M.O.T. DISCS

Cat. no. K9101 50 discs/bottle

PRINCIPLES/DISCUSSION

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

ONPG: Fermentation of lactose depends on the presence of two enzymes: permease, which allows the lactose to enter the bacterial cell, and beta-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-beta-D-galacto-pyranoside liberating ortho-nitrophenol with its characteristic yellow color.

MUG: Most Escherichia coli have the enzyme b-glucuronidase. This enzyme reacts with 4-methylumbelliferyl-beta-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 red ring in the supernatant layer after the addition of Kovac's reagent.

ACTIVE INGREDIENTS/MSDS

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

MATERIALS PROVIDED

M.O.T. discs are sold 50 per bottle. Consult a current reference manual for the correct media to use.

MATERIALS REQUIRED BUT NOT PROVIDED

PROCEDURE

1. Place one M.O.T. disc into a small non-fluorescing tube and add 2-3 drops of sterile water.

2. Inoculate the tube with growth from a Blood Agar (Cat. no. A10) or MacConkey (Cat. no. G35) plate. Asingle colony is sufficient, but a denser suspension will react faster. Mix well by using the loop to move the disc around in the tube.

3. Incubate for two-four hours. The test will begin to dry and be rendered invalid after four hours. ONPG can be read at one hour is a positive is easy to see. However, results are brighter and easier to read after longer incubation. Negatives must be held for the full four hours.

INTERPRETATION OF RESULTS

  1. ONPG: Observe for a yellow color indicating a positive test.
  2. MUG: Observe the tube for fluorescence, using a long-wave ultra-violet light. A positive MUG shows a bright blue 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: After observing the other tests, add one-two drops of Kovac's reagent to the tube. For a positive test, a red ring will form at the surface of the liquid within five minutes.

After tests are completed, refer to the chart for identification. Only E. coli is positive for all three tests. Negative results on any of the three tests may require futher testing to identify the organism.

Organism MOT / OMPI
ONPG MUG IND PYR
Cedecea spp. + - - -
Citrobacter spp. + - + e +
Edwardsiella spp. - - + -
Escherichia coli + + f + -
Escherichia spp. + - + +
Hafnia alvei - - - -
Klebsiella spp. + a - - b +
Kluyvera spp. + - + -
Leminorella spp. - - - -
Morganella morganii - - + -
Proteus spp. - - + c -
Providencia spp. - - + -
Salmonella spp. - d V - -
Serratia spp. + - - +
Shigella sonnei + + - -
Shigella spp. - V V -
Yersinia spp. V - - +

a

Klebsiella rhinoscleromatis is negative.

b

Klebsiella oxytoca and Klebsiella ornithinolytica are positive.

c Proteus vulgaris may be weakly positive.
d Salmonella arizonia is positive.
e

Citrobacter freundii is negative.


f Escherichia coli O157 is MUG negative.
Positive ONPG Reaction

Showing Positive ONPG Reaction
Escherichia coli (ATCC ® 25922) suspension with M.O.T. Disc (Cat. no. K9101). Incubated aerobically for 1 hour at 35ºC. The yellow color development was indicative of a positive reaction (ONPG positive). E. coli was transfered from Blood Agar (Cat. no. A10) after an aerobic incubation at 35ºC. for 24 hours.

Negative ONPG Reaction

Showing Negative ONPG Reaction
Proteus mirabilis (ATCC ® 12453) suspension with M.O.T. Disc (Cat. no. K9101). Incubated aerobically for 1 hour at 35ºC. No yellow color development was indicative of a negative reaction (ONPG negative). P. mirabilis was transfered from Blood Agar (Cat. no. A10) after an aerobic incubation at 35ºC. for 24 hours.



MUG Test

Left: Positive MUG Reaction; Escherichia coli (ATCC ® 25922) suspension with M.O.T. Disc (Cat. no. K9101).
Right: Negative MUG Reaction; Proteus mirabilis (ATCC ® 12453) suspension with M.O.T. Disc (Cat. no. K9101).

Organisms were incubated aerobically for 1 hour at 35ºC. Shown under long-wave UV light. Fluorescence is indicative a positive MUG test. Organisms were transfered from Blood Agar (Cat. no. A10) after an aerobic incubation at 35ºC. for 24 hours.











Positive Indole Test

Showing Positive Indole Reaction
Escherichia coli (ATCC ® 25922) suspension with M.O.T. Disc (Cat. no. K9101). Incubated aerobically for 1 hour at 35ºC. Red color development after addition of five drops Kovacs Indole Reagent (included) was indicative of a positive indole reaction. E. coli was transfered from Blood Agar (Cat. no. A10) after an aerobic incubation at 35ºC. for 24 hours.

Negative Indole Reaction

Showing Negative Indole Reaction
Proteus mirabilis (ATCC ® 12453) suspension with M.O.T. Disc (Cat. no. K9101). Incubated aerobically for 1 hour at 35ºC. No red color development after addition of five drops Kovacs Indole Reagent (included) was indicative of a negative indole reaction. P. mirabilis was transfered from Blood Agar (Cat. no. A10) after an aerobic incubation at 35ºC. for 24 hours.

REFERENCES

1. Jorgensen., et al. Manual of Clinical Microbiology, American Society for Microbiology, Washington, D.C.


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