ChatterBank1 min ago
presumptive coliform bacilli
what are these? are they gas forming bacteria?
any other info would be great!!
TIA
any other info would be great!!
TIA
Answers
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Presumptive coliform bacteria are those that pass the presumptive coliform test. That's it! So I think I'd better tell you about the test.
Going back to basics, coliform organisms are mainly members of the Escherichia and the Aerobacter genera. They are all aerobic and facultative gram negative non-spore-forming bacilli capable of fermenting lactose with the production of gas.
All coliform bacteria must be able to ferment the lactose in nutrient lactose broth or lauryl tryptose broth with the production of gas. After this, they must either be able to grow on eosin-methylene blue agar OR ferment the lactose in brilliant green bile lactose broth.
The growth of the bacilli in nutrient lactose broth with gas production when incubated at 37 degrees C over a 48 hour period is the first part of the presumptive test. Some laboratories add Bromocresol Purple to the broth which turns yellow as the lactic acid is produced. The actual volume of gas produced is irrelevant and most laboratories nowadays don't even bother measuring it. The second part of the presumptive test is the ability of the bacilli to form distinct colonies on the EMB agar when smeared using platinum loops or suchlike.
The reason this technique is used is that the coliform group can grow but any anaerobic spore-forming bacteria contaminating the sample perish as they cannot grow aerobically. In a similar fashion the Eosin and Methylene Blue present on the agar are bactericidal to many gram positive other species.
(continued)
Going back to basics, coliform organisms are mainly members of the Escherichia and the Aerobacter genera. They are all aerobic and facultative gram negative non-spore-forming bacilli capable of fermenting lactose with the production of gas.
All coliform bacteria must be able to ferment the lactose in nutrient lactose broth or lauryl tryptose broth with the production of gas. After this, they must either be able to grow on eosin-methylene blue agar OR ferment the lactose in brilliant green bile lactose broth.
The growth of the bacilli in nutrient lactose broth with gas production when incubated at 37 degrees C over a 48 hour period is the first part of the presumptive test. Some laboratories add Bromocresol Purple to the broth which turns yellow as the lactic acid is produced. The actual volume of gas produced is irrelevant and most laboratories nowadays don't even bother measuring it. The second part of the presumptive test is the ability of the bacilli to form distinct colonies on the EMB agar when smeared using platinum loops or suchlike.
The reason this technique is used is that the coliform group can grow but any anaerobic spore-forming bacteria contaminating the sample perish as they cannot grow aerobically. In a similar fashion the Eosin and Methylene Blue present on the agar are bactericidal to many gram positive other species.
(continued)
The final part of the technique is to return one or more of the agar colonies to a new nutrient lactose broth sample in a fermentation tube and also to a plain agar slant. If the organisms produce gas in the incubated fermentation tube, then the agar-slant is checked for spores and a negative gram's reaction. This test will eliminate aerobic, lactose-fermenting, gram positive spore-formers.
What you have left has passed the presumptive coliform test.
I would add that some laboratories look for gas production after 24 hours and there are other culture media available as well as the ones cited.
What you have left has passed the presumptive coliform test.
I would add that some laboratories look for gas production after 24 hours and there are other culture media available as well as the ones cited.
It's years since I done this stuff, so I can't be certain if my little grey cells have still retained this information properly.
Anyhow as I remember, in the methylene blue reduction test, 1.0ml of a freshly prepared solution of methylene blue thiocyanate is mixed with 10 ml of a liquid food sample,
such as milk. Tubes of the blue-coloured mixture are usually held at 0-4.4"C if it is not immediately convenient to incubate them. The tubes of samples are placed in a thermostatically controlled water bath with sufficient water to heat the samples to 36�C within 10 minutes of incubation. The water level is maintained above the level of the tubes' contents and the samples are protected from light. During incubation the samples are observed for colour change. The time taken for colour change in the test sample from blue to colourless is inversely proportional to the number of metabolically active organisms in the sample.
The same technique is used in reduction tests
involving or 2,3,5 triphenyl tetrazolium chloride (TTC) aka resazurin. Resazurin is used in two testing procedures
to assess the microbial quality of milk: the 1 hour test and the triple reading test. In the 1 hour test the extent of colour change is observed after 1 hour of incubation. The triple reading test involves measuring the time required (up to 3 hours) for reduction of resazurin to a specified colour end point. In reduction tests involving tetrazolium salts, TTC is most often used because it is less toxic to bacteria.
Anyhow as I remember, in the methylene blue reduction test, 1.0ml of a freshly prepared solution of methylene blue thiocyanate is mixed with 10 ml of a liquid food sample,
such as milk. Tubes of the blue-coloured mixture are usually held at 0-4.4"C if it is not immediately convenient to incubate them. The tubes of samples are placed in a thermostatically controlled water bath with sufficient water to heat the samples to 36�C within 10 minutes of incubation. The water level is maintained above the level of the tubes' contents and the samples are protected from light. During incubation the samples are observed for colour change. The time taken for colour change in the test sample from blue to colourless is inversely proportional to the number of metabolically active organisms in the sample.
The same technique is used in reduction tests
involving or 2,3,5 triphenyl tetrazolium chloride (TTC) aka resazurin. Resazurin is used in two testing procedures
to assess the microbial quality of milk: the 1 hour test and the triple reading test. In the 1 hour test the extent of colour change is observed after 1 hour of incubation. The triple reading test involves measuring the time required (up to 3 hours) for reduction of resazurin to a specified colour end point. In reduction tests involving tetrazolium salts, TTC is most often used because it is less toxic to bacteria.
When added to milk, TTC undergoes two colour changes
during reduction. In the first change the indicator turns pink due to the formation of resorufin. This change results from the loss of an oxygen atom loosely bound to the nitrogen on the phenoxazine structure and is not reversible. In the second change, the pink resorufin is reduced to dihydroresorufin, which is colourless. This change is easily reversed in the presence of atmospheric oxygen. TTC is colourless when oxidized but gives a red colour when reduced.
Traditionally, methylene blue and resazurin reduction tests have been utilized to determine the microbial quality of milk and ice cream. These tests are used in the grading of raw milk and can be adapted for use at dairy processing plants, receiving stations, cheese factories and similar dairy operations. they are easy to perform to perform and allow simultaneous testing of numerous food samples. The TTC reduction test has been applied in predicting shelf life of pasteurized milk and cream and for assessing contamination levels of food contact surfaces. The production of a red colour from reduction of TTC on areas of food contact surfaces represents sites of bacterial activity and/or soiled
areas.
during reduction. In the first change the indicator turns pink due to the formation of resorufin. This change results from the loss of an oxygen atom loosely bound to the nitrogen on the phenoxazine structure and is not reversible. In the second change, the pink resorufin is reduced to dihydroresorufin, which is colourless. This change is easily reversed in the presence of atmospheric oxygen. TTC is colourless when oxidized but gives a red colour when reduced.
Traditionally, methylene blue and resazurin reduction tests have been utilized to determine the microbial quality of milk and ice cream. These tests are used in the grading of raw milk and can be adapted for use at dairy processing plants, receiving stations, cheese factories and similar dairy operations. they are easy to perform to perform and allow simultaneous testing of numerous food samples. The TTC reduction test has been applied in predicting shelf life of pasteurized milk and cream and for assessing contamination levels of food contact surfaces. The production of a red colour from reduction of TTC on areas of food contact surfaces represents sites of bacterial activity and/or soiled
areas.
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