how galactosidase detects coliforms and glucuronidase detects most E. coli strains
Write a one to two page summary of the following learning objectives:
Understand:
- coliforms are indicators of fecal contamination and that most foodborne illness pathogens have fecal reservoirs
- the history of cholera and how it led to the need for sanitary water monitoring (why couldn’t we just look for V. cholera?)
- the risk of cholera today in US versus third world countries (not in materials – look it up)
- the differences in coliform, fecal coliform, and E. coli
- how galactosidase detects coliforms and glucuronidase detects most E. coli strains
- how Salmonella, Shigella, E. coli O157, and Enterobacter fit into the coliform, fecal coliform, E. coli indicator assay
- both the MPN and membrane filtration techniques for determining coliforms in samples
- how petrifilm products work to enumerate coliforms and E. coli
- coliform indicators are far from perfect and they have their deficiencies
This is module 20 – food safety microbiological indicators. While there may be many different indicators we will focus on coliforms. Coliform bacteria are a commonly used bacterial indicator of sanitary quality of foods and water.
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A majority of the foodborne illness bacteria have a fecal reservoir. [read genera]. This makes perfect sense, since many cause gastroenteritis as an infection in the small intestines.
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Cholera, a lethal diarrheal disease historically killed millions each year, especially during endemics. Robert Koch himself traveled to India during one such endemic to isolate the bacterial cause of the disease. In 1884, after over a year of work he was convinced the organism was Vibrio cholera. The organism proved difficult to culture.
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Just a year later, Escherichia coli, originally known as Bacterium coli, was identified in 1885 by the German pediatrician, Theodor Escherich. He noted that it was widely distributed in the intestine of humans and warm‐blooded animals. In 1892, Shardinger proposed the use of E. coli as an indicator of fecal contamination. This was based on the premise that E. coli is abundant in human and animal feces and not usually found in other niches. Furthermore, since E. coli could be easily detected by its ability to ferment lactose, it was easier to isolate than known gastrointestinal pathogens, especially Vibrio.
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The presence of E. coli in food or water became accepted as indicative of recent fecal contamination. Although the concept of using E. coli as an indicator of fecal presence, it was complicated in the lab, due to the presence of other bacteria including Citrobacter, Klebsiella and Enterobacter that can also ferment lactose. As a result, the term “coliform” was coined to describe this entire group of enteric bacteria. Coliform is not a taxonomic classification but simply a definition of this group of bacteria. In 1914, the U.S. Public Health Service adopted the enumeration of coliforms as a more convenient standard of sanitary significance.
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Coliforms are defined as rod‐shaped gram‐negative non‐spore forming bacteria that ferment lactose with the production of acid and gas when incubated at 35‐37°C. The table indicates the many media that have been created over the years to culture and enumerate coliforms. Most will permit growth in 24 hours and a few require 48 hour incubations. We’ll work with the yellow highlighted media in lab.
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Although coliforms were easy to detect, their association with fecal contamination was questionable because some coliforms are found naturally in environmental samples. This led to the introduction of the fecal coliforms as an indicator of contamination. Fecal coliforms were first defined based on the works of Eijkman. They are a subset of total coliforms that grow and ferments lactose at 44.5 to 45.5°C. Fecal coliform analyses are done at 45.5°C for food testing. Water, shellfish and shellfish harvest water analyses use 44.5°C. The photo depicts a filtered water sample placed over mFC medium. The petri dish was incubated at 45.5°C for 24 hours. The blue colonies are fecal coliforms. The blue is nitrophenol freed by the fecal coliforms beta‐galacotosidase enzyme.
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The fecal coliform group consists mostly of E. coli but some enterics such as Klebsiella can also ferment lactose at these temperatures and therefore, be considered as fecal coliforms. The inclusion of Klebsiella spp in the working definition of fecal coliforms diminished the correlation of this group with fecal contamination. As a result, E. coli itself has reemerged as an indicator, partly facilitated by the introduction of newer methods that can rapidly identify E. coli. The photo depicts a fecal coliform broth tube with 4‐methyl‐umbelliferone‐ glucuronide. Since only E. coli has the enzyme glucuronidase, it alone will cause the medium to fluoresce.
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Let’s summarize the three levels of coliform assays. The total coliform group will catch all lactose fermenters producing gas in 48h at 35‐37°C. This includes E. coli, Citrobacter, Enterobacter, and Klebsiella. A few Salmonella and Shigella can ferment lactose, but most cannot. Many pathogenic E. coli, including O157:H7 cannot ferment lactose. The next level are the fecal coliforms. A slightly more selective media is used together with higher incubation temperatures from 44.5 – 45.5°C. This narrows the group to E. coli and Klebsiella. Finally, newer specialty media can quickly isolate E. coli itself. These include media that use MUG.
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MPN is a procedure to estimate the numbers of viable microorganisms in a test sample using liquid media and the theory of probability. It is most often used for water. Water samples are inoculated into a series of media tubes usually in 3 or 5 replicates. Note that when more than 1 ml of water sample is used, the media is double strength to account for sample dilution of the media. Positive growth is monitored for the each replicate and recorded for each dilution. The most accurate data is obtained when the highest inoculum is all positive and the lowest inoculum is all negative. In this case we have an MPN of 3,2,1.
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Using the growth numbers from the assay of 3,2,1 for 10 ml, 1 ml and 0.1 ml respectively, click on the MPN for 100 mls of water.
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The MPN assay is suitable for water with expected coliform counts from 3‐1100 coliforms per 100 mls. However, it does not work as well statistically in the lower range of numbers. Another method to determine coliforms in water is the membrane filter method. This method is most often used to ensure potable water is free of coliforms. A special apparatus is required that incudes a vacuum pump and sample filters. First a sterile 0.45 micron filter membrane is placed on a grid where the clamp is. The clamp holds the top and bottom parts together. At the top, 100 mls of water is poured into the reservoir. A vacuum is applied and the water is pulled through the filter. Then, filters are removed and placed onto special membrane filter petri plates containing coliform media.
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Total coliforms are determined by placing the filters on mENDO broth soaked pads. These are incubated at 35 ºC for 24 hours. Fecal coliforms use mFC soaked pads and incubate these at 44.5 or 45.5 ºC. For 24 hours. Positive coliforms are dark red. Positive fecal coliforms are dark blue. Most potable water standards limit coliforms to none detected in 100 mls.
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Violet red bile agar (VRBA) is one of the most common coliform enumeration agars. Peptone and Yeast extract are a source of nitrogen, sulfur, carbon, vitamins and minerals. Bile salts and crystal violet are the inhibitors of gram‐positive microorganisms. Lactose is the fermentable carbohydrate. Neutral red dye changes to red‐purple due to acid formation from lactose fermentation. Finally, coliforms will precipitate bile salts around the colony. Pale colonies are negative.
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The 3 M company has created coliform pertrifilms. They are essentially violet red bile agar in a desiccated film. Either 1 ml or 5 ml of sample is added and the film is incubated for 24‐ 48 hours at 32 or 35 ºC. Red colonies with a gas bubble trapped are positive coliforms.
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The 3 M company also has created a more sensitive assay for just E. coli by adding a glucuronidase indicator dye. In this case glucuronidase positive colonies will be blue with a gas bubble. Glucuronidase negative colonies will be red with or without gas.
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Coliforms have been used as quality and sanitation standards for many years. Here are just some of the coliform standards for pasteurized milk, cooked frozen foods, and custard foods.
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Coliforms make a good indicator of fecal contamination, but they are far from perfect. The main problem is that coliforms can include soil Enterobacter. Salmonella and E. coli O157:H7 are actually fecal coliform negative. Therefore they may be present but not detected with tis assay. Coliforms are not halotolerant. Therefore they do not make a good choice for seafood. Despite this fact, coliforms are still used for seafood and seafood harvest water sanitation because its still the best indicator tool available.
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