LAB: PLATE COUNTS OF FOODS
PROCEDURE

     Total counts of bacteria able to grow aerobically are made as an aid in determining the quality of foods.
Colonies are counted on a dish of enrichment medium and reported as colony forming units (CFU) or simply
as bacterial number per unit weight or volume. CFU is perhaps more correct since more than one bacterium
may have initiated the colony, but number has long usage. High numbers of bacteria indicate poor methods
of production, handling, holding, or processing. Standards have been established for many foods, especially
milk, and ingredients of foods as a means of control.
      The objectives of this lab are to:
      1. Prepare and properly dilute a food sample.
      2. Pour agar and mix the food sample correctly.
      3. Count colonies (colony forming units) according to some basic rules of counting.

Day 1
A. Preparation of samples.
1. Obtain 3 petri dishes and mark each with the following information:
      a) Names of lab group members;
      b) Name of food being tested;
      c) Which of the appropriate dilutions will be completed in the dish.
2. Locate a digital pan balances on your lab table. Place your food sample on aluminum foil so
      it can be aseptically weighed. Use the table below to determine the amount of food needed.
      Place the food in the blender with water from 1 dilution jar if the table indicates blending is
      needed. If blending is not needed place the food directly into 1 dilution jar.

      FOOD             WEIGHT              DILUTIONS                 BLEND
    Hamburger           11 g               10-1  10-3  10-5                Yes
    Sausage              11 g               10-1  10-3  10-5                 Yes
    Fish                    11 g               10-1  10-3  10-5                 Yes
    Flour                   11 g               10-1   10-3  10-5                 No
    Pepper                  1 g               10-2  10-4  10-6                  No
    Chili                      1 g               10-2  10-4  10-6                  No
    Milk                    11 g                10-1  10-3  10-5                 No
    Milk Powder        11 g                10-1  10-3  10-5                 No

3. Complete common lab procedures for serial decimal dilutions, as shown in the following
      diagram. In this lab all dilutions will be completed using dilution jars filled with 99 ml of water.
4. Complete all dilutions needed, using as many dilution jars as necessary. Always shake each
      dilution bottle just before removing any food to resuspend the food particles that may have
      settled.
5. When the dilution jars of food samples are all ready, pour plate count agar (PCA) into all 3
      petri dishes. Let the dishes cool for a few minutes, but before they become solid pipette
      1 ml of each dilution needed into each of the appropriately labeled petri dishes.
           (Pipette patterns:
                For 11 g of food: 1 ml from 1st dilution jar into 1st dish = dilution of 10-1.
                                         1 ml from 2nd dilution jar into 2nd dish = dilution of 10-3.
                                         1 ml from 3rd dilution jar into 3rd dish = dilution of 10-5.
               For 1 g of food: follow the same procedure and:
                                         1st dish = 10-2. 2nd dish = 10-4. 3rd dish = 10-6.
6. Immediately and gently swirl each dish to mix the food sample and the agar.
7. Allow the agar to solidify, invert the petri dishes and incubate at 32°C for 48 hours (or 5 days
      at room temperature).

Day 2
B. Counting colonies.
      NOTE: The following rules are a simplified version of those used throughout the food industry.
1. The best dishes to count are those with between 30-300 colonies. Use a Quebec Colony
      Counter (see diagram below) to determine if any of your plates are between 30-300 colonies.
      If any are, use the following equation to determine the number of colonies per g or ml of food:
           # Colonies = NUMBER OF COLONIES COUNTED ON WHOLE DISH
                                 AMOUNT OF FOOD PLATED X DILUTION FACTOR
      Example: If there are 67 colonies on a plate that began with 11 g of food and was diluted to 10-3,
      then the total amount of bacteria is: 67 X.1 (reciprocal of 11) X 1000 (reciprocal of 10-3) = 6700.

2. If no plate has 30-300 colonies but one or more has more than 300, use the dilution with the
      count closest to 300. The best way to count such plates is to count all colonies in a single
      square and multiply by the total number of squares. Any plates counted in this manner must
      be written as an "Estimated" Plate Count.
      Example: Plate 1: Count equals approx. 8 colonies per square,1 g, and dilution equals 10-5.
                     Plate 2: Count equals approx 11 colonies per square, 1 g and dilution equals 10-4.
      Solution: Use Plate 1 -> 8 X 1 X 70 (# of squares) X 100,000 (reciprocal of 10-5) = 56,000,000.
3. If less than 30 colonies are found on any plate, record the actual number and multiply by the
      dilution factor. Report the results as an "Estimated" Plate Count.
      Example: Plate 1 (10-1)= 0 colonies, Plate 2 (10-3) = 19 colonies, Plate 3 (10-5)= 4 colonies.
            All plates began with 11 g of food.
      Solution: Use Plate 2 -> 19 X .1 X 1000 (reciprocal of (10-3) = Estimate 1900.
4. If there are no colonies at any dilution, report as "Estimated" less than one
      times the lowest dilution factor.
      Example: Plate 1 (10-1) = 0 colonies. 1 g. Solution: 1 X 1 X 10 = Estimated < 10
5. Other codes: Spr = spreader (if more than half of the plate is covered, do not use for
      counting). TNTC = too numerous to count (do not use unless the plate is truly
      uncountable at the highest dilution).