How to Calculate Dilutions , and some Example Problems

(Adapted with permission from Dr. William Lorowitz, Weber State Univ., http://faculty.weber.edu/wlorowitz/3053/DILUTION.pdf)



Concentration = Particles/Volume or,
C = P/V
For example, if 0.1 ml of culture is spread on a plate and 150 colonies are counted, and if we assume that the
number of separate colonies equals the number of viable cells (called colony forming units, or CFU) spread
on the plate, then: C = 150 CFU / 0.1 ml = 1500 CFU/ml
Where: C = the concentration of colony forming units (CFUs) in the culture plated
P = the number of particles (molecules, cells, etc.) - in this case CFUs - on the plate
V = the volume plated
Now, what if the aliquot plated was from a 10-fold dilution of the culture (so the plated material had 1/10 the
original concentration)? It seems obvious that the concentration of CFUs in the original culture would be
10X higher - or 15,000 CFU/ml - than the value calculated above for the plated material. How can this type
of relationship be expressed mathematically, in order to simplify making dilutions and performing
calculations later? Let’s take it step by step.


If a suspension is diluted with diluent, such as the sterile saline we used in lab, the total number of particles
doesn't change. So, the first thing we can say is P1 = P2 where P1 is the number of CFUs before dilution and
P2 is the number of CFUs after dilution. But, since the volume increases, the concentration (P/V) decreases.
Based on the equation for concentration above (C = P/V), we can also write P = CV. P = CV for both P1
and P2, so:
C1V1 = C 2V2

In other words, since the number of particles is the same before and after dilution, the concentration (C)
times the volume (V) will be the same. Now, to calculate dilutions:

Dilution = Volume of Sample Added/Total Volume =

______________Volume of Sample Added____________

(Volume of Diluent + Volume of Sample Added)



For example, if a culture contains 1 x 109 cells/ml and 0.1 ml is added to 9.9 ml of diluent,


Dilution = 0.1ml/(9.9 ml + 0.1 ml) = 0.1 ml/10 ml = 0.01 or 1/100

The dilution factor (DF) is the reciprocal of the dilution, or 1/dilution. The total dilution is the product of
all dilutions:
Dtotal = D1 x D2 x D3 . . .
The initial concentration C1 (that is, the concentration of cells in the undiluted culture) is equal to DF times
C2 (the concentration of cells in the diluted material spread on the plate). Recall that C2 is P2/V2. So, 











where C1 = initial concentration (or CFU/ml in the undiluted stock culture), C2 = CFU/ml of the diluted
material actually spread on the plate, P2 = CFU on the plate, V2= volume plated (ml), and DF = dilution
factor. The other side of this sheet shows how to use these concepts in solving typical problems.



1. If there are 2 x 1012 bacteria in 200 ml, what is the cell concentration?






2. If you have 175 ml of a crystal violet solution of 5 µg/ml, how much crystal violet do you have?


P
=
CV
=
(5
µ g/ml)(175
ml)
=
875
µ g
of
crystal
violet



3. If you add 2 ml of a bacterial suspension with a concentration of 2 x 109 cells/ml to 13 ml of saline,
what is the new cell concentration?










(2
x
109
cells/ml)(2
ml)
=

C2(15
ml)



C2
=
4
x
109
cells/15
ml
=
2.67
x
108
cells/ml





4. Diagram a scheme to make a 1:10,000 dilution



















5. Diagram a scheme to make a 1:7.2 dilution





6. You perform the following series of dilutions: 1:10, 1:3, 1:2. What is your total dilution? What is the
DF?
































7. After diluting your culture 1:5000, you have a cell concentration of 2.3 x 102 cells/ml. What was the initial
concentration?
C1
=
C2DF
=
(2.3
x
102
cells/ml)(5000)
=
1.5
x
106
cells/ml


8. After diluting your culture 1:2500, you plate 0.1 ml and get 154 colonies. What was the initial
concentration?
3.85 X 106 CFU/ml