RAPID MGP MEDIUM

Cat. no. Z225 Rapid MGP Medium, 0.5ml 20 tests/box

INTENDED USE

Hardy Diagnostics Rapid MGP Medium is a five hour test used to differentiate Enterococcus spp. 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 perform 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. (2)

FORMULA

Ingredients per liter of deionized water.*

Basal Medium 15.0gm
Indicator 50.0mg
Methyl-alpha-D-glucopyranoside 50.0gm

Final pH 7.4 +/- 0.2 at 25ºC.

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

STORAGE AND SHELF LIFE

Storage: Upon receipt store 2-8ºC 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, and freezing.

PRECAUTIONS

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 an inoculating needle, collect a paste from several colonies of a 18-24 hour pure culture of the organism to be tested. Stab the Rapid MGP Medium with the inoculating needle. There should be cell paste visible on the tip of 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 35ºC for up to five hours.

4. Observe for the development of a yellow color along the stab line, this is indicative of a positive test. Hold the negatives for at least five hours before discarding. 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

Negative Rapid MGP tests may change from their original color, becoming fainter shades of blue to blue-green. 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.

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

Test Organisms
Inoculation Method*
Incubation
Results
Time
Temperature
Atmosphere
Enterococcus gallinarum

ATCC ® 49573
* 5hr 35°C Aerobic Media turns yellow along line of inoculation
Enterococcus casseliflavus

ATCC ® 25788
* 5hr 35°C Aerobic Media turns yellow along line of inoculation
Enterococcus faecalis

ATCC ® 29212
* 5hr 35°C Aerobic Media remains blue or a shade of blue

* Rapid MGP Medium is inoculated using a needle to collect a paste from several colonies from a pure culture, and stabbing the medium once.

USER QUALITY CONTROL

PHYSICAL APPEARANCE

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

Positive MGP Test

Enterococcus gallinarum (ATCC ® 49573) growing in Rapid MGP Medium (Cat. no. Z225). The yellow color change along the stab line is indicative of a positive MGP test. Incubated aerobically for five hours at 35ºC.

Negative MGP Test

Enterococcus faecalis (ATCC ® 29212) growing in Rapid MGP Medium (Cat. no. Z225). No yellow color change along the stab line is indicative of a negative MGP test. Incubated aerobically for five hours at 35ºC.

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. Jorgensen., et al. Manual of Clinical Microbiology, 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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041116gr