Lab Four
Quantifying Bacteria & Population Growth
Plate count method
Viable vs. Microscope-Based Counting
Spectrophotometer-estimates
Fresh food and pasteurized milk typically should have low bacterial counts. Laboratory testing is
done for quality control and to ensure there was no contamination. As food products are handled and
stored, the bacterial concentrations may increase until it becomes a public health hazard. Pasteurized
Grade A milk is required to have less than 20,000 bacteria/ml, raw milk when sold could only contain
50,000 bacteria/ml, and ground beef may contain up to 50 million bacteria/gm. We will use the plate
count method to determine the number of bacteria in a sample. Because samples may contain
different levels of bacteria that differ by several orders of magnitude, samples of these foodstuffs
must be diluted. We will use serial dilutions plated out in order to achieve the desired range of
colonies per plate (50-500). The plate count method is a standard method to determine the quality or
contamination of a product. For pour plates, dilutions ranging from undiluted to 103 to 105 should be
plated.
Part One – Assay of bacteria in Milk (pasteurized and raw milk)
Remember to use aseptic technique when working with the milk, dilutions and agar.
Pasteurized Milk Protocol (since they should have less bacteria):
Work in pairs. Shake the milk 25X in an up and down motion to evenly mix up the milk.
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Label four empty plates with your initials, the date, specimen (milk), and dilution factor (1:10 or
1:100).
Take a 9 ml sterile dH2O.
Dilute the milk: pipet 1.0 ml milk into above dilution tube, vortex to mix.
Add 1.0 ml aliquots: pipet 1.0 ml into two plates (1:10)
Get another 9.0 ml sterile dH2O.
Pipet 1.0 mL milk from the 1: 10 dilution tube into above dilution tube, vortex to mix.
Add 1.0 ml aliquots: pipet 1.0 ml into two plates
Pick up a tube melted plate count agar, cooled to 45oC, (here in a 45oC water bath)
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ADD MELTED AGAR TO MAKE POUR PLATE: add about 15 ml
Swirl 20X to mix completely. Return flask of agar back into warm water before agar solidifies .
When the agar is solid, invert the plate and incubate 35o C for 48 hr.
Count colonies on the plates and calculate CFU per mL:
CFU on plate x dil'n factor (102) x aliquot factor (either 1/1.0 ml or 1/0.1 mL) = CFU/ mL milk
Example plate at left: 40 colonies on plate x 102 x 1/1.0 mL = 4000 CFU/mL of milk
Biology 318, Lab Four, 1
Protocol for raw milk
Work in pairs. Shake the milk 25X in an up and down motion to evenly mix up the milk.
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Label six empty plates with your initials, the date, specimen (milk), and dilution factor (1:100 or
1:1000 or 1:10000).
Take a 99 ml sterile dH2O.
Dilute the milk: pipet 1.0 ml milk into above dilution tube, vortex to mix.
Add aliquots: First pipet 1.0 ml into two plates (1:100) and then pipet 0.1 ml into two plates
(1:1000)
Get another 99 ml sterile dH2O.
Pipet 1.0 mL milk from 1: 100 dilution bottle into above dilution tube, vortex to mix.
Add 1.0 ml aliquots: Using a pipet, pipet 1.0 ml into two plates
Pick up a tube melted plate count agar, cooled to 45oC,
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ADD MELTED AGAR TO MAKE POUR PLATE: add about 15 ml
Swirl 20X to mix completely. Return flask of agar back into warm water before agar solidifies .
When the agar is solid, invert the plate and incubate 35o C for 48 hr.
Count colonies on the plates and calculate CFU per mL:
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Part Two - Microscopic Counting
DRY LAB EXERCISE
Review text about microscope-based
counting using a hemacytometer. Although
this method is rapid, it does not distinguish
between living and dead cells, and can be
difficult if cells are clumpy or moving. A
hemacytometer allows scientists to place
0.0001 ml into 1 counting square (i.e. your
count = cells per 0.0001 ml). Count the cells
in the adjacent square and determine how
many cells/ml were in the original sample.
Biology 318, Lab Four, 2
Part Three –Population Growth
Population growth in bacteria is an increase in the quantity of cells and is dependent upon the availability of
nutrients in the environment. In the lab, under favorable conditions, a growing bacterial population doubles at
regular intervals. This is called exponential growth. In reality, exponential growth is only a small part of the
bacterial life cycle, and not representative of the normal pattern of growth of bacteria in Nature. When a fresh
medium is inoculated with a given number of cells, and the population growth is monitored over a period of
time, plotting the data will yield a typical bacterial growth curve.
The generation time for E. coli in the laboratory is 15-20 minutes, but in the intestinal tract, the coliform's
generation time is estimated to be 12-24 hours. For most known bacteria that can be cultured, generation times
range from about 15 minutes to 1 hour.
Turbidity measurements employ a variety of instruments to determine the amount of light scattered by a
suspension of cells. Particulate objects such as bacteria scatter light in proportion to their numbers. The
turbidity or optical density of a suspension of cells is directly related to cell mass or cell number. Using
dilution plating for colony counts, one can construct and calibrate a standard curve. The method is simple and
nondestructive, but the sensitivity is limited to about 107 cells per ml for most bacteria
a) Growth Curve Using Turbidity Measurements:
Different age cultures of E. coli are available for analysis. You will take a 1 ml sample of various age cultures
and measure the optical density of liquid medium at 600 nm (OD600) which is an accurate means of evaluating
the density of bacterial cells in a sample of culture. Use sterile nutrient broth as a blank in a cuvette. Then
evaluate the OD600 of each of the staged cultures. The time and day that cultures were started is marked
on each flask. Record the OD600 of each culture.
Biology 318, Lab Four, 3
b) Counting Using a Spectophotometer - DRY LAB EXERCISE
Review text about spectrophotometer-based counting. Although this method is rapid, it also does not
distinguish between living and dead cells. Also, you need to run appropriate control standards so that
you can graph a standard curve (X = cells/ml and Y = absorbance). By locating unknown
absorbances on the graph, you can determine cells/ml. Graph the following data on the worksheet to
determine the standard curve, from which you can determine unknown concentrations.
Sample
Standard 1
Standard 2
Standard 3
Standard 4
Standard 5
Standard 6
Dirty Sponge Water
UTI Urine Sample
Ground Beef Water
Concentration
10,000 cells/ml
8,000 cells/ml
6,000 cells/ml
4000 cells/ml
2,000 cells/ml
1,000 cells/ml
Unknown
Unknown
Unknown
Absorbance
20
16
12
8
4
2
5
17
15
Biology 318, Lab Four, 4
Biology 318 Worksheet Due Next Lab - Turn in Individually
Name:
(1) 6 pts. Viable Count Data. 300 or more = TMTC (too many to count).
Pasteurized Milk
1:10
1:10
1:100
1:100
Observed Number of Colonies
Plate
1:100
1:100
1:1000
1:1000
1:10000
1:10000
Observed Number of Colonies
(2) 4 pts. Using the count above that is closest to 100 to calculate the number of organisms per ml in
the original sample.
Pasteurized Milk _____________________________
Raw Milk
_____________________________
(3) 4 pts. Microscope-Based Counting.
Number of Microbes In Square: ______________________________________
Estimated #microbes per ml: ______________________________________
a) 10 pts.
Sample
0h
4h
8h
16 h
24h
32 h
48 h
Absorbance
Biology 318, Lab Four, 5
Graph the data from the above table and try to determine the doubling time of the E. coli
population.
Biology 318, Lab Four, 6
b) (6 pts) Spectrophotometer-Based Counting. Properly label entire graph, draw standard curve
in RED; circle each unknown along curve. Use this information to complete the table on the
next page.
Unknown
Dirty Sponge Water
UTI Urine Sample
Ground Beef Water
Estimated Concentration (Cells/ml)
Biology 318, Lab Four, 7