Bacteria enumeration

Instructor Terry Wiseth
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YOU CAN CLICK ON “VIEW” AT THE TOP OF THE
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KEY.

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Click on the blackboard
to view a larger board for
discussion.Loops
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Bunsen burner
Microbe Samples Pencil
SwabsAntiseptic Dispenser

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ENUMERATION OF BACTERIA
As part of daily routine, 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. Knowing the bacterial count in
drinking water, fresh milk, buttermilk, yogurt, can be useful in
many aspects of industrial microbiology. Bacteria are so small
and numerous, counting them directly can be very difficult.
Some of the methods used involve diluting the sample to a
point at which the number of bacteria has been reduced to very
small numbers. This enables an estimate to be established for
quantifying the bacteria. Direct counts of
bacteria require a dye to be introduced to the
populations of bacteria to allow the observer
to view the bacteria.

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Observe the three links given below to bring
you to the VIRTUAL LAB that you wish to
perform. If you have performed all of the
exercises, you can click on END LAB.
Viable Plate Count
Direct Count End Lab
Turbidity Count

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VIABLE PLATE COUNT
Viable Plate Count (also called a Standard Plate Count) is one
of the most common methods, for enumeration of bacteria.
Serial dilutions of bacteria are plated onto an agar plate.
Dilution procedure influences overall counting process. The
suspension is spread over the surface of growth medium. The
plates are incubated so that colonies are formed. Multiplication
of a bacterium on solid media results in the formation of a
macroscopic colony visible to naked eye. It is assumed that
each colony arises from an individual viable cell. Total number
of colonies is counted and this number multiplied by the
dilution factor to find out concentration of cells in the original
sample. Counting plates should have 30-300 colonies at least.
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.

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A major limitation in this method is selectivity. The
nature of the growth medium and the incubation
conditions determine which bacteria can grow and thus
be counted. Viable counting measures only those cells
that are capable of growth on the given medium under
the set of conditions used for incubation. Sometimes
cells are viable but non-culturable.
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 in
such a manner that single isolated bacteria form visible
isolated colonies, the number of colonies can be used as
a measure of the number of viable (living) cells in that
known dilution. The viable plate count method is an
indirect measurement of cell density and reveals
information related only to live bacteria.

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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 (colony forming units) 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 was 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 x 1,000,000
as shown in the formula given on the next page.

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CFUs per ml of sample =
The # of colonies X The dilution factor of the plate counted
In the case of the example given on the previous page:
150 x 1,000,000 = 150,000,000 CFUs per ml
At the end of the incubation period, select all of the agar plates
containing between 30 and 300 colonies. Plates with more than
300 colonies cannot be counted and are designated too
numerous to count (TNTC). Plates with fewer than 30 colonies
are designated too few to count (TFTC).

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PROCEDURE: VIABLE PLATE COUNT
We will be testing four samples of water for the Viable Count.
The samples include:
1) Water from a drinking fountain
2) Boiled water from a drinking fountain
3) Water from the local river
4) Boiled water from the local river
You will need DATA TABLE 1 to input your data and calculate
the number of CFU per ml. Use the link given below to access a
printable version of DATA TABLE 1.
DATA TABLE 1

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1) Take 6 dilution tubes, each containing 9 ml of sterile saline.
2) Dilute 1 ml of a sample by withdrawing 1 ml of the sample
and dispensing this 1 ml into the first dilution tube.
3) Using the same procedure, withdraw 1 ml from the first
dilution tube and dispense into the second dilution tube.
Subsequently withdraw 1 ml from the second dilution tube and
dispense into the third dilution tube. Continue doing this from
tube to tube until the dilution is completed.

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4) Transfer 1 ml from each of only the last three dilution tubes
onto the surface of the corresponding agar plates.
5) Incubate the agar plates at 37°C for 48 hours.
6) Choose a plate that appears to have between 30 and 300
colonies.

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7) Count the exact number of colonies on that plate
8) Calculate the number of CFUs per ml of original sample as
follows:
CFUs per ml of sample
=
The # of colonies
X
The dilution factor of the plate counted

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Click on the DILUTION TUBE rack of
test tubes to bring them to the table.
Each of the dilution tubes contain 9 ml
of sterile saline solution. Next Click on
the WATER SAMPLES to bring the
samples to the table. Now Click on the
Eye Droppers to withdraw 1 ml of
sample #1 (Fountain Water) and
dispense this to the first dilution tube.
Click on NEXT when this initial transfer
is finished.
LoopsSwabsAntiseptic Dispenser
Bunsen burnerEye Droppers
Sterile Dilution TubesWater Samples Pencil
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Sample #1 Fountain Water

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Click again on the EYE DROPPER to
withdraw 1 ml from the first dilution
tube and dispense into the second
dilution tube and subsequently
withdraw 1 ml from the second dilution
tube and dispense into the third dilution
tube. Continue doing this from tube to
tube until the dilution is completed
through dilution tube #6. Click on NEXT
when the dilutions are complete.
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6
54
1
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The dilutions for each of the 6 dilutions
tubes can be summarized in the image
below. Dilution tube #1 has a 1/10 dilution
with a dilution factor of 10. The dilution
factor for each of the tubes is listed below.
Tube #1 = 10
Tube #2 = 100
Tube #3 = 1000
Tube #4 = 10,000
Tube #5 = 100,000
Tube #6 = 1,000,000
Click on NEXT when you are ready for the
next step in the exercise
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Next we will be inoculating agar plates with
the last three broth culture dilutions. Click
on the agar plates on the shelf to bring them
to the table. Now click on the EYE
DROPPERS to transfer 1 ml of dilution #4 to
plate # 1, 1 ml of dilutions #5 to plate #2 and
1 mil of dilution #6 to plate #3. Next click on
the pencil to label agar plate #1 with a
dilution factor 10,000; plate #2 with a dilution
factor 100,000 and plate #3 with a dilution
factor 1,000,000. Click on NEXT when
finished.
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Lactose Broth Culture TubesWater Samples Pencil
4
1
2
3 4
1
2
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6
5
Eye Droppers
10,000 100,000 1,000,000

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Click on the agar plates to place them in the
incubator at 37 0
C for 48 hours. We will now
need to perform these same dilution and
inoculation steps for each of the test
samples. The process is the same for each
sample and we will assume the process of
dilution and inoculation has been completed
for all four of the water samples and the 48
hours of incubation time has now been
completed. Click on NEXT when you are
ready to view the incubated plates.
Bunsen burner
4
1
2
3 4
1
2
3
6
5
Eye Droppers
10,000 100,000 1,000,000

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Click on the incubator to bring all of the
inoculated agar plates to the table. Each of
the groups of inoculated plates is labeled
with the source of their respective samples.
A key for the sample #s is given below. Click
on one of the sample groups to view the
bacterial growth of the individual dilutions.
Bunsen burner
Eye Droppers
#1 #2 #3 #4
1) Fountain Water
2) Boiled Fountain Water
3) River Water
4) Boiled River Water

Sample 1
Viable Plate Count
10,000
100,000
1,000,000

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You are viewing the agar plates that were
inoculated with FOUNTAIN WATER. Click on
each of the three inoculated agar plates to
view the bacterial colony growth. Count the
number of colonies that are present and
enter the data in DATA TABLE 1. If the count
is less than 30 colonies, the notation will be
“TFTC”. If the count is more than 300
colonies, the notation will be “TNTC”.
Bunsen burner
Eye Droppers
#2 #3 #4
1) Fountain Water
3) River Water
10,000 100,000 1,000,000
#1
Click here if you have
viewed all the agar plates
from all four of the
samples

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The dilution factor for the plate
you are viewing is 10,000.
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Eye Droppers
#2 #3 #4
10,000 100,000 1,000,000
10,000
samples

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Eye Droppers
#2 #3 #4
10,000 100,000 1,000,000
100,000
samples

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you are viewing is 1,000,000.
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Eye Droppers
#2 #3 #4
10,000 100,000 1,000,000
1,000,000
samples

Sample 2
Viable Plate Count
1,000,000
100,000
10,000

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inoculated with BOILED FOUNTAIN WATER.
Click on each of the three inoculated agar
plates to view the bacterial colony growth.
Count the number of colonies that are
present and enter the data in DATA TABLE 1.
If the count is less than 30 colonies, the
notation will be “TFTC”. If the count is more
than 300 colonies, the notation will be
“TNTC”.
Bunsen burner
Eye Droppers
#2 #3 #4
1) Fountain Water
3) River Water
10,000 100,000 1,000,000
#1
samples

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“TNTC”.
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Eye Droppers
#1 #3 #4
10,000 100,000 1,000,000
10,000
samples

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“TNTC”.
Bunsen burner
Eye Droppers
#1 #3 #4
10,000 100,000 1,000,000
100,000
samples

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“TNTC”.
Bunsen burner
Eye Droppers
#1 #3 #4
10,000 100,000 1,000,000
1,000,000
samples

Sample 3
Viable Plate Count
10,000
1,000,000
100,000

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inoculated with RIVER WATER. Click on
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Eye Droppers
#2 #3 #4
1) Fountain Water
3) River Water
10,000 100,000 1,000,000
#1
samples

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Bunsen burner
Eye Droppers
#1 #2 #4
10,000 100,000 1,000,000
10,000
samples

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Bunsen burner
Eye Droppers
#1 #2 #4
10,000 100,000 1,000,000
100,000
samples

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Bunsen burner
Eye Droppers
#1 #2 #4
10,000 100,000 1,000,000
1,000,000
samples

Sample 4
Viable Plate Count
1,000,000
100,000
10,000

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inoculated with BOILED RIVER WATER.
“TNTC”.
Bunsen burner
Eye Droppers
#2 #3 #4
1) Fountain Water
3) River Water
10,000 100,000 1,000,000
#1
samples

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“TNTC”.
Bunsen burner
Eye Droppers
#1 #2 #3
10,000 100,000 1,000,000
10,000
samples

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“TNTC”.
Bunsen burner
Eye Droppers
#1 #2 #3
10,000 100,000 1,000,000
100,000
samples

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“TNTC”.
Bunsen burner
Eye Droppers
#1 #2 #3
10,000 100,000 1,000,000
1,000,000
samples

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Observe the three links given below to bring
you to the VIRTUAL LAB that you wish to
perform. If you have performed all of the
exercises, you can click on END LAB.
Viable Plate Count
Direct Count
Turbidity Count
End Lab

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DIRECT MICROSCOPIC COUNT
In the direct microscopic count, a counting chamber with a
ruled slide is employed. It is constructed in such a manner that
the ruled lines define a known volume. 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.

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The Petroff-Hausser counting chamber for example, has small
etched squares 1/20 of a millimeter (mm) by 1/20 of a mm and
is 1/50 of a mm deep. The volume of one small square therefore
is 1/20,000 of a cubic mm or 1/20,000,000 of a cubic centimeter
(cc). There are 16 small squares in the large double-lined
squares that are actually counted, making the volume of a large
double-lined square 1/1,250,000 cc. The normal procedure is to
count the number of bacteria in five large double-lined squares
and divide by five to get the average number of bacteria per
large square. This number is then multiplied by 1,250,000 since
the square holds a volume of 1/1,250,000 cc, to find the total
number of organisms per ml in the original sample.
Petroff-Hausser
counting chamber

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The Petroff-Hausser counting chamber as viewed through low
power of the microscope

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If the bacteria are diluted, such as by mixing the bacteria 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:
# bacteria per cc (ml)
=
# of bacteria per large square
X
dilution factor of large square (1,250,000)
X
dilution factor (dye)

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PROCEDURE: DIRECT MICROSCOPIC COUNT
We will be testing four samples of water for the Direct
Microscopic Count. The samples include:
1) water from a drinking fountain
2) boiled water from a drinking fountain
3) water from the local river
4) boiled water from the local river
You will need DATA TABLE 2 to input your data and calculate
the number of bacteria per ml. Click below to access a
printable version of Data Table 2.
DATA TABLE 2

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1) Add 1 ml of the sample into a tube containing 1 ml of the dye
methylene blue. This gives a 1/2 dilution of the sample.
2) Fill the chamber of a Petroff-Hausser counting chamber with
this 1/2 dilution.
3) Place the chamber on a microscope and focus on the
squares using 400X.
4) Count the number of bacteria in one of the large double-
lined squares. Count all organisms that are on or within the
lines.
5) Calculate the number of bacteria per cc (ml) as follows:
The number of bacteria per cc (ml)
=
The number of bacteria per large square
X
The dilution factor of the large square (1,250,000)
X
The dilution factor after mixing it with dye (2 in this case)

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The large, double-lined square holds a volume of 1/1,250,000 of
a cubic centimeter. Using a microscope, the bacteria in the
large square are counted. Count all organisms that are on or
within the darker double lines.

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Data Table 2
Direct Count
Sample
# of
Bacteria
Dilution Factor
(Large Square)
Dilution
Factor
(Dye)
DF (large square) X
DF (Dye) X # of Colonies
# of
Bacteria / ml
Faucet Water 1,250,000 2 1,250,000 X 2 X ______
River Water 1,250,000 2 1,250,000 X 2 X ______
Boiled Faucet Water 1,250,000 2 1,250,000 X 2 X ______
Boiled River Water 1,250,000 2 1,250,000 X 2 X ______
# bacteria per ml = # of bacteria in square X dilution factor (Large Square) (1,250,000) X dilution factor (dye)
Printable Version of
DATA TABLE 2

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Click on the WATER SAMPLES to bring
the samples to the table. Next, click on
the Methylene Blue bottle to bring the
dye to the table. Now Click on the top of
the Methylene Blue dye to withdraw 1
ml of the dye and dispense this to 1 ml
of each of the Water Samples. Click on
NEXT when dye has been added to all of
the Water Samples.
LoopsAntiseptic Dispenser
4
1
2
3
MicroscopeMethylene Blue
Slides
1) Fountain Water
3) River Water

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Click on any one of the numbered
WATER SAMPLES to add 1 ml of the
sample to the Petroff-Hausser counting
chamber for viewing and counting using
the microscope under 400 X. You will
need to view all four of the Water
Samples.
4
1
2
3
Slides
1) Fountain Water
3) River Water
Click Here if You
Have Viewed All of
the Water
Samples

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Click on the Microscope to bring it to
the table. Next click on the SLIDES to
bring one of them to the microscope.
Now click on the EYE DROPPERS to
transfer 1 ml of Water Sample #1 to the
slide. Click on NEXT when you have
added the sample to the slide on the
microscope.
4
1
2
3
Slides

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You are viewing bacteria from Sample
#1 (Fountain Water). Count all
organisms that are on or within the
darker double lines. Record your count
in TABLE 2. Calculate the number of
bacteria per ml. Click on the EYEPIECE
of the microscope to view the slide
under High Power (400 X).
4
1
2
3
Slides
Click Here to View
a Different Water
Sample

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microscope.
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1
2
3
Slides

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#2 (Boiled Fountain Water). Count all
4
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2
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Slides
Click Here to View
a Different Water
Sample

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microscope.
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1
2
3
Slides

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#3 (River Water). Count all organisms
that are on or within the darker double
lines. Record your count in TABLE 2.
Calculate the number of bacteria per ml.
Click on the EYEPIECE of the
microscope to view the slide under High
Power (400 X).
4
1
2
3
Slides
Click Here to View
a Different Water
Sample

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microscope.
4
1
2
3
Slides

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#4 (Boiled River Water). Count all
4
1
2
3
Slides
Click Here to View
a Different Water
Sample

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TURBIDITY COUNT
When you 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.
Spectrophotometric analysis is based on turbidity and
indirectly measures all bacteria (cell biomass), dead and alive.
The Spectrophotometer
used to analyze turbidity
of bacteria

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The instrument used to measure turbidity is a
spectrophotometer. It consists of a light source, a filter which
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 the amount of
light absorbed in the tube. The absorbance (or optical density)
is directly proportional to the cell concentration. (The greater
the absorbance, the greater the number of bacteria.) Light
entering a cloudy solution will be absorbed. A clear solution
will allow almost all of the light through.
A Description of How
the Spectrophotometer
Works

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The amount of absorbance measures what fraction of the light
passes through a given solution and indicates on the
absorbance display the amount of light absorbed compared to
that absorbed by a clear solution.
Inside, a light shines through a filter (which can be adjusted by
controlling the wavelength of light), then through the sample
and onto a light-sensitive phototube. This produces an
electrical current. The absorbance meter measures how much
light has been blocked by the sample and thereby prevented
from striking the phototube. A clear tube of water or other clear
solution is the BLANK and has zero absorbance. The amount
of substance in the solution is directly proportional to the
absorbance reading. A graph of absorbance vs. concentration
will produce a straight line. As the number of bacteria in a
broth culture increases, the absorbance increases.

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A standard curve comparing absorbance to the number of
bacteria can be made by plotting absorbance versus the
number of bacteria per ml. 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 ml.
A Standard Curve Chart
For Bacterial Count

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PROCEDURE: TURBIDITY COUNT
We will be testing only two samples of water for the
turbidity enumeration test. One of the samples has been
drawn from a drinking water faucet while the other was
taken from the local river. You will need DATA TABLE 3
and a printable version of the STANDARD CURVE
CHART to enumerate your samples bacteria. Click on
NEXT when you have the DATA TABLE 3 and
STANDARD CURVE CHART.
Click Here for a
Printable Version
of DATA TABLE 3
Click Here for a
Printable Version
of the STANDARD
CURVE CHART

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1) Place the ORIGINAL tube of the sample and four tubes of the
sterile broth in a test-tube rack. Each tube of broth contains 5
ml of sterile broth.
2) Use four of these tubes (tubes 2 to 5) of broth to make four
serial dilutions of the culture.
3) Transfer 5ml of the ORIGINAL sample to the first broth tube.
Transfer 5ml from that tube to the next tube, and so on until the
last of the four tubes has 5ml added to it. These tubes will be
1/2, 1/4, 1/8, and 1/16 dilutions.
Turbidity dilution tubes

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4) Set the display mode on the Spectrophotometer to
ABSORBANCE by pressing the MODE control key until the
appropriate red LED is lit.
5) Set the wavelength to 520 nm by using the WAVELENGTH
dial.
6) Standardize the spectrophotometer by using a BLANK. The
BLANK used to standardize the machine is sterile nutrient
broth: it is called the BLANK because it has a sample
concentration equal to zero (# of bacteria = 0).
7) Place the original bacterial specimen into the
spectrophotometer.
8) Next insert the 1/2 dilution and read it. Repeat this with the
1/4, 1/8, and 1/16 dilutions. Read to the nearest thousandth
(0.001) on the absorbance digital display.
Spectrophotometer

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9) Record your values in DATA TABLE 3 for each of the
individual samples, along with the dilutions that they came
from.
10) Using the standard curve table given below, calculate the
number of bacteria per milliliter for each dilution.
Click Here for a
Printable Version
of DATA TABLE 3
Click Here for a
Printable Version
of the STANDARD
CURVE CHART

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**Review the example of absorbance counts acquired and
the determinations of # of bacteria for the dilutions using
the STANDARD CURVE CHART given on the next page. Be
sure to keep track of all of the zeros in your calculations of
the subsequent calculations for average bacteria per ml.
SAMPLE NAME: EXAMPLE
Dilutions Absorbance # of Bacteria
Dilution
Factor
Dilution factor X Bacteria #
Original 0.130 26,000,000 1 1 X 26,000,000 = 26,000,000
1/2 0.066 12,900,000 2 2 X 12,900,000 = 25,800,000
1/4 0.034 6,500,000 4 4 X 6,500,000 = 26,000,000
1/8 0.018 3,200,000 8 8 X 3,200,000 = 25,600,000
1/16 0.010 1,750,000 16 16 X 1,750,000 = 28,000,000
Total = 131,400,000
Average # of Bacterial Cells per ml (Total / 5) = 26,306,280 bacteria per ml
EXAMPLE
DATA TABLE 3
TURBIDITY COUNT

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Standard Curve for
Bacterial Count
Click Here for a
Printable Version
of the STANDARD
CURVE CHART

Spectrophotometer
Click on the WATER SAMPLES to bring
the rack of samples to the table. There
are only two samples of water we will
test. Sample A is Faucet or Fountain
Water and Sample B is River Water.
Click on the STERILE DILUTION TUBES
to bring them to the table. Click on
NEXT when both of the test tube racks
are on the table.
A
B 1/8
1/2
1/4
1/16
Blank

Spectrophotometer
Next click on Sample A to move the sample to
the Dilution rack. Next we will perform the
dilution by transferring 5 ml of the original
sample to the first dilution tube labeled as 1/2.
Then we will transfer 5 ml of the 1/2 dilution to
the tube labeled as 1/4 and so on until the last
tube labeled as 1/16 has 5 ml added to it. Click
on the blue EYE DROPPERS to perform the
dilution. There is a tube labeled as BLANK
which contains only pure sterile broth with no
bacteria (population = 0). Click on NEXT when
the dilutions have been made.
A
B 1/8
1/2
1/4
1/16
A
Blank
A

Spectrophotometer
Click on the SPECTROPHOTOMETER to
bring the machine to the table. Next click on
the MODE button to set the machine to
ABSORBANCE mode. Then click on the DIAL
to set the wavelength to 520 nm. Click on the
BLANK to insert it into the
Spectrophotometer. Next click on READ to
view the ABSORBANCE for the BLANK. The
BLANK will read “0” as there are no bacteria
in the solution and thus no absorbance of
light. Click on NEXT when the BLANK has
been read.
1/8
1/2
1/4
1/16
A
SPECTROPHOTOMETER
722-2000
MODE
Absorbance
Transmittance
000
READ
520
0.000
Blank

Spectrophotometer
Click on one of the dilutions in the dilution
rack. Once the dilution has been inserted
into the Spectrophotometer, click on READ
to view ABSORBANCE for that dilution.
Record your value for the dilution that you
have selected in DATA TABLE 3 for
FOUNTAIN WATER. One at a time click on
each of the other dilutions and then click on
READ to view each of the ABSORBANCE
values for the individual dilutions. Click on
NEXT when you have viewed all of the
dilutions.
SPECTROPHOTOMETER
722-2000
MODE
Absorbance
Transmittance
000
READ
520
0.000
1/8
1/2
1/4
1/16
A
0.0440.0230.0140.0080.005

Spectrophotometer
Click on Sample B to move the sample to the
Dilution rack. Next we will perform the dilution
by transferring 5 ml of the original sample to
the first dilution tube labeled as 1/2. Then we
will transfer 5 ml of the 1/2 dilution to the tube
labeled as 1/4 and so on until the last tube
labeled as 1/16 has 5 ml added to it. Click on
the blue EYE DROPPERS to perform the
dilution. There is a tube labeled as BLANK
which contains only pure sterile broth with no
bacteria (population = 0). Click on NEXT when
the dilutions have been made.
A
B 1/8
1/2
1/4
1/16
B
Blank
B

Spectrophotometer
Click on the SPECTROPHOTOMETER to
bring the machine to the table. Next click on
the MODE button to set the machine to
ABSORBANCE mode. Then click on the DIAL
to set the wavelength to 520 nm. Click on the
BLANK to insert it into the
Spectrophotometer. Next click on READ to
view the ABSORBANCE for the BLANK. The
BLANK will read “0” as there are no bacteria
in the solution and thus no absorbance of
light. Click on NEXT when the BLANK has
been read.
1/8
1/2
1/4
1/16
B
SPECTROPHOTOMETER
722-2000
MODE
Absorbance
Transmittance
000
READ
520
0.000
Blank

Spectrophotometer
Click on one of the dilutions in the dilution
rack. Once the dilution has been inserted
into the Spectrophotometer, click on READ
to view ABSORBANCE for that dilution.
Record your value for the dilution that you
have selected in DATA TABLE 3 for
FOUNTAIN WATER. One at a time click on
each of the other dilutions and then click on
READ to view each of the ABSORBANCE
values for the individual dilutions. Click on
NEXT when you have viewed all of the
dilutions.
SPECTROPHOTOMETER
722-2000
MODE
Absorbance
Transmittance
000
READ
520
0.000
1/8
1/2
1/4
1/16
B
0.1210.0730.0350.0180.010

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You have now entered the Data required for DATA TABLE #3.
Calculate the number of bacteria for each of the two water
samples by using the formulas given.
If you have performed all of the enumeration exercises you can
click on END LAB given below. If you would like to review or
perform any of the other exercises for this lab click on the
appropriate link given below.
Viable Plate Count
Turbidity Count
End Lab
Direct Count

SPECTROPHOTOMETER
722-2000
MODE
Absorbance
Transmittance
000
READ
SPECTROPHOTOMETER
722-2000
MODE
Absorbance
Transmittance
520
READ

Mouth Culture
Square in which
bacteria are counted

Bacteria enumeration

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