 g
4
62.5 CFUs / 1 X 10 g
4
62.5 X 10 CFUs / g
5
6.25 CFUs X 10 CFUs / g
This solution almost makes me consider throwing away the formulas. I'm still thinking about changing the
term "plated dilution" to "virtual dilution" or better yet "virtual amount."
19. One-tenth ml of drinking water was added to a petri dish to which 19.9 ml of melted Plate Count Agar
were added. After incubation, 240 colonies arose on the plate. What was the count of CFUs per one ml
of the water.
As 240 colonies arose from the 0.1 ml inoculum, then there had been 2400 or 2.4 X 103 CFUs per ml of the
spring water. You have ten times the number of CFUs in ten times the amount of sample. No Appendix C
formulas are needed but if they are used, the "dilution factor" would be 10. Note that there is no dilution in
this problem, as the whole 0.1 ml of the sample gets inoculated into the plate. We could have used twice the
amount of medium, and the answer would still be the same.
