GENERAL: Milk is one of the most common food products in the world. Fresh milk is a staple for people of all ages, and is frequently called "the ideal food product". In today's world, this product is frequently collected in one site, processed in another and transported another distance before it reaches the consumer. All of these transfers of the product introduce the chance of contamination by disease causing microorganisms. A low number of microorganisms in milk is acceptable, as it is not a sterile product; but in the United States, strict guidelines are followed to monitor the quality of the product.

All raw milk must be processed within 72 hours of arriving at the plant. It is such a nutritious food that numerous naturally occurring bacteria are always present. The milk is pasteurized, which is a process of heating the raw milk to kill all of the pathogens that may be present. Pasteurization is not sterilization; some bacteria may survive the process, and it is these bacteria that will cause milk to "go sour". Keeping milk refrigerated is the best way to slow the growth of these bacteria. (Some bacteria do not cause spoilage, but are actually added to milk or cream after pasteurization to make "cultured" products such as cheese, cottage cheese, yogurt, buttermilk, acidophilus milk and sour cream.

Testing the foods we consume for the presence of pathogenic microorganisms is very important. Although 100% of the food cannot be tested, it can be deemed "safe" through proper audit of the food supply. In many instances, the pathogenic microorganisms are present in very small numbers, but for many of these pathogens, small numbers are all that are necessary to transmit disease or illness. For that reason, the presence of other microorganisms is monitored. These microorganisms provide an index of the sanitary quality of the product and may serve as an indicator of potential for the presence of pathogenic species. (E. coli is commonly employed as an indicator organism.)

Total counts of microorganisms are also an indication of the sanitary quality of a food. Referred to as the Standard Plate Count (SPC), this total count of viable microbes reflects the handling history of the product, including production, transport and storage of the food. This is especially true of milk.

The following bacterial counts are standards for milk, as recommended by the US Public Health Service:

Grade A raw milk for pasteurization: Not to exceed 100,000 bacteria per ml prior to commingling with other produced milk; and not exceeding 300, 000 per ml as commingled milk prior to pasteurization.

Grade A pasteurized milk: Not to exceed 20,000 bacteria per ml, and not over 10 coliforms (E.coli and other bacteria in its family) per ml.

OBJECTIVES: At the conclusion of this exercise, you will have learned additional microbiology techniques (quantitative culturing) and a modification of a standardized method used to check food products for quality. You will learn how to make calculations from dilutions and understand that relationships of the quantity of microorganisms to the quality of a food product.

INTRODUCTION: In this experiment, you will test milk for total number of viable microorganisms using a modification of the Standard Plate Count assay. This involves spreading a specific amount of diluted samples of milk onto a Standard Methods Agar petri plate, incubating the plate for 48 hours, and then counting the number of colonies on each plate and calculating the number of microorganisms per ml in the original sample. This method is similar to the one used by dairy quality assurance inspectors, to determine if the milk sample meets the specification for Grade A milk. You will be working in pairs.

EQUIPMENT AND MATERIALS:

Milk sample #1, 1:10 dilution (one bottle per table)
Milk sample #2, 1:10 dilution (one bottle per table)
Sterile disposable inoculating loops: 2 1 ul loops (Blue: holds 0.001 ml of liquid) and 2 10 ul loops (Yellow: holds 0.01 ml liquid) per 2 students
Four sterile Standard Methods Agar plates per 2 students
Felt-tip, black, marking pen
Masking tape
Microbiological incubator, 35C
Squeeze bottle of disinfectant
Paper towels

PROCEDURE:

1. Wash your hands and disinfect your work bench.

2. Invert your four agar plates. Using as little space as possible, towards an edge, put the following information on the plates:

Plate 1: Your names; the date; Milk A --1:100 dil.
Plate 2: Your names; the date; Milk A -- 1:1000 dil.
Plate 3: Your names; the date; Milk B --1:100 dil.
Plate 4: Your names; the date; Milk B-- 1:1000 dil.

3. Using the correct loops for each dilution and following the technique shown by your instructor, inoculate one loopful of the correct milk mixture on the appropriate plate. DO NOT LET THE LOOP TOUCH ANYTHING OTHER THAN THE MILK OR THE AGAR! After emptying the loop, streak the specimen all over the agar surface.

Plate #1: Place one YELLOW loopful (0.01 ul) of Milk A on the agar; streak out
Plate #2: Place one BLUE loopful (0.001 ul) of Milk A on the agar; streak out
Plate #3: Place one YELLOW loopful (0.01 ul) of Milk B on the agar; streak out
Plate #4: Place one BLUE loopful (0.001 ul) of Milk B on the agar; streak out

4. Allow the specimens to "rest" for 5-10 minutes, then invert the petri plates and tape together.

5. Place your inverted plates in the holding cans provided by your instructor. They will be incubated at 35C for 48 hours, and then refrigerated, halting any further microbial growth, until next week.

The Following Week:

(After 48 hours of incubation, your instructor will remove the petri plates from the incubator, and refrigerate them, to inhibit any further growth of microorganisms).

1. Find your particular plates and take them to your lab area. Be careful not to turn the plates right-side up if there is excessive condensation on the cover plate.

2. Count all colonies using your marking pen. Record the total number of colonies for each plate on the Data Sheet.

3. Calculate the number of microorganisms per milliliter of milk sample by multiplying the total number of colonies for a particular plate times the inverse of the plate's dilution factor. For example, if you have 83 colonies on a 1:1000 dilution plate, you will need to multiply by 10 (for the original dilution of the milk samples) and then by 1000 (for the amount of inoculum placed on the plate), or, 83 x 10,000. This would give you a total of 83,000 organisms per milliliter of milk. (If there are so many colonies that you cannot count them, use the term TNTC --- this means "too numerous to count".) *The best results are obtained from plates containing 20 to 200 colonies per plate.

4. Record your answers on the Data Sheet.

(This activity is adapted from an internet article by The Institute for Food Technologists: http:www.ift.org)

Plate #1 ---

Count:_____________________________________________________

Calculation:_________________________________________________

Your answer: ________________________________ microbes per ml

Plate # 2---

Count:_____________________________________________________

Calculation:_________________________________________________

Your answer: ________________________________ microbes per ml

Plate # 3 ---

Count:_____________________________________________________

Calculation:_________________________________________________

Your answer: ________________________________ microbes per ml

Plate # 4 ---

Count:_____________________________________________________

Calculation:_________________________________________________

Your answer: ________________________________ microbes per ml

What do these figures tell you? Is either milk sample suitable for drinking? (Refer back to the GENERAL section to refresh yourself on acceptable bacterial loads in fresh milk.)

Click here to fly back to the home page!