TEST PRINCIPLE:
Some bacteria produce enzymes which hydrolyze various chromogenic substrates. Because the organism produces these enzymes during the growth process and the presence of the enzymes can be detected rapidly, more substrates can be used in a wide variety of applications which would normally require special growth conditions.
When bound to nitrophenol, the hydrolysis of the colourless aryl-substituted glycoside or phosphoester releases the nitrophenol base with its yellow colour. The most common test using this principle is o-Nitrophenol-β-d-galactopyranoside (ONPG) used for enterics and Neisseria species.
ACTIVE INGREDIENTS:
The tablets contain approximately 0.05 mg of one or more of the following substrates in a Sodium Chloride and Dicalcium phosphate base:
p-Nitrophenol-β-d-glucopyranoside (βGLU)
SINGLE TEST SETUP:
(1) Add 3-5 drops of water to the test tube.
(2) Inoculate heavily with a loopful of organism from a fresh pure 24 hour culture plate or slant. Mix with the loop until the organism is in suspension. Go to step 3 below.
MULTIPLE TEST SETUP:
1) Harvest sufficient colonies into 1-2 ml of water to make a suspension equal to #5 McFarland. This suspension may be stored aerobically for later use. (up to 5 days).
2) Add 3-5 drops of this solution to the tube containing the tablet, shaking to disintegrate the tablet.
3) Incubate all tests aerobically, uncovered, at 34-37C for 2 hours. Tests may be incubated up to 24 hours.
TIPS:
Vortexing or shaking the test vigorously will enhance colour development. A denser suspension will also produce brighter and faster reactions.
INTERPRETATION OF RESULTS:
No colour change at 2 hours is negative. The appearance of a yellow colour at any time during the 2 hours is a positive glycosidase test. At 24 hours only very bright yellow should be considered positive.
QUALITY CONTROL:
Each lot of tablets should be tested with known positive and negative organisms. Some suggested strains for beta Glucosidase are:
Positive: Bacteroides fragilis
Negative: Bacteroides ureolyticus
REFERENCES:
(1) Manual of Clinical Microbiology, Fifth Edition, Chapter 36, Enterobacteriaceae
(2) Kilian, M and Bulow, P. 1976. Rapid Diagnosis of Enterobacteriacae, Acta path. microbio. Scan, Sect B, 84: 245-251
(3) Wadsworth Anaerobic Bacteriology Manual, 5th Edition, 1993,Glucosidase tests, page 152.