9.1: Introduction

Learning Objectives

• Learn how to use serial dilutions.
• Learn how to do a standard plate count.
• Understand the importance of standard microbiological tests for food safety

Pathogens can be introduced into foods at any stage: during growth/production at the farm, during processing (grinding, chopping, milling, etc.), during handling and packaging, and when the food is prepared in the kitchen. In many cases, small numbers of pathogenic bacteria are not dangerous, but improper storage and/or cooking conditions can allow these bacteria to multiply to dangerous levels.

Fecal contamination of water (and through water, contamination of food materials) is another one of the ways in which pathogens can be introduced. Coliform bacteria are Gram-negative non-spore forming bacteria that are capable of fermenting lactose to produce acid and gas. A subset of these bacteria are the fecal coliforms, which are found at high levels in human and animal intestines. Fecal coliform bacteria such as E. coli, are often used as indicator species, as they are not commonly found growing in nature in the absence of fecal contamination. The presence of E. coli suggests feces are present, indicating that serious pathogens, such as Salmonella species and Campylobacter species, could also be present.

In this lab, you will examine bacteria found in milk, chicken, and in other assorted food materials.

Milk

Milk contains carbohydrates, minerals, fats, vitamins, and proteins, and is therefore susceptible to breakdown by a wide variety of microorganisms. Several different kinds of bacteria may be present in milk, most commonly the genera Lactobacillus, Micrococcus, and Streptococcus. As discussed in Lab 7, regular pasteurization is the process used to reduce microbial loads to acceptable levels in foods like milk and fruit juices. Milk that has undergone a regular pasteurization procedure can still contain bacteria. If this milk is stored at 4ºC (refrigerator temperature) the bacteria are prevented from multiplying, but if the milk is left out at room temperature, the bacteria will reproduce and the milk will spoil.

If done aseptically, UHT (ultra high temperature) pasteurization can sterilize foods. This is why UHT milk can be left unopened at room temperature for long periods of time.

Microorganisms of concern in milk include Campylobacter jejuni, Escherichia coli O157:H7, Listeria monocytogenes, Mycobacterium bovis, Mycobacterium tuberculosis, Salmonella species (most common: S. enteritidis and S. typhimirium), and Yersinia enterocolitica.

Chicken

Raw poultry products are frequently contaminated by pathogens (e.g. Salmonella, Staphylococcus aureus, Clostridium perfringens, Campylobacter jejuni, etc.). Salmonellosis is one of the most common serious foodborne infection caused by contaminated poultry. The prevalence of Salmonella in raw fresh and frozen poultry approaches 80% in some countries.

Fruits and Vegetables

Fruits and vegetables may become contaminated with potential pathogens from the soil or water they come in contact with while growing. They may also pick up harmful bacteria during processing, storage and preparation. While the use of potable (uncontaminated, drinkable) water to wash and freshen harvestable fruits and vegetables is useful both commercially and in the home, residual water may support extensive microbial growth during further storage.

Standard Plate Count and Serial Dilution Techniques

One of the most common methods of determining the amount of bacteria in a food product is a standard plate count. In this method, serial dilutions of the food are plated on general purpose and/or differential/selective growth media. Bacterial colonies are then counted, and the number of CFUs (colony forming units) in the original undiluted sample is calculated. (CFUs are as a measure of the number of bacteria to take into account that one colony might be the product of more than a single bacterium).

Serial dilution is a technique that is used to produce very dilute solutions without the necessity of measuring very small quantities of liquids. It is a series of stepwise dilutions, in which one first dilutes a solution, then dilutes the dilution, then dilutes the dilution of the dilution and so forth. The dilution factor at each step is usually constant, resulting in a geometric progression of concentration. An example of a serial dilution is seen below. In this example, each dilution is a 10-fold dilution (transferring 1 ml into 9 ml of H₂O results in a 1/10 dilution; i.e., 1 ml in a total volume of 10 ml).

Serial dilutions are often used in standard plate counts because the number of bacteria in a sample (water, food, or a medical sample such as a urine or a fecal sample) is unknown. The sample is diluted to obtain a number of CFUs that supplies statistically significant results, yet is still easily countable. The general recommendation for a countable plate is between 30 – 300 CFUs/plate.

Figure 9.1.1: Serial Dilution

After dilutions are prepared, a set amount of liquid (typically between 0.1- 1 ml) is spread out over the surface of an agar plate, and then incubated to allow for bacterial growth. CFU counts from these diluted plates are used to calculate the number of bacterial cells/ml in your original (undiluted) sample. If you plate a full milliliter (ml) of your dilution, you would simply multiply the number of CFU’s counted by the dilution factor of the plate you counted.

• Example: I count 55 CFU’s on a plate diluted 1:1000 (10^-3 ) in which I added a full ml of my dilution. My calculation is 55 x 1000 = 55,000 (5.5 x 10⁴ ) cells/ml.

However, it is important to note that we do not always plate a full ml (it can be difficult to get that much liquid to be absorbed into an agar plate). Therefore an additional calculation is often necessary to be able to express your results as cells/ml. If you plate 0.1 ml of sample, you will need to multiply the number of CFU’s by 10 to determine # CFU’s/ml. If you plate 0.5 ml of a sample, you will need to multiply by 2 to determine the number of CFU’s/ml.

• Here’s another example: you set up serial dilutions for a milk sample and plate out 0.5 ml of your dilutions. The countable plate that came from your 1:100 (10^-2 ) dilution has 114 colonies.

Your calculation:

1. Multiply: #CFU’s x dilution factor = 114 x 100 = 11400 cells/0.5 ml.
2. Multiply by 2 to express your result as cells/ml 11400 x 2 = 22800 cells/ml (or 2.28 x 10⁴ ) If dilutions are done properly, we should expect to see a geometric progression in the number of cells in each dilution as shown below.

	Dilution	Number of bacteria/ml in each tube after dilution	Number of CFU’s (1 ml plated)	Number of CFU’s (0.1 ml plated)
Undiluted sample		1,000,000/ml = 10^6/ml	1,000,000 (10^6)	100,000 (10^5)
Tube 1	1/10 =10^-1	100,000/ml = 10^5/ml	100,000 (10^5)	10,000 (10^4)
Tube 2	1/100 = 10^-2	10,000/ml = 10^4/ml	10,000 (10^4)	1,000 (10^3)
Tube 3	1/1000 = 10^-3	1,000/ml = 10^3/ml	1,000 (10^3)	100 (10^2)

Of course your results would not turn out as perfectly as those shown above. Slight inaccuracies in pipetting would cause variations in the final results. So in the real world, the results of multiple dilutions are averaged together when determining the final number of bacteria/ml in a sample.

For a review of scientific notation and additional practice problems to help you understand serial dilution, see Appendix II. For additional information about the use of pipettors for measuring small volumes, see Appendix III.

Key Terms

CFU, indicator bacteria, serial dilution, coliform bacteria, fecal coliform bacteria, standard plate count, UHT pasteurization