K9101 MOT DISCS (50)

PRINCIPLES/DISCUSSION:

It is known that approximately 50% of all clinical isolates and 80% of gram-negative bacilli are from the family Enterobacteriaceae(1); the most common of which is Escherichia coli. Most Enterobacteriaceae can be recognized by their reactions to a few chemical compounds. M.O.T. discs provide the tests (ONPG, Indole, MUG and Indol-pyruvic acid) needed to identify approximately 80% of such organisms; the others requiring further testing.

ONPG: Fermentation of lactose depends on the presence of two enzymes: permease, which allows the lactose to enter the bacterial cell, and ß-galactosidase, which splits lactose into glucose and galactose, which are subsequently metabolized. Organisms which ferment lactose slowly are deficient in permease. The demonstration of beta-galactosidase is accomplished by the hydrolysis of ortho-nitrophenol-ß-D-galactopyranoside liberating ortho-nitrophenol with its characteristic yellow color.

MUG: Most Escherichia coli have the enzyme ß-glucuronidase. This enzyme reacts with 4-methylumbelliferyl-ß-D-glucuronide and releases 4-methylumbelliferone which is fluorescent under long-wave ultra-violet light. Rare strains of Salmonella, Shigella, and Yersinia may also possess the enzyme but these organisms are easily differentiated by their lack of ability to ferment lactose.

Indole: The oxidation of tryptophane forms indole which is indicated by the formation of a green color from Spot Indole (PEP) reagent.

IPA: Some enterobacteriacae (such as Proteus, Providencia, and Morganella) are able to break down tryptophan to indol-pyruvic acid (see also Phenylanine Deaminase tests) with accompanying pink color reactions upon addition of Ferric Chloride.

ACTIVE INGREDIENTS / MSDS

Each disc is impregnated with a solution of 4-methylumbelliferyl-ß-D-Glucuronide, tryptophane, and ONPG in water. None of these are known at this time to be hazardous.

MATERIALS REQUIRED:

M.O.T. discs are sold 50 per bottle with K980190,Ferric Chloride and K982375 PEP provided. Usage requires 24 hours growth on media not available from KEY. Consult a current reference manual for the correct media to use. The following items are also required but not provided:

Non-fluorescent test tubes

Microbiology loop or needle

Distilled water, neutral pH

Long-wave fluorescent light KS1699 or equal

PROCEDURE:

1) Place one M.O.T. disc into a non-fluorescing tube and add 2 drops of sterile water.

2) Inoculate the tube with a single colony from a primary agar plate (eg. Blood agar or MacConkey). Mix well by using the loop to move the disc around in the tube. If a pure culture is available, inoculate heavily for faster results. (15-30 minutes).

3) Incubate for at least 1 hour from blood agar or 2 hours from MacConkey. *see LIMITATIONS AND TIPS

INTERPRETATION OF RESULTS:

1) ONPG: Observe for a yellow color indicating a positive test. If positive proceed to MUG results. If the ONPG is colorless reincubate 2 more hours.

2) MUG: Observe the tube for fluorescence, using a long-wave ultra-violet light. A positive MUG shows a bright bluish green fluorescence and indicates Escherichia coli. Some test tubes fluoresce so disregard other colors or pale fluorescence. If in doubt, compare the fluorescence of the completed test to an identical tube of inoculated water only.

3) Indole: Drop 1 drop of PEP reagent into the tube. A positive reaction will be green or blue. Pink or yellow is negative. *See LIMITATIONS AND TIPS

4) IPA: It is crucial to perform the IPA test after the indole. Add 1 drop of 50% Ferric chloride to the tube to which the PEP has already been added. Observe for the formation of a pink color. Colorless, yellow, or orange is negative. It may take up to 5 minutes for the color to develop fully.

After tests are completed, refer to the chart for identification. Only E. coli is positive for all 3 tests. If 3 of the 4 tests are negative further testing is recommended before ruling out any organism. Supplemental tests are also listed.

LIMITATIONS AND TIPS

ONPG may work in 15 minutes but MUG, IND, and IPA require longer to develop. For best results, allow 24 hours for indole and IPA if the culture is from MacConkey. Indole and IPA from blood agar works within the 2 hour time frame but the results are pale in comparison to a 4 to 24 hour incubation. Heavy inoculum (at least a #5 McFarland) from a blood agar plate will work within 2 hours for all tests.

Please note that this procedure is the newer version. If you have been following the previous inserts, those methods are still valid. The product has not changed.

References/footnotes:

1) Manual of Clinical Microbiology, 5th Edition, Chapter 36.

a) K. rhinoscleromatis is negative.

b) K. oxytoca and K. ornithinolytica are positive.

c) K. ornithinolytica is positive.

d) K. ozaenae and K. rhinosclermatis are negative.

e) Lem. grimonti is positive.

f) P. vulgaris may be weakly positive.

g) Only Prov. stuarti is positive.

h) Salmonella arizonia is positive.

i) Ser. rubidea is usually positive.

j) Yer. enterocolitica is negative.

k) Yer enterocolitica is positive.

m) C. freundi is negative.

n) E. coli 0157 is MUG negative.