Coliscan MF for the Identification and Differentiation of E. coli and General Coliforms from Liquid Samples
The patented Coliscan method is a means to separate and identify E. coli (fecal coliforms) and general coliform members of the family Enterobacteriaceae from other bacterial types.
Coliscan MF (membrane filter) medium is a nutrient liquid formulation which 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). A 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.
Therefore, the growth and identification of E. coli and general coliforms is a simple one step procedure with the Coliscan MF method. Results can generally be read in 18-20 hours if incubation is in the range of 34-37° C, and the overall accuracy and ease of use surpasses the widely used m-TEC method.
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.
There are other tests available for either E. coli or coliforms which use single chromogens and which will identify either E. coli or coliforms, but not both in the manner which Coliscan does. The use of our patented process gives the best assurance that the colonies counted as E. coli are correct because Coliscan tests for the presence of the two enzymes produced by the great majority of E. coli, while single chromogen tests for E. coli detect only the single enzyme glucuronidase. Therefore, with Coliscan, only deep blue-purple colonies are counted as E. coli, and the color is a combination of the two chromogens. General coliforms (which produce only galactosidase) are pink-red and glucuronidase only producers are a teal-green color.
Sometimes waste waters or general surface waters also contain bacteria closely related to the coliforms which 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 which 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.
Coliscan has numerous advantages over the other methods for water testing.
- Incubation temperature is not critical. Accurate results occur over a range from ambient to 37° C.
- Coliscan visually differentiates and identifies E. coli and general coliforms from other members of the Enterobacteriaceae in one simple test procedure.
- Test results in 24 hours or less.
- May be used for other indicator organisms such as Pseudomonas and Aeromonas species without interfering with E. coli and general coliform growth and expression.