Summer Science Academy Experiment:
Determining the Amount of Bacteria in Food
TIME NEEDED: 1 hour (day 1); 1 hour (day 2)
Bacteria can be found in nearly any location on the earth's surface that has a supply of moisture - in lakes and ponds, in soil, on the surfaces of plants and skin, and in the digestive tract of humans and animals. While the vast majority of bacteria pose no threat to us, and in some cases offer considerable benefits, a few types of bacteria may produce serious disease. In order to reduce the incidence of such disease, various substances which routinely harbor potentially harmful organisms are tested to determine their bacterial content or titer.
Three methods are used to determine bacterial titer:
1. When bacterial content is very low (no more than a few cells per liter), a known volume of sample is run through a small-pore (0.2 micrometer) filter which traps bacterial cells. The filter is then placed on an agar plate, and each cell grows up to form a visible colony. The number of colonies is divided by the sample volume to give bacterial concentration. For example if a 10 liter sample yielded 140 colonies, the titer would be:
140 colonies = 14 bacteria per liter
2. For samples of intermediate bacterial content (drinking water, samples from public beaches), various volumes of sample (10 ml, 1 ml, 0.1 ml) are added to growth medium and incubated or 24 hours. Bacterial content is determined statistically, based on the smallest sample size which will produce growth in liquid culture. The current standard for drinking water is less than 1 coliform bacterium per 100 ml sample. (1 coliform per 100 ml is the lowest titer which can be reliably detected using this method.)
3. Food samples and liquid bacterial cultures have such a high bacterial content that even one drop spread on an agar plate will produce too many colonies to count. The serial dilution method is used to determine the bacterial titer of such cultures. A series of 1:10 dilutions (1 part sample diluted with 9 parts liquid) of such samples is plated and the resulting colonies are counted. the number of colonies on a plate is then multiplied by the dilution factor (the number of times that the 1:10 dilution was done) for that plate to obtain the bacterial count in the original sample. In the experiment that you will be doing today, the serial dilution plating method will be used to determine the bacterial content of ground beef.
The intestinal tract of warm-blooded animals contains a variety of bacterial species. When an animal is butchered, these bacteria may be spread from the gut to the edible portions of the carcass. While such bacteria generally are not harmful to humans (beef is most often contaminated with E. coli, a normal inhabitant of the human intestine), some types of bacteria (such as Salmonella, found frequently in chickens) can cause food poisoning. It is, therefore, essential that meats be thoroughly cooked prior to eating, The non-pathogenic varieties of bacteria in foods may cause chemical changes which alter the flavor and texture of food. Because of this, meats are refrigerated or frozen to retard the growth of bacteria. Since some bacteria can grow over a wide range of temperatures (from less then 10° C to greater than 45° C for E. coli), refrigeration is adequate for only short term storage of meats.
tryptic soy agar plates (6)
large sterile tubes (2)
tubes with 9 ml of sterile nutrient broth (11)
sterile transfer pipets
sterile sticks (2)
ground beef samples (Sample A has been stored in a refrigerator for 4 days;
Sample B has been stored frozen)
1. Label 9 dilution broth tubes as follows:
A 10- 2 , A 10-3, A 10-4, A 10-5, A 10-6, A 10-7,
B 10-2, B 10-3, B 10-4
2. Label 6 agar plates as follows:
A 10-6, A 10-7, A 10-8
B 10-3, B 10-4, B 10-5
3. Label one large sterile tube A and the other large sterile tube B. Use a sterile stick to measure out approximately 1 gram of sample A (about the size of a pea), and place it in sterile tube A. Add the contents of one tube of dilution broth to the meat sample, and shake the sample until the suspension appears fairly uniform. Repeat this with sample B, using a new sterile stick, and sterile tube B.
4. Serial Dilutions
NOTE: YOU MUST USE A NEW STERILE PIPET FOR EACH OF THE DILUTION STEPS!!
Use a sterile pipet to transfer 1 ml of the suspension from large tube A to the culture tube labeled A 10-2. Mix the contents thoroughly by pipeting up and down several times. Use a new pipet to transfer 1 ml from tube A 10-2 to tube A 10-3 and mix thoroughly as before. Continue this series of dilutions into tubes A 10-4, A 10-5, A 10-6 and A 10-7.
Repeat this series of dilution using sample B. Transfer 1 ml of suspension from large tube B into tube B 10-2 and mix thoroughly. Serially transfer, as before, into tubes B 10-3 and B 10-4.
5. Plating bacteria
Use a micropipettor to withdraw 100 m l (0.1 ml) of liquid from tube A 10-5 and squirt it onto the surface of the agar plate labeled A 10-6. (NOTE: Plating 0.1 ml of a 10-5 dilution will give you the same number of colonies as plating 1 ml of a 10-6 dilution; the agar plate cannot absorb 1 ml of liquid, so the smaller volume is used.)
NOTE: YOUR INSTRUCTOR WILL DEMONSTRATE THIS NEXT PART
Sterilize the bacterial spreader by dipping it into a beaker of alcohol. Remove and shake off the excess. Carefully run the spreader through the flame of a Bunsen burner and allow the alcohol to burn off. Cool the spreader by holding it against the condensation on the inside of the petri dish lid. Gently spread the liquid culture onto the surface of the agar by moving the spreader in a circular manner while rotating the plate. This will ensure an even distribution of bacteria.
6. Repeat step 5 with the remainder of the A cultures:
Spread 100 m l (0.1 ml) from culture tube A 10-6 onto plate A 10-7
Spread 100 m l (0.1 ml) from culture tube A 10-7 onto plate A 10-8
7. Repeat step 5 with the B cultures:
Spread 100 m l (0.1 ml) from culture tube B 10-2 onto plate B 10-3
Spread 100 m l (0.1 ml) from culture tube B 10-3 onto plate B 10-4
Spread 100 m l (0.1 ml) from culture tube B 10-4 onto plate B 10-5
8. Allow plates to absorb the cultures, then turn plates upside-down and incubate overnight at 37 ° C.
Choose one plate from each series (A or B) on which there are at least 20 well-separated colonies. Enter your results in the table below. Count the colonies on the plates and multiply by the dilution factor for that plate to obtain the bacterial count in the original sample. (Example: If a plate labeled 10-7 has 87 colonies, then the sample has 87 x 107 = 8.7 x 108 colonies per gram)
PLATE # COLONIES
Bacterial count in sample A (refrigerated ground beef):
PLATE # COLONIES
Bacterial count in sample B (frozen ground beef):
Based on your results, answer the following questions:
1. New York City sets an upper limit of 100,000 bacteria/gram for some foods. In Massachusetts the limit is 500,000 bacteria/gram. Do your samples meet these guidelines (yes or no)?
Sample A Sample B
New York City ________ ________
Massachusetts ________ ________
2. Pick several colonies to make gram stain smears of (follow your lab book procedures for gram staining). Most colonies should be composed of gram-negative rods. Gram-positive colonies probably indicate contamination by water or dust. Record your results below.