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MEMBRANE LACTOSE GLUCURONIDE AGAR (MLGA)

Code: CM1031

A medium for the differentiation and enumeration of Escherichia coli and other coliforms by a single membrane filtration technique.

Typical Formula*

gm/litre

Peptone

40.0

Yeast extract  

6.0

Lactose

30.0

Phenol red     

0.2

Sodium lauryl sulphate

1.0

Sodium pyruvate  

0.5

Agar

10.0

X-Glucuronide (BCIG)  
0.2

pH 7.4± 0.2 @ 25°C

* Adjusted as required to meet performance standards 

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.

Appearance
Dehydrated medium: Straw coloured, free-flowing powder
Prepared medium: Bright red coloured gel

Quality control

Positive controls:

Expected results

Escherichia coli ATCC® 25922 *

Good growth; green coloured colonies

Enterobacter aerogenes ATCC®13048 *

Good growth; yellow coloured colonies

Pseudomonas aeruginosa ATCC®27853 *Good growth; pink coloured colonies

Negative control:

 

Bacillus subtilis ATCC®6633 *

Inhibited

References
1. The Environment Agency - Methods for Examination of Waters and Associated Material - The Microbiology of Drinking Water 2002.

 
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