Another common test in our laboratory is the detection of Escherichia coli. From Ying Ying’s first posting, we know that is LTB tubes are positive (gas production), we will have to continue by inoculating a loop of the positive LTB tube into a BGB and a EC tube.
If the food sample is positive for EC tube (gas production), we can conclude that the bacterium is a lactose fermenter. Further tests to confirm whether it is Escherichia coli needs to be carried out.
A loopful of the culture from the EC tube is being streaked onto an Eosin Methylene Blue agar.
Eosin Methylene Blue agar (EMB)
- Consists of Eosin Y and Methylene Blue dyes which inhibit gram-positive bacteria growth and differentiates lactose fermenting and non-lactose fermenting bacteria
- Lactose fermenting bacteria will appear blue-black
- Non-lactose fermenting bacteria will appear colourless and transparent.
- If positive for Escherichia coli, purple colour colonies with green metallic sheen will grow.
E.coli on EMB plate
After incubating for 1 day at 35◦C, the EMB plate is taken out. If positive for Escherichia coli, purification of a single colony for the EMB is done and incubated.
After purification, biochemical tests are being carried out.
Indole test
- If positive for Escherichia coli, a red ring is being formed when Kovac’s reagent is added. This is because Escherichia coli produces tryptophanase which cleaves tryptophan to produce indole and other products. Kovac’s reagent will then react with indole to form a red ring. (Positive indole test)
Methyl Red test
- If positive for Escherichia coli, the methyl red indicator will turn red due to the acids produce by the degradation of glucose, lowering the pH. (Positive MR test)
Voges-Proskauer test
- If positive for Escherichia coli, there will be no change. This is because E.coli does not produce acetyl methyl carbinol which will react with alpha-napthol and potassium hydroxide to produce pink colour after standing for about 1 hour. (Negative VP test)
Simmon’s Citrate test
- If positive for Escherichia coli, there will be growth on the surface of the Simmon’s Citrate slant but no colour change from green to blue.
Gram-stain
- If positive for Escherichia coli: gram-negative bacillus. (pink, rod-shaped)
Gram stain of E.coli
Results of the biochemical tests are then recorded and compared with the results of the control to check whether it is positive for Escherichia coli.
After incubating for 1 day at 35◦C, the EMB plate is taken out. If positive for Escherichia coli, purification of a single colony for the EMB is done and incubated.
After purification, biochemical tests are being carried out.
Indole test
- If positive for Escherichia coli, a red ring is being formed when Kovac’s reagent is added. This is because Escherichia coli produces tryptophanase which cleaves tryptophan to produce indole and other products. Kovac’s reagent will then react with indole to form a red ring. (Positive indole test)
Methyl Red test
- If positive for Escherichia coli, the methyl red indicator will turn red due to the acids produce by the degradation of glucose, lowering the pH. (Positive MR test)
Voges-Proskauer test
- If positive for Escherichia coli, there will be no change. This is because E.coli does not produce acetyl methyl carbinol which will react with alpha-napthol and potassium hydroxide to produce pink colour after standing for about 1 hour. (Negative VP test)
Simmon’s Citrate test
- If positive for Escherichia coli, there will be growth on the surface of the Simmon’s Citrate slant but no colour change from green to blue.
Gram-stain
- If positive for Escherichia coli: gram-negative bacillus. (pink, rod-shaped)
Gram stain of E.coli
Results of the biochemical tests are then recorded and compared with the results of the control to check whether it is positive for Escherichia coli.
~Jeremy~
TG01
9 comments:
Heyy,
For the Simmon’s Citrate test, can all organisms grow on the slant? What is the requirement for a colour change to occur?
Thanks!
Kent Lieow
TG01
hey
hmm... why is EMB agar used instead of normal blood agar or MAC? do u isolate E.coli on EMB after comfirmation from biochemical tests or from the start? In that case, would it be expensive?
Hi Jemmy,
Here's a simple question. What do you do with the results (i.e. Do you inform the company that their food/water sample contains Escherichia coli) and if so, what can they do about it?
Desmond Heng
0503179D
TG02
Hey jeremy,
What is the purpose of inoculating a loop of the positive LTB tube into a BGB and a EC tube?
Andre, TG01
Hey
How is purification of a single colony for the EMB is done? Care to explain more. Thanks :-)
Eugene Wong
TG02
HELLO! =D
What chemical reaction/interaction is involve in the formation of green metallic sheen in EMB?
Tang Jiaxin
TG01
to kent:
not all organisms can grow on the slant. only organisms that are able to utilize ammonium dihydrogen phosphate and sodium citrate as the sole source of nitrogen and carbon will produce an alkaline reaction by changing the bromthymol blue indicator from green to blue.
to medbankers:
this is because our lab follows the regulations from the FDA. we perform a streak on the EMB plate once the EC tube shows a positive results. as EMB is a selective media, it should be quite an expensive plate. (last answer was the staff. lol)
To desmond:
lol stop calling such gay names. we just record down those results. If they fail the testing, most probably we will collect the same food next time if possible. But if it is a food outbreak case, we will report back to MOH. they will decide what action to take but we are not informed... haha...
to andre:
that is to test for the presence of coliforms. BGB is for coliforms while EC is for faecal coliforms. as e.coli belongs to the faecal coliform family, streaking on EMB is only done to EC positive tubes.
To eugene:
its just picking one colony from the 1st plate that u streaked from the EC tube. this is to ensure only one colony is grown. only purified colony can be used for biochemical tests. But if they do not have the characterisitics of e.coli on the EMB plate, no purification is needed. result is recorded as e.coli negative.
to jia xin:
any gram-negative bacilli that is a fermentor of sucrose and lactose would produce a green metallic sheen... haha... hope this helps...
~Jeremy~
TG01
hi jeremy,
can i know what if some tests give positive and some give negative results? does it means it has E coli anot?
Ai Tee
TG 01
to ai tee:
nope. the biochemical test results must be similar to conclude that it is e.coli. if its only one result that doesnt match. u should check the control and see whether it gives the same result because that batch of reagent might not be working. if control is normal, then it can be concluded that it is not e.coli.
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