Introduction (Plate Count)
The laboratory microbiologist often has to determine the number of bacteria in
a given sample, as well as having to compare the amount of bacterial growth
under various conditions. Enumeration of microorganisms is especially important
in dairy microbiology, food microbiology, and water microbiology.
Since the enumeration of microorganisms involves the use of extremely
small dilutions and extremely large numbers of cells, scientific notation is
routinely used in calculations.
The number of bacteria in a given sample is usually too great to be counted
directly. However, if the sample is serially diluted and then plated out on an
agar surface, single isolated bacteria can form visible isolated colonies.

Figure 26 Single isolated colonies obtained during the plate count. 
The number of colonies can be used as a measure of the number of viable
(living) cells in that known dilution. owever, keep in mind that if the organism
normally forms multiple cell arrangements, such as chains, the colonyforming
unit ay consist of a chain of bacteria rather than a single bacterium. In
addition, some of the bacteria may be clumped ogether. Therefore, when doing
the plate count technique, we generally say we are determining the number of
colonyforming units (CFUs) in that known dilution. By extrapolation, this
number can in turn be used to calculate the number of CFUs in the original
sample.
Normally, the bacterial sample is diluted by factors of 10 and plated on
agar. After incubation, the number of colonies on a dilution plate showing
between 30 and 300 colonies is determined. A plate having 30–300 colonies is
chosen, because this range is considered statistically significant. If there are less
than 30 colonies on the plate, small errors in dilution technique or the presence
of a few contaminants will have a drastic effect on the final count. Likewise,
if there are more than 300 colonies on the plate, there will be poor isolation and
colonies will have grown together.
Generally, one wants to determine the number of CFUs per milliliter (mL)
of sample. To find this, the number of colonies (on a plate having 30–300
colonies) is multiplied by the number of times the original mL of bacteria were
diluted (the dilution factor of the plate counted). For example, if a plate containing
a 1/1,000,000 dilution of the original mL of sample shows 150 colonies, then
150 represents 1/1,000,000 the number of CFUs present in the original mL.
Therefore, the number of CFUs per mL in the original sample is found by
multiplying 150 × 1,000,000, as shown in the formula below:
Number of CFUs per ml of sample = number of colonies (30–300 plate) ×
the dilution factor of the plate counted
In the case of the example above, 150 × 1,000,000 = 150,000,000 CFUs
per mL.
For a more accurate count, it is advisable to plate each dilution in duplicate
or triplicate and then find an average count.
Direct Microscopic Method (Total Cell Count)
In the direct microscopic count, a counting chamber consisting of a ruled slide
and a coverslip is employed. It is constructed in such a manner that a known
volume is delimited by the coverslip, slide, and ruled lines. The number of
bacteria in a small known volume is directly counted microscopically and the
number of bacteria in the larger original sample is determined by extrapolation.

Figure 27 Large doublelined square of a PetroffHausser counter.


Figure 28 PetroffHausser Counter as seen through a microscope. 
The doublelined “square” holding 1/20,000,000 cc is shown by the bracket.
The arrow shows a bacterium.
The square holds a volume of 1/20,000,000 of a cubic centimeter. Using a
microscope, the bacteria in the square are counted. For example, has squares
1/20 of a millimeter (mm) by 1/20 of a mm and is 1/50 of a mm deep. The
volume of 1 square, therefore, is 1/20,000 of a cubic mm or 1/20,000,000 of a
cubic centimeter (cc). The normal procedure is to count the number of bacteria
in 5 large doublelined squares and divide by 5 to get the average number of
acteria per large square. This number is then multiplied by 20,000,000, since
the square holds a volume of 1/20,000,000 cc, to find the total number of
organisms per cc in the original sample.
If the bacteria are diluted (such as by mixing with dye) before being placed
in the counting chamber, then this dilution must also be considered in the final
calculations.
The formula used for the direct microscopic count is:
number of bacteria per cc = 
the average number of bacteria per large
doublelined square × the dilution factor of
the large square (20,000,000) × the dilution
factor of any dilutions made prior to placing
the sample in the counting chamber, e.g.,
mixing the bacteria with dye. 
Turbidity
When we mix the bacteria growing in a liquid medium, the culture appears
turbid. This is because a bacterial culture acts as a colloidal suspension that blocks and
reflects light passing through the culture. Within limits, the light absorbed by the
bacterial suspension will be directly proportional to the concentration of cells in
the culture. By measuring the amount of light absorbed by a bacterial suspension,
one can estimate and compare the number of bacteria present.

Figure 29 A spectrophotometer. 
The instrument used to measure turbidity is a spectrophotometer.
It consists of a light source, a filter that allows only a single wavelength
of light to pass through, the sample tube containing the bacterial suspension,
and a photocell that compares the amount of light coming through the tube
with the total light entering the tube.
The ability of the culture to block the light can be expressed as either
percentage of light transmitted through the tube or amount of light absorbed in
the tube.

Figure 30 A spectrophotometer. 
The percentage of light transmitted is inversely proportional to the bacterial
concentration. (The greater the percent transmittance, the lower the number of
bacteria.) The absorbance (or optical density) is directly proportional to the cell
concentration. (The greater the absorbance, the greater the number of bacteria.)
Turbidimetric measurement is often correlated with some other method of
cell count, such as the direct microscopic method or the plate count. In this
way, turbidity can be used as an indirect measurement of the cell count. For
example:
 Several dilutions can be made of a bacterial stock.
 A PetroffHausser counter can then be used to perform a direct microscopic
count on each dilution.
 Then, a spectrophotometer can be used to measure the absorbance of each
dilution tube.
 A standard curve comparing absorbance to the number of bacteria can be
made by plotting absorbance versus the number of bacteria per cc.

Figure 31 A standard curve plotting the number
of bacteria per cc versus absorbance. 
 Once the standard curve is completed, any dilution tube of that organism
can be placed in a spectrophotometer and its absorbance read. Once the
absorbance is determined, the standard curve can be used to determine the
corresponding number of bacteria per cc.

Figure 32 Using a standard curve to determine the number
of bacteria per cc in a sample by measuring the sample’s absorbance. 
Materials: 6 tubes each containing 9.0 mL of sterile saline, 3 plates of
trypticase soy agar, 2 sterile 1.0mL pipettes, pipette filler, turntable, bent glass
rod, dish of alcohol.
Organism: Trypticase soy broth culture of Escherichia coli.
Procedure
Plate Count
 Take 6 dilution tubes, each containing 9.0 mL of sterile saline. Aseptically
dilute 1.0 mL of a sample of E. coli, as shown in and described as follows:

Figure 33 Plate count dilution procedure. 
 Remove a sterile 1.0mL pipette from the bag. Do not touch the portion
of the pipette tips that will go into the tubes and do not lay the pipette
down. From the tip of the pipette to the “0” line is 1 mL; each numbered
division (0.1, 0.2, etc.) represents 0.1 mL.
 Insert the cottontipped end of the pipette into a blue 2mL pipette filler.
 Flame the sample flask, insert the pipette to the bottom of the flask, and
withdraw 1.0 mL (up to the “0” line of the sample) by turning the filler
knob towards you. Draw the sample up slowly so that it isn’t accidentally
drawn into the filler itself. Reflame and cap the sample.
 Flame the first dilution tube and dispense the 1.0 mL of sample into the
tube by turning the filler knob away from you. Draw the liquid up and
down in the pipette several times to rinse the pipette and help mix.
Reflame and cap the tube.
 Mix the tube thoroughly by either holding the tube in one hand and
vigorously tapping the bottom with the other hand or by using a vortex
mixer. This is to assure an even distribution of the bacteria throughout
the liquid.
 Using the same procedure, aseptically withdraw 1.0 mL from the first
dilution tube and dispense into the second dilution tube. Continue
doing this from tube to tube as shown in until the dilution is completed.
Discard the pipette in the biowaste disposal containers at the front of
the room and under the hood.
These pipetting and mixing techniques will be demonstrated by your
instructor.
 Using a new 1.0mL pipette, aseptically transfer 0.1 mL from each of the
last 3 dilution tubes onto the surface of the corresponding plates of trypticase
soy agar as shown in figure. Note that since only 0.1 mL of the bacterial
dilution (rather than the desired 1.0 mL) is placed on the plate, the bacterial
dilution on the plate is 1/10 the dilution of the tube from which it came.
Using a turntable and sterile bent glass rod, immediately spread the solution
over the surface of the plates as follows:

Figure 34 Using a bent glass rod and a turntable
to spread a bacterial sample. 
 Place the plate containing the 0.1 mL of dilution on a turntable.
 Sterilize the glass rod by dipping the bent portion in a dish of alcohol
and igniting the alcohol with the flame from your burner. Let the flame
burn out.
 Place the bent portion of the glass rod on the agar surface and spin the
turntable for about 30 seconds to distribute the 0.1 mL of dilution
evenly over the entire agar surface.
 Replace the lid and resterilize the glass rod with alcohol and flaming.
 Repeat for each plate.
 Discard the pipette in the biowaste disposal containers at the front of
the room and under the hood.
 Incubate the 3 agar plates upside down at 37°C until the next lab period.
Place the used dilution tubes in the disposal baskets in the hood.
Direct Microscopic Method
 Pipette 1.0 mL of the sample of E. coli into a tube containing 1.0 mL of the
dye methylene blue. This produces a ½ dilution of the sample.
 Using a Pasteur pipette, fill the chamber of a PetroffHausser counting
chamber with this ½ dilution.
Turntable
 Place a coverslip over the chamber and focus on the squares using 400X
(40X objective).
 Count the number of bacteria in 5 large doublelined squares. For those
organisms on the lines, count those on the left and upper lines, but not
those on the right and lower lines. Divide this total number by 5 to find
the average number of bacteria per large square.
 Calculate the number of bacteria per cc as follows:
Number of bacteria per cc = 
the average number of bacteria per large
square × the dilution factor of the large
square (20,000,000) × the dilution factor of
any dilutions made prior to placing the
sample in the counting chamber, such as
mixing it with dye (2 in this case). 
Turbidity
Your instructor will set up a spectrophotometer demonstration illustrating that
as the number of bacteria in a broth culture increases, the absorbance increases
(or the percent of light transmitted decreases).
Results
Plate Count
 Choose a plate that appears to have between 30 and 300 colonies.
 Sample 1/100,000 dilution plate
 Sample 1/1,000,000 dilution plate
 Sample 1/10,000,000 dilution plate.
 Count the exact number of colonies on that plate using the colony counter
(as demonstrated by your instructor).
 Calculate the number of CFUs per mL of original sample as follows:
Number of CFUs per mL of sample = 
Number of colonies (30–300 plate)
× the dilution factor of the plate
counted 
____________
= Number of colonies
____________
= Dilution factor of plate counted
____________
= Number of CFUs per mL.
 Record your results on the blackboard.
Direct Microscopic Method
Observe the demonstration of the PetroffHausser counting chamber.
Turbidity
Observe your instructor’s demonstration of the spectrophotometer.
Performance Objectives
Discussion
 Provide the formula for determining the number of CFUs per mL of sample
when using the plate count technique.
 When given a diagram of a plate count dilution and the number of colonies
on the resulting plates, choose the correct plate for counting, determine the
dilution factor of that plate, and calculate the number of CFUs per mL in
the original sample.
 Plate count practice problems
 A sample of E.coli is diluted according to the above diagram. The
number of colonies that grew is indicated on the petri plates. How
many CFUs are there per mL in the original sample?

Figure 35 Plate count: Practice problem A. 
The correct dilutions are shown on the tubes and plates above.
The formula to be used is:
Number of CFUs per mL of sample = 
Number of colonies (30–300 plate)
× the dilution factor of the plate
counted 
 First choose the correct plate to count, that is, one with between 30 and
300 colonies.
 The correct plate is the one having 63 colonies on the 1/1,000,000
or 10^{–6} dilution.
 Multiply 63 by the dilution factor of that plate.
 Since the dilution factor is 1/1,000,000 or 10^{–6}, the dilution factor or
inverse is 1,000,000, or 10^{6}.
 63 × 1,000,000 = 63,000,000 CFUs per ml (6.3 × 10^{7} in scientific notation.)

Figure 36 Plate count: Answer to practice problem A. 
 A sample of E.coli is diluted according to the above diagram. The number
of colonies that grew is indicated on the petri plates. How many CFUs are
there per mL in the original sample?

Figure 37 Plate count: Practice problem B. 
The correct dilutions are shown on the tubes and plates above.
The formula to be used is:
Number of CFUs per mL of sample = 
Number of colonies (30–300 plate)
× the dilution factor of the plate
counted 
 First choose the correct plate to count, that is, one with between 30 and
300 colonies.
 The correct plate is the one having 161 colonies on the 1/1,000,000
or 10^{–6} dilution.
 Multiply 161 by the dilution factor of that plate.
 Since the dilution factor is 1/1,000,000 or 10^{–6}, the dilution factor or
inverse is 1,000,000, or 10^{6}.
 161 × 1,000,000 =161,000,000 CFUs per mL (1.61 × 10^{8} in scientific
notation.)

Figure 38 Plate count: Answer to practice problem B. 
 Explain the principle behind the direct microscopic method of enumeration.
 Provide the formula for determining the number of bacteria per cc of
sample when using the direct microscopic method of enumeration.
 When given the total number of bacteria counted in a PetroffHausser
chamber, the total number of large squares counted, and the dilution of the
bacteria placed in the chamber, calculate the total number of bacteria per
cc in the original sample.
 Describe the function of a spectrophotometer.
 Explain the relationship between absorbance (optical density) and the
number of bacteria in a broth sample.
 Explain the relationship between percent of light transmitted and the
number of bacteria in a broth sample.
Procedure
Perform a serial dilution of a bacterial sample, according to instructions in the
lab manual, and plate out samples of each dilution using the spinplate technique.
Results
Using a colony counter, count the number of colonies on a plate showing
between 30 and 300 colonies and, by knowing the dilution of this plate, calculate
the number of CFUs per mL in the original sample.