Bacteria Enumeration

1
Bacteria Enumeration
IF YOU CAN SEE THIS MESSAGE YOU ARE NOT IN SLIDE
SHOW MODE. PERFOMING THE LAB IN THIS MODE WILL
NOT ALLOW FOR THE ANIMATIONS AND INTERACTIVITY OF
THE EXERCISE TO WORK PROPERLY. TO CHANGE TO
SLIDE SHOW MODE YOU CAN CLICK ON VIEW AT THE
TOP OF THE PAGE AND SELECT SLIDE SHOW FROM THE
PULL DOWN MENU. YOU CAN ALSO JUST HIT THE F5
KEY.
Instructor Terry Wiseth
2
Click on the blackboard to view a larger board
for discussion.
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3
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 ofbacteria require a dye to be introduced
to thepopulations of bacteria to allow the
observerto view the bacteria.
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4
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.
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pH 9
pH 11
pH 5
pH 3
Viable Plate Count
Direct Count
End Lab
Turbidity Count
5
VIABLE PLATE COUNT
6
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.
Agar Plates
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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.
Agar Plates
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8
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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9
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
page150 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).
 
Agar Plates
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10
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
fountain3) Water from the local river4) 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.
Agar Plates
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pH 9
pH 11
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pH 3
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 37C for 48 hours.6) Choose a plate
that appears to have between 30 and 300 colonies.
 
Agar Plates
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7) Count the exact number of colonies on that
plate8) Calculate the number of CFUs per ml of
original sample as follows CFUs per ml of
sample The of coloniesXThe 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.
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Sterile Dilution Tubes
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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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Sterile Dilution Tubes
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Sample 1 Fountain Water
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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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Sterile Dilution Tubes
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Sample 1 Fountain Water
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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 Tubes
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10,000
100,000
1,000,000
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Click on the agar plates to place them in the
incubator at 37 0C 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.
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Lactose Broth Culture Tubes
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Eye Droppers
19
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.
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Water Samples
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1) Fountain Water2) Boiled Fountain Water3)
River Water4) Boiled River Water
Bunsen burner
Eye Droppers
20
Sample 1Viable Plate Count
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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.
Agar Plates
Loops
Swabs
Antiseptic Dispenser
Lactose Broth Culture Tubes
Water Samples
Pencil
Bunsen burner
Eye Droppers
1) Fountain Water2) Boiled Fountain Water3)
River Water4) Boiled River Water
Click here if you have viewed all the agar plates
from all four of the samples
22
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. The dilution factor for the plate
you are viewing is 10,000.
Agar Plates
Loops
Swabs
Antiseptic Dispenser
Lactose Broth Culture Tubes
Water Samples
Pencil
Bunsen burner
Eye Droppers
Click here if you have viewed all the agar plates
from all four of the samples
23
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. The dilution factor for the plate
you are viewing is 100,000.
Agar Plates
Loops
Swabs
Antiseptic Dispenser
Lactose Broth Culture Tubes
Water Samples
Pencil
Bunsen burner
Eye Droppers
Click here if you have viewed all the agar plates
from all four of the samples
24
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. The dilution factor for the plate
you are viewing is 1,000,000.
Agar Plates
Loops
Swabs
Antiseptic Dispenser
Lactose Broth Culture Tubes
Water Samples
Pencil
Bunsen burner
Eye Droppers
Click here if you have viewed all the agar plates
from all four of the samples
25
Sample 2Viable Plate Count
26
You are viewing the agar plates that were
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.
Agar Plates
Loops
Swabs
Antiseptic Dispenser
Lactose Broth Culture Tubes
Water Samples
Pencil
Bunsen burner
Eye Droppers
1) Fountain Water2) Boiled Fountain Water3)
River Water4) Boiled River Water
Click here if you have viewed all the agar plates
from all four of the samples
27
You are viewing the agar plates that were
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. The dilution factor for the plate
you are viewing is 10,000.
Agar Plates
Loops
Swabs
Antiseptic Dispenser
Lactose Broth Culture Tubes
Water Samples
Pencil
Bunsen burner
Eye Droppers
Click here if you have viewed all the agar plates
from all four of the samples
28
You are viewing the agar plates that were
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. The dilution factor for the plate
you are viewing is 100,000.
Agar Plates
Loops
Swabs
Antiseptic Dispenser
Lactose Broth Culture Tubes
Water Samples
Pencil
Bunsen burner
Eye Droppers
Click here if you have viewed all the agar plates
from all four of the samples
29
You are viewing the agar plates that were
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. The dilution factor for the plate
you are viewing is 1,000,000.
Agar Plates
Loops
Swabs
Antiseptic Dispenser
Lactose Broth Culture Tubes
Water Samples
Pencil
Bunsen burner
Eye Droppers
Click here if you have viewed all the agar plates
from all four of the samples
30
Sample 3Viable Plate Count
31
You are viewing the agar plates that were
inoculated with RIVER 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.
Agar Plates
Loops
Swabs
Antiseptic Dispenser
Lactose Broth Culture Tubes
Water Samples
Pencil
Bunsen burner
Eye Droppers
1) Fountain Water2) Boiled Fountain Water3)
River Water4) Boiled River Water
Click here if you have viewed all the agar plates
from all four of the samples
32
You are viewing the agar plates that were
inoculated with RIVER 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. The dilution factor for the plate
you are viewing is 10,000.
Agar Plates
Loops
Swabs
Antiseptic Dispenser
Lactose Broth Culture Tubes
Water Samples
Pencil
Bunsen burner
Eye Droppers
Click here if you have viewed all the agar plates
from all four of the samples
33
You are viewing the agar plates that were
inoculated with RIVER 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. The dilution factor for the plate
you are viewing is 100,000.
Agar Plates
Loops
Swabs
Antiseptic Dispenser
Lactose Broth Culture Tubes
Water Samples
Pencil
Bunsen burner
Eye Droppers
Click here if you have viewed all the agar plates
from all four of the samples
34
You are viewing the agar plates that were
inoculated with RIVER 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. The dilution factor for the plate
you are viewing is 1,000,000.
Agar Plates
Loops
Swabs
Antiseptic Dispenser
Lactose Broth Culture Tubes
Water Samples
Pencil
Bunsen burner
Eye Droppers
Click here if you have viewed all the agar plates
from all four of the samples
35
Sample 4Viable Plate Count
36
You are viewing the agar plates that were
inoculated with BOILED RIVER 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.
Agar Plates
Loops
Swabs
Antiseptic Dispenser
Lactose Broth Culture Tubes
Water Samples
Pencil
Bunsen burner
Eye Droppers
1) Fountain Water2) Boiled Fountain Water3)
River Water4) Boiled River Water
Click here if you have viewed all the agar plates
from all four of the samples
37
You are viewing the agar plates that were
inoculated with BOILED RIVER 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. The dilution factor for the plate
you are viewing is 10,000.
Agar Plates
Loops
Swabs
Antiseptic Dispenser
Lactose Broth Culture Tubes
Water Samples
Pencil
Bunsen burner
Eye Droppers
Click here if you have viewed all the agar plates
from all four of the samples
38
You are viewing the agar plates that were
inoculated with BOILED RIVER 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. The dilution factor for the plate
you are viewing is 100,000.
Agar Plates
Loops
Swabs
Antiseptic Dispenser
Lactose Broth Culture Tubes
Water Samples
Pencil
Bunsen burner
Eye Droppers
Click here if you have viewed all the agar plates
from all four of the samples
39
You are viewing the agar plates that were
inoculated with BOILED RIVER 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. The dilution factor for the plate
you are viewing is 1,000,000.
Agar Plates
Loops
Swabs
Antiseptic Dispenser
Lactose Broth Culture Tubes
Water Samples
Pencil
Bunsen burner
Eye Droppers
Click here if you have viewed all the agar plates
from all four of the samples
40
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.
Agar Plates
pH 7
pH 9
pH 11
pH 5
pH 3
Viable Plate Count
Direct Count
End Lab
Turbidity Count
41
Direct Count
42
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.
Agar Plates
pH 7
pH 9
pH 11
pH 5
pH 3
43
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.
Agar Plates
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pH 9
pH 11
pH 5
pH 3
Petroff-Hausser counting chamber
44
Agar Plates
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pH 9
pH 11
pH 5
pH 3
The Petroff-Hausser counting chamber as viewed
through low power of the microscope
45
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)
Agar Plates
pH 7
pH 9
pH 11
pH 5
pH 3
46
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.
Agar Plates
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pH 9
pH 11
pH 5
pH 3
DATA TABLE 2
47
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 squareXThe dilution factor
of the large square (1,250,000)XThe dilution
factor after mixing it with dye (2 in this case)
Agar Plates
pH 7
pH 9
pH 11
pH 5
pH 3
48
Agar Plates
pH 7
pH 9
pH 11
pH 5
pH 3
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.  
49
Data Table 2Direct Count Data Table 2Direct Count Data Table 2Direct Count Data Table 2Direct Count Data Table 2Direct Count Data Table 2Direct Count
Sample of Bacteria Dilution Factor(Large Square) DilutionFactor (Dye) DF (large square) XDF (Dye) X of Colonies ofBacteria / 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) bacteria per ml    of bacteria in square  X dilution factor (Large Square) (1,250,000)  X dilution factor (dye) bacteria per ml    of bacteria in square  X dilution factor (Large Square) (1,250,000)  X dilution factor (dye) bacteria per ml    of bacteria in square  X dilution factor (Large Square) (1,250,000)  X dilution factor (dye) bacteria per ml    of bacteria in square  X dilution factor (Large Square) (1,250,000)  X dilution factor (dye) bacteria per ml    of bacteria in square  X dilution factor (Large Square) (1,250,000)  X dilution factor (dye)
Agar Plates
pH 7
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pH 11
pH 5
pH 3
Printable Version ofDATA TABLE 2
50
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.
Agar Plates
Microscope
Methylene Blue
Loops
Antiseptic Dispenser
Slides
Sterile Dilution Tubes
Water Samples
Pencil
Bunsen burner
Eye Droppers
1) Fountain Water2) Boiled Fountain Water3)
River Water4) Boiled River Water
51
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.
Agar Plates
Microscope
Methylene Blue
Loops
Antiseptic Dispenser
Slides
Sterile Dilution Tubes
Water Samples
Pencil
1) Fountain Water2) Boiled Fountain Water3)
River Water4) Boiled River Water
Bunsen burner
Eye Droppers
Click Here if You Have Viewed All of the Water
Samples
52
Water Sample 1
53
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.
Agar Plates
Microscope
Methylene Blue
Loops
Antiseptic Dispenser
Slides
Sterile Dilution Tubes
Water Samples
Pencil
Bunsen burner
Eye Droppers
54
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).
Agar Plates
Microscope
Methylene Blue
Loops
Antiseptic Dispenser
Slides
Sterile Dilution Tubes
Water Samples
Pencil
Bunsen burner
Eye Droppers
Click Here to View a Different Water Sample
55
Water Sample 2
56
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 2 to the slide.
Click on NEXT when you have added the sample to
the slide on the microscope.
Agar Plates
Microscope
Methylene Blue
Loops
Antiseptic Dispenser
Slides
Sterile Dilution Tubes
Water Samples
Pencil
Bunsen burner
Eye Droppers
57
You are viewing bacteria from Sample 2 (Boiled
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).
Agar Plates
Microscope
Methylene Blue
Loops
Antiseptic Dispenser
Slides
Sterile Dilution Tubes
Water Samples
Pencil
Bunsen burner
Eye Droppers
Click Here to View a Different Water Sample
58
Water Sample 3
59
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 3 to the slide.
Click on NEXT when you have added the sample to
the slide on the microscope.
Agar Plates
Microscope
Methylene Blue
Loops
Antiseptic Dispenser
Slides
Sterile Dilution Tubes
Water Samples
Pencil
Bunsen burner
Eye Droppers
60
You are viewing bacteria from Sample 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).
Agar Plates
Microscope
Methylene Blue
Loops
Antiseptic Dispenser
Slides
Sterile Dilution Tubes
Water Samples
Pencil
Bunsen burner
Eye Droppers
Click Here to View a Different Water Sample
61
Water Sample 4
62
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 4 to the slide.
Click on NEXT when you have added the sample to
the slide on the microscope.
Agar Plates
Microscope
Methylene Blue
Loops
Antiseptic Dispenser
Slides
Sterile Dilution Tubes
Water Samples
Pencil
Bunsen burner
Eye Droppers
63
You are viewing bacteria from Sample 4 (Boiled
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).
Agar Plates
Microscope
Methylene Blue
Loops
Antiseptic Dispenser
Slides
Sterile Dilution Tubes
Water Samples
Pencil
Bunsen burner
Eye Droppers
Click Here to View a Different Water Sample
64
Turbidity Count
65
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.
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pH 3
The Spectrophotometer used to analyze turbidity
of bacteria
66
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.
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pH 9
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pH 3
A Description of How the Spectrophotometer Works
67
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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pH 7
pH 9
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pH 5
pH 3
68
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.
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pH 7
pH 9
pH 11
pH 5
pH 3
A Standard Curve Chart For Bacterial Count
69
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.
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pH 7
pH 9
pH 11
pH 5
pH 3
Click Here for a Printable Version of DATA TABLE 3
Click Here for a Printable Version of the
STANDARD CURVE CHART
70
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.
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Turbidity dilution tubes
71
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.  
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pH 9
pH 11
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pH 3
Spectrophotometer
72
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.  
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pH 9
pH 11
pH 5
pH 3
Click Here for a Printable Version of DATA TABLE 3
Click Here for a Printable Version of the
STANDARD CURVE CHART
73
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.
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pH 9
pH 11
pH 5
pH 3

SAMPLE NAME EXAMPLE SAMPLE NAME EXAMPLE SAMPLE NAME EXAMPLE SAMPLE NAME EXAMPLE SAMPLE NAME EXAMPLE
Dilutions Absorbance of Bacteria DilutionFactor 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 Total 131,400,000 Total 131,400,000 Total 131,400,000 Total 131,400,000
Average of Bacterial Cells per ml (Total / 5)      26,306,280 bacteria per ml Average of Bacterial Cells per ml (Total / 5)      26,306,280 bacteria per ml Average of Bacterial Cells per ml (Total / 5)      26,306,280 bacteria per ml Average of Bacterial Cells per ml (Total / 5)      26,306,280 bacteria per ml Average of Bacterial Cells per ml (Total / 5)      26,306,280 bacteria per ml
EXAMPLE DATA TABLE 3TURBIDITY COUNT  
74
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pH 5
pH 3
Standard Curve for Bacterial Count
Click Here for a Printable Version of the
STANDARD CURVE CHART
75
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.
Spectrophotometer
Loops
Swabs
Antiseptic Dispenser
Sterile Dilution Tubes
Water Samples
Pencil
Bunsen burner
Eye Droppers
76
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.
Spectrophotometer
Loops
Swabs
Antiseptic Dispenser
Sterile Dilution Tubes
Water Samples
Pencil
Bunsen burner
Eye Droppers
1/8
1/4
1/2
77
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.
Spectrophotometer
Loops
Swabs
Antiseptic Dispenser
Sterile Dilution Tubes
Water Samples
Pencil
Bunsen burner
Eye Droppers
0.000
520
78
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
Loops
Swabs
Antiseptic Dispenser
Sterile Dilution Tubes
Water Samples
Pencil
Bunsen burner
Eye Droppers
0.000
0.044
0.023
0.014
0.008
0.005
520
79
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.
Spectrophotometer
Loops
Swabs
Antiseptic Dispenser
Sterile Dilution Tubes
Water Samples
Pencil
Bunsen burner
Eye Droppers
1/8
1/4
1/2
80
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.
Spectrophotometer
Loops
Swabs
Antiseptic Dispenser
Sterile Dilution Tubes
Water Samples
Pencil
Bunsen burner
Eye Droppers
0.000
520
81
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
Loops
Swabs
Antiseptic Dispenser
Sterile Dilution Tubes
Water Samples
Pencil
Bunsen burner
Eye Droppers
0.000
0.121
0.073
0.035
0.018
0.010
520
82
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.
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Viable Plate Count
End Lab
Direct Count
Turbidity Count
83
STUFF
84
MODE
READ
Transmittance
722-2000
Absorbance
520
SPECTROPHOTOMETER
85
(No Transcript)
86
STUFF






87
Boiled River Water
88
River Water
89
Faucet Water
90
Boiled Faucet Water
91
Mouth Culture