FOOD SAFETY ENV 450 ________________________________________________________________ PRACTICAL 1 1.

1 OBJECTIVES To enumerate bacteria by the standard plate count method, i.e. spread plate and pour plate techniques. To formulate and prepare a series of dilutions. Isolation of E.coli from beef (minced).

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STANDARD PLATE COUNT METHOD AND DILUTION TECHNIQUES

The standard plate count (SPC) method is the most widely used method in food microbiological testing i.e. for determining the numbers of viable cells or colonyforming units (c.f.u.) in a food product. The pour plate and spread plate techniques are the techniques commonly employed in SPC method. The pour plate technique allows for better discrete colonies allowing easier colony counts. The spread plate technique offers advantages in determining the numbers of heat-sensitive psychrotrophs in a food product because the organisms do not come in contact with melted agar. It is also the method of choice when the colonial features of a colony are important to its presumptive identification and for most selective media. Strict aerobes are obviously favoured by surface plating, but microaerophilic organisms tend to grow slower. The disadvantage however, is the problem of spreaders (especially when the agar surface is not adequately dry prior to plating) and the crowding of colonies, which makes enumeration more difficult. This practical session is to allow students to review these pure culture techniques. Different series of quantitative dilutions will be also prepared and the final dilution and dilution factors will be determined. The number of colonyforming units (c.f.u.) will then be determined.

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MATERIALS (per group of students)

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PROCEDURES
First period: Preparing sample 1. Use a sterile utensil to measure 12.5g of the beef (minced) into a sterile stomacher bag. 2. Add 112.5 ml peptone water (this is your first 1/10 dilution, or 10-1). 3. Place this bag in the stomacher and homogenize the sample for 2 minutes. 4. Prepare serial dilutions (10-2 to 10-4). Transfer this sample into 9ml of peptone water (test tubes) and mix well. Before actually starting the dilution series, plan out your dilution plan and show your calculations. Pour plate 1. Label an empty Petri dish (group names, plated dilution). You will be plating each dilution. 2. Pipette 0.1ml of the diluted samples on the EMB semi solid (10 -2 and 10-3) and swirl the plates gently. 3. Leave pour plates on the bench until solidified. 4. After the agar solidifies, invert the plates and incubate at 32 C to 37C for 24 to 48 hours.

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Spread plate 1. Label an empty Petri dish (group names, plated dilution). You will be plating each dilution. 2. Pipette 0.1ml of the diluted samples on the EMB agar plates (10-4 and 10-5). 3. Use a flamed hockey stick to spread the liquid. 4. Then, incubate at 32C to 37C for 24 to 48 hours.

Second Period 1. Arrange each plate in order from lowest to highest dilution. 2. Select the plate with 30 to 300 colonies. Record data for plates with fewer than 30 colonies as too few to count (TFTC) and those with more than 300 colonies, too numerous to count (TNTC). 3. Count the number of colonies on the plate selected. 4. Multiply the number of colonies by the dilution of the plate to determine the number of bacteria in the original food. For example, if 129 colonies were counted on 1 : 103 dilution: 129 colonies = 129,000 1 ml x 10-3 = 1.29 x 10' bacteria / ml or gram of food

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FOOD SAFETY ENV 450 ________________________________________________________________ Name : _____________________________________ Group : _____________________________________ Date : _____________________________________ RESULTS AND OBSERVATIONS Food sample: ___________________________
Dilution Colonies per plate No. of organisms per ml/gm Count the typical coliform colonies (colour:______________)

DISCUSSION Questions: What do EMB stands for? What is an EMB principle of selectivity? ______________________________________________________________________ ______________________________________________________________________
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What could be their sources of contamination? ______________________________________________________________________ ______________________________________________________________________ ______________________________________________________________________ ______________________________________________________________________ Why used peptone water? ______________________________________________________________________ ______________________________________________________________________

CONCLUSION ______________________________________________________________________ ______________________________________________________________________ ______________________________________________________________________ ______________________________________________________________________

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