RAPID MGP MEDIUM

INTENDED USE

Hardy Diagnostics Rapid MGP Medium is a five hour test used to differentiate enterococci based on the ability to acidify the carbohydrate methyl-alpha-D-glucopyranoside (MGP).

SUMMARY

Enterococcus species are common nosocomial pathogens. Enterococcus faecalis and Enterococcus faecium, components of the human intestinal tract, are the two most frequently encountered enterococci in the clinical laboratory.⁽⁵⁾ Other species, such as Enterococcus casseliflavus and Enterococcus gallinarum, are not commonly encountered in the clinical setting but are significant because they display intrinsic resistance to vancomycin. E. faecalis, E. faecium, E. gallinarum, and E. casseliflavus have similar phenotypic traits, and can be difficult to differentiate, particularly in the case of vancomycin-resistant E. faecium and E. gallinarum.^(3-6)

Biochemical methods for the differentiation of Enterococcus species have been presented, but are not entirely reliable as a result of strain to strain variation.^(3-6) PCR identification of these species based on vancomycin-resistant genes has shown to be reliable.⁽⁴⁾ Unfortunately, the time and resources are not always available for labs to perfrom PCR identifications. MGP Broth was introduced as a simple, inexpensive means of differentiating enterococci as reliably as PCR. MGP Broth, developed in 1996 by Devriese, et al., is a simple 24 hour carbohydrate acidification test that can distinguish different species of Enterococcus that are vancomycin-resistant. Enterococcus casseliflavus and Enterococcus gallinarum are MGP positive. Enterococcus faecalis and Enterococcus faecium are MGP negative.

This rapid medium is a modification of the MGP Broth formula presented by Devriese, et al.⁽²⁾ The Rapid MGP Medium can be read in five hours, a significant decrease from the 24 hour incubation of the original MGP Broth. Rapid MGP has a sensitivity of 100 percent and a specificity of 96.5 percent.

FORMULA

Ingredients per liter of deionized water.*

Final pH 7.4 +/- 0.2 at 25 degrees C.

* Adjusted and/or supplemented as required to meet performance standards.

STORAGE AND SHELF LIFE

Storage: Upon receipt store 2-8 degrees C. away from direct light. Product should not be used if there are any signs of contamination, deterioration, discoloration, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.

The expiration date applies to the product in its intact packaging when stored as directed.

This product has the following shelf life from the date of manufacture:

Refer to the keyword "Storage", in the Hardy Diagnostics software program HUGO, for more information on storing culture media.

PRECAUTIONS

For in vitro diagnostic and laboratory use only. Observe approved biohazard precautions and aseptic techniques. This product is to be used only by adequately trained and qualified laboratory personnel. Sterilize all biohazard waste before disposal.

Refer to the keyword "Precautions", in the Hardy Diagnostics software program HUGO, for more information regarding general precautions when using culture media.

PROCEDURE

Note: Rapid MGP Medium gives similar results when inoculated from Tryptic Soy Agar, Tryptic Soy Agar with 5% Sheep Blood, Brain Heart Infusion Agar with Vancomycin, Bile Esculin Azide Agar with Vancomycin, and Columbia CNA Agar.

1. Using a "sweep" of colonies from an 18-24 hour pure culture of the organism to be tested, stab the Rapid MGP Medium with an inoculating needle. There should be cell paste visible on the needle as the media is being inoculated.

2. Prepare positive and negative control tests with known cultures as described above.

3. Incubate the tubes aerobically at 35 degrees C. for five hours.

4. Observe for the development of a yellow color along the stab line, this is indicative of a positive test. When examining test results, compare results of the unknown culture(s) to results obtained with the known controls to ensure proper reading of the test.

5. Replace weak or inconclusive reactions in the incubator for a total of 24 hours, and read as described in step 4.

INTERPRETATION OF RESULTS

A positive test occurs when a yellow color develops along the line of inoculation. Stronger positive reactions will exhibit a yellow color change diffusing into the medium away from the stab line. A positive test is indicative of E. casseliflavus and E. gallinarum.

A negative test, indicative of E. faecalis and E. faecium, will remain blue. Some isolates will show a degree of lightening (from blue to light blue) in the medium due to the heavy inoculum. The discoloration is easily distinguished from the yellow positive reaction, particularly when seen in comparison to the positive and negative controls.

LIMITATIONS

It is recommended that biochemical and/or serological tests be performed on colonies from pure culture for complete identification.

Negative Rapid MGP tests may change from their original color, becoming fainter shades of blue. This color change is easily distinguished if positive and negative control tubes are performed in parallel with each test being performed. If multiple unknowns are being performed at one time, one positive and one negative control will suffice for all tests being run.

Do not over or under inoculate Rapid MGP Medium. A lack of sufficient inoculum can lead to false-negative results. Over inoculating the tubes can overwhelm the media, causing false-positive results. Use of an inoculating needle is recommended, as inoculums from a loop can overwhelm the media.

Inoculating Rapid MGP Medium with mixed cultures may result in erroneous results.

Reactions that are weakly positive, or difficult to interpret after five hours should be replaced in the incubator for a total of 24 hours.

Refer to the keyword "Limitations", in the Hardy Diagnostics software program HUGO, for more information regarding general limitations on culture media.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, other culture media, swabs, applicator sticks, incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

The following organisms are routinely used for testing at Hardy Diagnostics:

User Quality Control: User Quality Control: Check for signs of contamination and deterioration. Users of commercially prepared culture media may be required to perform quality control testing with at least one known organism to demonstrate growth or a positive reaction; and at least one organism to demonstrate inhibition or a negative reaction (where applicable). Refer to the following keywords in HUGO for more information on QC: "Introduction to QC", "User QC", "QC of Finished Product", "Licensing Requirements for Media QC", and "Media not Requiring User QC". Also, see listed references for more information.^(5,6)

* Rapid MGP Medium is inoculated using a needle to obtain a "sweep" of colonies from a pure culture, and stabbing the medium once.

Physical Appearance: Rapid MGP Medium should appear slightly opalescent, and blue in color.

REFERENCES

1. Carvalho, M. G. S., et al. 1998. Use of tests for acidification of methyl-alpha-D-glucopyranoside and susceptibility to efrotomycin for differentiation of strains of Enterococcus and related genera. J. Clin. Microbiol. 36:1584-1587.

2. Devriese, L A., et al. 1996. Acidification of methyl-alpha-D-glucopyranoside: a useful test to differentiate Enterococcus casseliflavus and Enterococcus gallinarum from Enterococcus faecium species group and Enterococcus faecalis. J. Clin. Microbiol. 34:2607-2608.

3. Murray, P. R., et al. 1999. Manual of Clinical Microbiology, 7th ed. American Society for Microbiology, Washington D.C.

4. Facklam. R. R., and M. D. Collins. 1989. Identification of Enterococcus species isolated from human infections by a conventional test scheme. J. Clin. Microbiol. 27:731-734.

5. Gin, A. S., and G. G. Zhanel. 1996. Vancomycin-resistant enterococci. Ann Pharmacother. 30:615-624.

6. Hanson, K. L. and C. P. Cartwright. 1999. Comparison of simple and rapid methods for identifying enterococci intrinsically resistant to vancomycin. J. Clin. Microbiol. 35:2526-2530.

7. Turenne, C. Y., et al. 1998. Screening of stool samples for identification of vancomycin-resistant Enterococcus isolates should include the methyl-alpha-D-glucopyranoside test to differentiate non-motile Enterococcus gallinarum from E. faecium. J. Clin. Microbiol. 36:2333-2335.

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