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MINERALS MODIFIED MEDIUM BASE WITH SODIUM GLUTAMATE

Code: CM0607

* Adjusted as required to meet performance standards

Sodium glutamate is provided along with the base medium under the CM0607 order code

Directions
Double Strength Dissolve 5g of ammonium chloride in 1 litre of distilled water. To this add 22.7g of Minerals Modified Medium Base, and 12.7g of Sodium glutamate LP0124. Mix to dissolve completely. Sterilise by autoclaving for 10 minutes at 116°C; alternatively heat to 100°C for 30 minutes on three successive days.

Single strength Dissolve 2.5g of ammonium chloride in 1 litre of distilled water. To this add 11.4g of Minerals Modified Medium Base, and 6.4g of Sodium glutamate LP0124. Mix to dissolve completely. Sterilise by autoclaving for 10 minutes at 116°C; alternatively heat to 100°C for 30 minutes on three successive days.

Note
To improve the stability of the dehydrated medium on storage the sodium glutamate LP0124 is supplied separately and must be added to the basal medium CM0607.

The pH of the final medium is critical for optimum performance and the sterilised broth should be checked to confirm that it is at pH 6.7 before use.

Differences in heating procedures cause differences in final pH value. If necessary the heating procedure should be adjusted so that the final pH, after sterilisation is 6.7¹.

Description
A chemically defined medium based on glutamic acid was first advocated by Folpmers² for the enumeration of the coliform group of bacteria in water.

The Public Health Laboratory Service³ carried out a trial and concluded that glutamic acid media containing glucose gave too many false positives in 48 hours. Gray⁴ modified a glutamate medium containing lactose and later published a formulation for an improved Formate Lactose Glutamate Medium⁵.

This latter medium was incorporated in another large trial carried out by the PHLS⁶ in which three glutamate media were compared with Teepol Broth (Jameson & Emberly⁷) and MacConkey Broth. The results showed that Gray’s improved formate lactose glutamate medium was superior to the other glutamate media on trial.

The report carried criticism of the mineral content of the medium and it was considered that it could be improved by modifying the amounts of minerals.

A co-operative investigation was carried out between the Metropolitan Water Board Laboratories and Oxoid Laboratories which resulted in a Minerals Modified Glutamate Medium CM0289.

The Oxoid Minerals Modified Glutamate Medium was used in further PHLS⁶ trials and the results with the Oxoid medium confirmed the superior performance of glutamate media reported previously (PHLS⁶).

The superior performance of Minerals Modified Glutamate Medium over MacConkey Broth is due mainly to improved detection of Escherichia coli. The table (adapated from PHLS⁸) illustrates the results obtained in the trial.

The table shows that for chlorinated water, incubation for >18 hours is required for glutamate media to demonstrate their superiority.
The medium and method are fully described in The Microbiology of Drinking Water2002¹.

More recently further trials showed Minerals Modified Glutamate Medium to be the medium of choice for the detection of Escherichia coli in chlorinated waters, especially where the numbers of organisms concerned were small.

It was also found better than Lauryl Tryptose Lactose Broth for detection of small numbers of Escherichia coli in other water, although the latter medium gave quicker results (18-24 hours compared to the 48 hours required by Minerals Modified Glutamate Medium).

Papadakis¹⁰ investigated the isolation of Escherichia coli from sea-water and found Minerals Modified Glutamate Medium to be better than MacConkey Broth formulations. However, to avoid high salt concentrations in the broth he recommended 1ml only of sea-water to be added to 10ml of single-strength MMG medium. Higher volumes of sea-water must be diluted out 1/10 with MMG medium.

Technique
The technique known as the Multiple Tube Method, Dilution Method or the Most Probable Number (MPN) method is used with Minerals Modified Glutamate Medium. A trial comparing membrane filtration and multiple tube methods showed glutamate medium to be unsatisfactory for use with membranes for enumerating coliform organisms in water¹¹.

With waters expected to be of good quality, the medium should be inoculated with one 50ml volume and five 10ml volumes. With waters of more doubtful quality, five 1ml volumes should be used in addition to the 50ml and 10ml volumes. Dilutions of the 1ml volumes may be required for polluted water and the 50ml volume may be omitted.

The larger volumes of water (10ml and 50ml) are added to equal volumes of double-strength medium, whereas the 1ml volumes (or dilutions of them) are added to 5ml of single-strength medium.

The tubes are incubated at 35°C and examined after 18-24 hours. All those tubes showing acid (yellow colour in the medium) and gas in the inverted inner (Durham) tube should be regarded as `presumptive positive’ tubes, including those in which gas appears after tapping the tube. The tube may only have a bubble of gas after tapping. The remaining tubes should be re-incubated and examined after another 24 hours. Any further tubes becoming `positive’ should be treated as `presumptive positives’.

Each `presumptive positive’ tube should be sub-cultured to a tube of Brilliant Green Bile (2%) Broth CM0031 and incubated for 24 hours at 44°C.
At the same time a tube of 1% Tryptone Water CM0087, should be inoculated for the production of indole after 24 hours at 44°C.
The production of gas from lactose at 44°C and the production of indole at 44°C are accepted in the United Kingdom as evidence of Escherichia coli.

Samples of chlorinated water giving `presumptive positive’ tubes must be tested to exclude false positive results due to aerobic or anaerobic spore-bearing organisms that produce gas. Sub-cultures are made into Brilliant Green Bile (2%) Broth and incubated at 35°C for 48 hours. Production of gas within 48 hours can be taken as sufficient confirmation that coliform organisms are present. If the tubes are sub-cultured to MacConkey Agar CM0007 at the same time, the colonial morphology of the organisms can easily be obtained for further differential tests.
A further multi-laboratory trial has demonstrated the efficiency of Lauryl Tryptose Mannitol Broth as a single tube confirmatory test of Escherichia coli¹².

The Most Probable Number of organisms can be calculated from the tables in Appendix C of HMSO Report 71¹.

Comparison of Minerals Modified Glutamate Medium and MacConkey Broth by Number of Positive Tubes

Modified direct plate method for counting Escherichia coli in food
A direct plate method (DPM) for the rapid enumeration of Escherichia coli. in foods has been described¹³. This method was modified by a resuscitation procedure using Minerals Modified Glutamate Agar¹⁴. In the modified method 15g of agar per litre is added to Oxoid Minerals Modified Glutamate Broth CM0607. Using this resuscitation stage the authors have recovered damaged cells from frozen, dried, heat-processed or low pH foods.

Abbiss et al.¹⁵ made a comparative assessment of the performance of Minerals Modified Glutamate Medium against three other enrichment broths in the enumeration of coliform organisms present in soft cheese, cooked meat and paté. Minerals Modified Glutamate Medium was superior in sensitivity to Lauryl Sulphate Tryptose Broth, MacConkey Broth and Brilliant Green Bile Broth.

Quality control of the medium includes testing in accordance with ISO 111333:2014^16.

Storage conditions and Shelf life
Store the dehydrated medium at 10-30°C and use before the expiry date on the label.
Store the prepared medium at 2-8°C.

Appearance
Dehydrated medium: Mauve coloured, free-flowing powder
Prepared medium: Purple coloured solution

Quality Control

* This organism is available as a Culti-Loop®

Precautions
Presumptive positive tubes must be sub-cultured to Lauryl Tryptose Mannitol Broth CM0831 and incubated at 44°C to detect indole formation at this temperature before the identification of Escherichia coli can be made.

References
1. The Environment Agency (2002) The Microbiology of Drinking Water 2002. Methods for the Examination of Waters and associated Materials.
2. Folpmers T. (1948) Ant. v. Leeuwenhoek, J. Microbiol. Serol. 14. 58-64.
3. PHLS Water Sub-Committee (1958) J. Hyg. Camb. 56. 377-388.
4. Gray R. D. (1959) J. Hyg. Camb. 57. 249-265.
5. Gray R. D. (1964) J. Hyg. Camb. 62. 495-508.
6. PHLS Standing Committee on Bacteriological Examination of Water Supplies (1968) J. Hyg. Camb. 66. 67-82.
7. Jameson J. E. and Emberly N. W. (1956) J. Gen. Microbiol. 15. 198-204.
8. PHLS Standing Committee on the Bacteriological Examination of Water Supplies (1969) J. Hyg. Camb. 67. 367-374.
9. Joint Committee of the PHLS and Standing Committee of Analysts (1980) J. Hyg. Camb. 85. 35-48.
10. Papadakis J.A. (1982) 6th Workshop on Marine Pollution of the Mediterranean, Cannes.
11. PHLS Standing Committee on the Bacteriological Examination of Water Supplies (1972) J. Hyg. Camb. 70. 691-705.
12. Joint Committee of the PHLS and Standing Committee of Analysts (1980) J. Hyg. Camb. 15. 51-57.
13. Anderson J. M. and Baird-Parker A. C. (1975) J. Appl. Bact. 39. 111-117.
14. Holbrook R., Anderson J. M. and Baird-Parker A. C. (1980) Food Technology in Australia, 32. 78-83.
15. Abbiss J. S., Wilson J. M., Blood R. M. and Jarvis B. (1981) J. Appl. Bact. 51. 121-127.
16. ISO 11133:2014 Microbiology of food, animal feed and water - Preparation, production, storage and performance testing of culture media