Is it possible to get a high count of E. coli than faecal coliforms when using the m-LGA (Membrane Lactose Glucuronide Agar) which is used for the differentiation and enumeration of Escherichia coli and other coliforms through the membrane filtration technique?
Directions
Suspend 88g in 1 litre of distilled water. Mix well and sterilize by autoclaving at 121°C for 15 minutes. Cool the medium to 50°C and pour into sterile Petri dishes.
Description
Tests for coliforms and Escherichia coli are the most important routine microbiological examinations carried out on drinking water. They provide the most sensitive means for detection of faecal contamination, for assessing the effectiveness of water treatment and disinfection, and for monitoring water quality in distribution1.
Organisms are isolated on a membrane filter and placed onto MLGA. The medium contains lauryl sulphate to inhibit Gram-positive organisms. Identification of Escherichia coli and coliforms is facilitated through two biochemical reactions within the medium:-Lactose fermentation is detected by the dye phenol red which gives yellow colonies when acid is produced.
The chromogenic substrate 5-bromo-4-chloro-3-indolyl-ß-D-glucuronide (BCIG) is cleaved by the enzyme glucuronidase and produces a blue chromophore which builds up in the bacterial cells.
Coliforms are lactose-positive so colonies will be yellow; Escherichia coli is both lactose-positive and possesses glucuronidase so will appear as green colonies1
Technique
For the full methodology refer to The Environment Agency - Methods for Examination of Waters and Associated Material – ‘The Microbiology of Drinking Water 2002, Part 4, Method B: The enumeration of coliforms and Escherichia coli by single membrane filtration technique.’
Filter the water sample to be analysed through a membrane filter (47mm diameter, cellulose-based 0.45µm nominal pore size). The volume and dilution of water filtered should be chosen to give the number of colonies to be counted on the membrane as 20-80. For treated waters 100ml should be filtered. For polluted waters a smaller volume or a diluted sample should be used.
Place the membrane filter onto a MLGA plate ensuring that no air-bubbles are trapped under the membrane. Incubate the plates at 30°C for 4 hours, then at 37°C for 14 hours. For an early indication of results, plates may be examined for colonies at 12 hours but must be re-incubated for the full 18 hours.
Count all yellow and green colonies. The yellow colonies are presumptive non-Escherichia coli coliform bacteria and the green colonies are Escherichia coli. The combined count can be regarded as the number of coliform bacteria. Results are expressed in colony forming units per volume of sample.
The specificity of the reactions within the medium means the likelihood of green colonies on MLGA being Escherichia coli is very high. Following suitable confirmation of performance within the laboratory, confirmation of green colonies may not be needed. If confirmation tests are required please refer to The Environment Agency document for the full methodology1
Storage conditions and Shelf life
The dehydrated medium should be stored at 10-30°C and used before the expiry date on the label.
Store the prepared medium may be stored for up to 1 week at 2-8°C.
Thanks Mehdi Rostami Rad. I already have the method. I was wondering if it is possible to get a high E. coli count compared to faecal coliforms on the same petri dish using MLGA. Rephrasing the question, are the yellow colonies (FC) always expected to be more than the green colonies (E. coli)?
Hi Bernice,
It is important to note that E. coli are faecal coliforms. In order to get a true total of faecal coliforms, you would need to add the E. coli and faecal coliforms. See the attached venn diagram link.
I've done thousands of bacteriological tests on environmental water samples and I can honestly say that there are some days when the majority of fecal coliforms that are present on my media are E. coli. There are also some days when I have high amounts of fecal coliforms and none of these fecal coliforms are E. coli. *Generally speaking, when E. coli are the dominant fecal coliform present, some researchers speculate that the water is more likely impacted by fecal contamination as E. coli are viewed as a better indicator of fecal contamination than fecal coliforms as a broad group since many fecal coliforms survive better in nature than E. coli - however, E. coli can also survive well in nature too outside the GI tract.
In brief, if you have more yellow colonies on this type of media you reference, then the majority of fecal coliforms are non-E. coli.
If you have more green colonies on this type of media (agar) that you reference, then the majority of fecal coliforms present are E. coli.
It is impossible for E. coli to outnumber total coliforms or fecal coliforms since E. coli are actually in those larger groups.
Let me know if you need any further clarification.
*Also - I'm not sure that your agar is measuring fecal coliforms or total coliforms too. There is a difference between these two groups. Total coliforms represent a larger group inclusive of the fecal coliforms. https://www.ncbi.nlm.nih.gov/pubmed/18492047
http://www.whatcomcd.org/sites/default/files/water-quality/FC_EColi_Ven_Diagram.png
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