Using Most Probable Number (MPN)Analysisand the Coliscan MF Kit to Assess Food and Water Safety

Introduction:

Bacteria are an essential part of our world. They perform vital roles in the environment; cycling nutrients such as carbon, nitrogen and sulfur, and also helping to degrade the waste that other life forms generate. They are capable of living in every known environment that contains water – even if that water is almost boiling hot or has a pH as low as hydrochloric acid! Our own bodies teem with thriving bacterial communities that help protect us from pathogens and help us digest our food.

Of course, bacteria are not always the good guys. They are responsible for many of the most famous disease outbreaks throughout human history, from the Black Plague to Tuberculosis. Pathogens in our food and water can cause serious disease outbreaks as well. During the summer of 2008, for example, hundreds of people in almost every state in the nation got food poisoning that was ultimately traced back to Salmonella typhimurium contamination of peanut products from the Peanut Corporation of America1.

There are two major forms of food-borne illness: food-borne infections and food-borne intoxications. In food-borne infections, the pathogen must be ingested and must then grow inside the host to cause disease. Salmonella and E. coli are two good examples of bacteria that cause food-borne infections. Food-borne intoxications are caused when the pathogenic bacteria make a toxin that gets ingested and causes the symptoms. Botulism, for example, is caused by the ingestion of a neurotoxin that is produced by Clostridium botulinum.

During this lab, you will learn to identify and enumerate some of the bacteria that commonly cause food- and water-borne infections.

Learning Objectives:

After performing this laboratory, participants will be able to:

  1. Describe several types of bacteria that are found in normal food and water samples, as well as those that are common pathogenic contaminants.
  2. Describe the biochemical and metabolic reactions that allow coliforms, E. coli and other bacteria to be distinguished in culture.
  3. Discuss how human activities can impact food and water safety.

Introduction:

In order to generate energy and create new biomolecules, all living things must obtain energy, carbon, electron sources and electron acceptors from their environment. Humans, for example, get their carbon, energy and electrons from organic chemical sources (hooray, pizza!), and ultimately generate energy as they pass these electrons along an electron transport chain to oxygen (the final electron acceptor). Because we use oxygen as our final electron acceptor, the process is referred to as aerobic respiration. Some bacteria can use a variety of non-oxygen acceptors (sulfate, nitrate, etc.) during a process called anaerobic respiration. Finally, some species can use organic molecules as the final electron acceptor, forgoing the electron transport chain altogether. This final process is referred to as fermentation, and generates very little energy. Fermentations are incredibly complex, and give us wonderful products such as beer, wine and cheese. A few bacterial fermentations are shown in the figure on the next page.

Bacterial fermentations can be used in the lab to identify food and water contaminants. Escherichia coli (E. coli), for example,is a Gram-negative, rod-shaped, facultative anaerobe (it prefers to use oxygen, but can do just fine with other types of metabolism) that is capable of fermenting lactose to produce a variety of acids (lactic, acetic, formic and others) and gas (CO2 and H2) within 48 h at 35°C. In food and water samples, it is generally considered to be an indicator of recent human fecal contamination because E. coli is present in the digestive tracts of humans, but not as frequently in other animals. Unfortunately, other enteric bacteria, such as Citrobacter, Klebsiella and Enterobacter, are also capable of fermenting lactose and are similar to E. coli in several phenotypic characteristics, so that they are not easily distinguished. As a result, the term "coliform" was coined to describe this group of enteric bacteria. In 1914, the U.S. Public Health Service adopted the enumeration of coliforms as a more convenient standard of sanitary significance.

Using Coliscan MF for the Identification and Differentiation of E. coli and General Coliforms from Liquid Samples:

Coliscan MF (membrane filter) medium is a nutrient liquid formulation that uses two color producing chemicals, one for the detection of the enzyme glucuronidase (produced by E. coli strains but not by general coliforms) and one for the detection of galactosidase (produced by all coliforms, including E. coli).

Galactosidase normally converts lactose into glucose and galactose.

In this method, a diluted liquid sample containing E. Coli and other coliforms is passed through a membrane filter to leave individual bacterial cells on the membrane filter. The filter is then placed on a pad soaked with the Coliscan MF medium. When incubated at a suitable temperature, the cells will grow into colony forming units (CFU) on the surface of the filter. If coliform (including E. coli) colonies are present, they will produce the enzyme galactosidase, which will react with its specific color-producing substrate in the medium and a water insoluble pink pigment will color the colony. If E. coli colonies are present, they will also produce the enzyme glucuronidase, which will react with its specific color-producing substrate in the medium and a water insoluble teal-green pigment will color the colony. However, since E. coli produces both galactosidase and glucuronidase, those colonies will be a combination of the teal-green and pink pigments and will appear as some shade of blue-purple.

There is, however, always the chance that an unusual strain of bacteria may be present and result in false negative or false positive readings. For example, the E. coli 0157:H7 does not produce the enzyme glucuronidase and so will appear as pink colonies indistinguishable from other general coliforms. And certain strains of other members of the family Enterobacteriaceae (some Salmonella and some Shigella) may produce the enzyme glucuronidase, although they do not produce galactosidase and therefore appear as teal-green colonies. These teal-green colonies do appear different than the general blue-purple E. coli, and fortunately another easy test screen for these various unusual bacterial types is available and may be used for verification after the initial readings. This is a spot test for the presence of indole, which is produced by virtually all strains of E. coli, but very few general coliforms or other members of the family Enterobacteriaceae. If the membrane filter containing the colonies that grew on the Coliscan MF medium is carefully lifted from the pad and placed on another pad which has been soaked with a reagent to detect the presence of indole, colonies that are indole positive will be indicated by the formation of a red (or magenta) zone around them within five minutes. Thus, all E. coli (including the non-purple O157:H7) will have the red zone, and non-E. coli (such as the teal-green Salmonella or Shigella) will not have the red zone. This simple additional test eliminates almost all possible false readings and adds further precision to the already excellent Coliscan MF method.

Sometimes waste waters or general surface waters also contain bacteria closely related to the coliforms that may grow on the Coliscan medium. Two that are common are Pseudomonas and Aeromonas species. Pseudomonas species are galactosidase and glucuronidase negative so colonies are not colored pink or purple, but they generally produce their own water-soluble bluish, greenish or yellowish pigments that form a diffused colored zone around colonies. A heavy growth over a membrane filter may give the entire normally white filter a colored appearance. Colonies producing the soluble pigments described are very easily verified as non-coliforms with the oxidase test. A small drop of colorless reagent on the colony gives a dark purple zone around the Pseudomonas colony within 15 seconds. Coliforms are negative and develop no color.

Aeromonas species are galactosidase positive and therefore grow as pink colonies like the general coliforms. However, if Aeromonas is suspected, they too can easily be differentiated from coliforms with the oxidase test. With the test reagent, Aeromonas colonies will develop a deep purple zone like Pseudomonas.

It is possible to eliminate these organisms from growing on the Coliscan medium through the use of various inhibitors and temperature control. However, they do not generally cause a problem with the general analysis. E. coli will still grow as deep blue-purple colonies. Debate continues on the importance of Aeromonas with many persons believing that it is as important an indicator as general coliforms. Likewise, the presence of Pseudomonas in water can be important from a sanitation and medical perspective.

Procedure:

1)Weigh 50 g food into sterile high-speed blender jar. Frozen samples can be softened by storing it for <18 h at 2-5°C, but do not thaw. This step should not be done with liquid samples.

2)Add 450 mL of Butterfield's phosphate-buffered water and blend for 2 min. If <50 g of sample are available, weigh portion that is equivalent to half of the sample and add sufficient volume of sterile diluent to make a 1:10 dilution. The total volume in the blender jar should completely cover the blades. This step need not be done with liquid samples.

3)Thaw the desired number of bottle(s) of Coliscan MF by leaving at room temperature overnight. For rapid same-day thawing, stand in warm water until liquid. All unused bottles should be left in freezer. Collect the water or food to be tested in the appropriate volume and dilution (see table below for water, and procedure above for food). It is best to do this within a couple hours prior to filtering or, if this is not possible, samples may be stored in refrigerator for no more than 24 hours.

4)Open a dropper or pipette and sterilely add 1.75-2 mL of Coliscan MF to a pad in the petri dish that is to be used.

5)Set up the filter apparatus and filter your sample:

If you are using a Coliscan MF filter apparatus:

  • The filter apparatus comes in a sterile pack. Open the pack and remove the apparatus. The clear top of the apparatus is the funnel, which is calibrated for 100 and 150 mL samples and is covered with a lid. It fits on the bottom collection container and is sealed with an O-ring. There is a side port with a tip for the attachment of the vacuum syringe. Twist it and it can also be removed. It contains a plug in its tip that can be removed. The contents of the bottom collection container are most easily poured out when the tip is removed. It is easily replaced by twisting back on.
  • To prepare the apparatus for use, remove the funnel and using a clean forceps place a sterile pad on the top grid-work (in the blue circle) of the container.
  • Open a sterile filter envelope and with the clean forceps, carefully remove the membrane filter from the pack. Be sure to separate the filter from the protective backing and handle the filter carefully so it is not torn or damaged. Place the filter, grid side up, on top of the sterile pad. Push the funnel down so that it is held and sealed by the O-ring and the filter and pad are held firmly in place. The funnel must be pushed down as far as possible to obtain a good seal.
  • Attach the syringe to the filter apparatus by pushing the end of the hose on to the side port tip of the funnel contained. Be sure that the syringe plunger is not pulled out.
  • Pour the diluted water or food sample into the funnel, swirl to mix and create a vacuum by pulling out the plunger of the syringe. The water will be pulled through the filter, depositing any microorganisms present onto the filter surface.
  • When the sample has been completely passed through the filter, disconnect the syringe, remove the funnel and with the clean forceps remove the filter and place grid side up directly on top of the pad of a dish prepared earlier. Make sure that there are no air spaces (bubbles) between the pad and the membrane filter. Place the lid back on the dish.
  • The filtered water in the collection container should be emptied and the filter apparatus prepared for repeat use. Before the funnel is used again it should cleaned. This may be done by rinsing with alcohol or radiating for 1 minute with germicidal UV. The absorbent pad can generally be reused as it will only contain filtered water (sterile).

If you are using a Nalgenefilter apparatus:

  • The filter apparatus comes in a sterile pack. Open the pack and remove the apparatus. The clear top of the apparatus is the funnel, which is calibrated for 150 mL samples and is covered with a lid. At the base of the funnel you will see a white filter. Do NOT touch this filter with your fingers or other unsterile items.
  • Open a sterile filter envelope and with the clean forceps, carefully remove the membrane filter from the pack. Be sure to separate the filter from the protective backing and handle the filter carefully so it is not torn or damaged. Place the filter, grid side up, in the center of the white filter in the base of the Nalgene apparatus funnel.
  • Attach the syringe or the vacuum supply to the filter apparatus by pushing the end of the hose on to the side port tip of the funnel contained. Be sure that the syringe plunger is not pulled out.
  • Using a sterile dropper pipet, drop the diluted water or food sample into the funnel covering the entire surface of the filter paper, as you create a vacuum by pulling out the plunger of the syringe or turning on the vacuum source. The water will be pulled through the filter, depositing any microorganisms present onto the filter surface. It is critical that you do NOT allow any of your sample to contact the outer white filter, or your bacteria will be lost and you will not be able to reuse the filter.
  • When the sample has been completely passed through the filter, disconnect the syringe or vacuum source, and with the clean forceps remove the filter and place grid side up directly on top of the pad of a dish prepared earlier. Make sure that there are no air spaces (bubbles) between the pad and the membrane filter. Place the lid back on the dish.
  • The filtered diluent in the collection container should be emptied and the filter apparatus prepared for repeat use. Before the funnel is used again it should cleaned. This may be done by rinsing with alcohol or radiating for 1 minute with germicidal UV.

6)Incubate in an incubator or a warm place. If using an incubator, incubate at 35̊ for 18- 24 hours. If an incubator is not available, find a place that will be warm for a 24 hour period. DO NOT place in direct sunlight or over a direct heat source, radiator, furnace duct etc. You may place them near one of these sources or in a warm spot in the kitchen. Allow 24-48 hours for growth to begin. Once growth begins you can incubate another 24 hours for complete growth to take place.

7)Once the incubation period is complete, a count of the colonies can be done. Count all blue colonies as E. coli (fecal coliform) and all pink colonies as general coliforms. The sum of these is the total coliform population. You can then use your dilution coefficients to calculate the total number of bacteria/coliforms/E.coli per mL of sample.