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. 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) 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

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Protocol for raw milk Work in pairs. Shake the milk 25X in an up and down motion to evenly mix up the milk. 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, 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:

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.

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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.

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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

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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 Plate 1:100 1:100 1:1000 1:1000 1:10000 1:10000 Observed Number of Colonies

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

Graph the data from the above table and try to determine the doubling time of the E. coli
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population.

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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)

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