Microbiological Food Safety

Testing for Bacterial Contamination of Food

Bacteria are incredibly diverse and abundantly found in most of the natural world. The majority are beneficial to us in ways we may not fully realize or appreciate. A few, however, are not and will cause disease when we cross paths with them. Pathogenic (harm-causing) and potentially pathogenic bacteria may be found in unexpected places, such as in the food we eat, the water we drink or use for recreation, in soil, on surfaces in your home, and elsewhere.

Unfortunately for us, the things we eat and drink are fairly common vehicles for disease transmission. And, because food and drink pass through our digestive tract, the most common symptoms of a foodborne disease are abdominal discomfort or pain, nausea, diarrhea, and/or vomiting. Gastrointestinal illnesses caused by foodborne microbes range in severity from mild to extremely debilitating, even fatal. The biological agents responsible for this type of disease may be viruses, bacteria, fungi, protozoa, or helminthes.

To protect the public from disease, manufacturers and distributors of food consumed in the United States must prove that their food is pathogen-free before it can be offered for sale. Regulatory agencies at the local, state and federal levels (such as the Department of Agriculture and the Food and Drug Administration) require routine bacteriological testing to protect the public from acquiring a foodborne illness. Although many types of microbes may cause foodborne disease, the CDC and FDA currently considers the bacteria Bacillus cereus, Campylobacter jejuni, Clostridium spp., pathogenic strains of Escherichia coli, Listeria monocytogenes, Salmonella spp., Shigella spp., Staphylococcus aureus, Vibrio spp., and Yersinia spp.; the parasites Cryptosporidium and Cyclospora; and the Norwalk virus (norovirus) (http://www.cdc.gov/foodnet/index.html) to be the most common and of the greatest concern in the United States.

Although there are “rapid” methods available to detect bacterial contaminants in food that rely on DNA and antibody testing, plating samples on differential and selective culture media is a tried and true method. The disadvantage is that culture methods take more time, but the advantages include the simplicity of the tests and a higher level of both specificity and sensitivity.

The relatively low number of bacteria present in a food sample limits the sensitivity of all of the various types of tests available to evaluate food safety, including those based on culture. A preliminary step called enrichment culture may be used to amplify the number of bacterial pathogens, by pre-incubating the food sample in a non-selective medium that promotes growth of any bacteria that might be in the sample.

Many standard methods include a two-stage enrichment culture. The first step, or pre-enrichment, involves adding a specific amount (determined by weight, typically 10–25 grams) of the food to be tested in a large (100–250ml) volume of a non-selective broth medium. After an incubation period of 18–24 hours at 37°C, a small sample of the enrichment culture is transferred to one or more types of selective media designed to inhibit growth of competing microbes while allowing the target pathogen to multiply. Many such formulations are also differential, in that growth of the bacterial target will cause a characteristic chemical change in the appearance of the medium, thus “differentiating” the pathogen from other possible contaminants, such as spoilage organisms, that might also be in the food.

We will be conducting our own investigation of food safety using a modified and scaled down adaptation of the standard laboratory methods, beginning with a pre-enrichment culture of food samples, followed by plated on several types of selective and differential media. Our determination of food contamination will be based on (a) growth of bacteria on the selective media and (b) observation of a specific biochemical reaction (usually a color change) characteristic for a particular type of pathogen. Note that these methods are based on bacterial phenotypes (traits), and more than one species of bacteria may have the same selective/differential traits. Therefore, definitive identification of a bacterium isolated from food requires additional testing.

Numerous media formulations are available that permit the isolation and identification of pathogenic bacteria in food. Using the media described in Table 1, we will be testing food for contamination with EHEC (enterohemorrhagic E. coli) and other strains of E. coli, S. aureus, B. cereus, Salmonella, and Shigella.

Table 1
Medium Selective Agent(s) Differential Agent(s) Detection
MacConkey Agar

(MAC)

Bile salts and crystal violet inhibits the growth of most Gram positive, non-enteric bacteria. Lactose and pH indicator Gram negative enteric bacilli will grow; E. coli will produce pink colonies, Salmonella and Shigella spp. do not ferment lactose and colonies are colorless.
Sorbitol MacConkey Agar

(SMAC)

Bile salts and crystal violet inhibits the growth of most Gram positive, non-enteric bacteria. Sorbitol and pH indicator Gram negative enteric bacilli will grow; E. coli 0157:H7 does not ferment sorbitol and colonies are colorless
Mannitol Salt Agar

(MSA

7.5% sodium chloride inhibits the growth of most Gram negative bacteria Mannitol and pH indicator Salt tolerant bacteria grow; S. aureus ferments mannitol and colonies are yellow; B. cereus does not ferment mannitol and colonies are deep red.

Pre-enrichment to Promote Bacterial Growth

Note: this will require time in addition to your regular lab period to complete. Because both the presence and type of bacteria that may be in the food is unknown, BSL2 containment practices should be used throughout the entire procedure.

Samples of foods will be available in the laboratory up to a week before your scheduled lab period.

Transfer 5 ml of Tryptic Soy Broth to a disposable plastic culture tube. You should select one food sample for testing. Prepare an enrichment culture of the selected food item, by transferring a small amount of the food to the broth in the culture tube, using aseptic technique. Place a cap on the tube, mix the contents fully, and place in the incubator at 37°C.

A minimum of 18 hours after starting the enrichment culture (one day after the enrichment culture is started is preferred), and no later than the day before the scheduled period for this investigation, return to the lab, and use an inoculating loop to subculture samples from the enrichment culture to each of the three types of selective and differential media described in Table 1. Use the streak plate method for all of the plates, so isolated colonies will form. Appropriately dispose of the enrichment culture as a potential biohazard.

Return the streak plates to the incubator for observation and further investigation during the lab period.

Food sample tested _____________________________________________________________

Cooked or raw? ________________________________________________________________

How was this food item stored before testing? ________________________________________

Medium Growth on medium? (yes or no) Appearance of colonies/medium if growth occurred
MacConkey Agar (MAC)
Sorbitol MacConkey Agar (SMAC)
Mannitol Salt Agar (MSA)

For each of the selective and differential media on which bacterial colonies are observed, indicate what the appearance of the colonies indicates in terms of the possible type(s) of bacteria present in the food sample.

Medium Observations that indicate contamination of food sample with potential food borne pathogens
MacConkey Agar (MAC)
Sorbitol MacConkey Agar (SMAC)
Mannitol Salt Agar (MSA)

The growth and appearance of colonies on selective and differential media is an indicator of the presence of specific bacterial pathogens, but these results must be confirmed before reporting that food is contaminated and ingestion may initiate a foodborne disease. Therefore, perform additional tests to confirm that the colonies observed on the selective media are potentially pathogenic bacteria. These tests include:

Gram stain of representative colonies (all)

Coagulase test to confirm Staphylococcus aureus (S. aureus is coagulase +)

Colonial morphology and endospore stain to confirm Bacillus cereus

TSI test to confirm E. coli, Salmonella, and Shigella

Expected and Experimental Results

For each bacterium, indicate the EXPECTED outcome of the Gram and Endospore stains and TSI tests which would confirm the identity of the potential foodborne pathogen.

Expected Gram stain reactions:

E. coli ______________________ Salmonella spp. _____________________

S. aureus ___________________ Shigella spp. ________________________

B. cereus ___________________

Expected Endospore stain reactions:

B. cereus ________________________________________

Expected TSI reactions:

E. coli ________________ Salmonella spp. _______________ Shigella spp. _______________

For each type of colony found growing on the various types of selective and differential media, perform the appropriate confirmatory (or indicatory) tests, and record those results in the results table.

Results of Confirmatory Tests

Potential Pathogen Found on which medium? Additional test(s) Outcome of test(s) Present in Food Sample?
E. coli
E. coli 0157:H7
S. aureus
B. cereus
Salmonella spp.
Shigella spp.

Once you know whether the pathogens we tested for in the lab were present in the food sample you tested, compare your results with those of others in your lab who tested other types of food.

Approximately how many of the food samples that were tested by your lab group were contaminated with one or more of these bacteria? List the foods with contaminants below:

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Reflect on the significance of the outcome of this investigation, and write your thoughts below. Before the class ends you may be asked to share your reflection as part of a larger discussion on what it means to find bacteria (pathogenic or otherwise) in food you might eat.

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Microbiological Food Safety by Holly Ahern is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License, except where otherwise noted.