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Material Safety Data Sheet

Organisms

Organisms this product works with:

Oxoid Biochemical Identification System (O.B.I.S.)

OXOID BIOCHEMICAL IDENTIFICATION SYSTEM - ALBICANS

Code: ID0700

A rapid colorimetric test for the differentiation of Candida albicans from other yeast species.

INTENDED USE
The Oxoid Biochemical Identification System (O.B.I.S.) albicans is a simple, non-hazardous colorimetric test for the differentiation of Candida albicans from other yeast species.

PRINCIPLE OF THE TEST
The O.B.I.S. albicans is a two-stage biochemical test that detects the presence of two enzymes, ß-galactosaminidase and L-proline aminopeptidase, using chromogenic substrates. Both these enzymes are produced by Candida albicans whereas one or both enzymes are absent in other yeast species1,2. The test offers a rapid diagnostic alternative to traditional, time-consuming and subjective physiological methods such as the germ-tube test3.
Other biochemical test kits have traditionally been based on the use of a ß-naphthylamide peptide, which is a potent carcinogen4. Oxoid has developed a new system using a non-carcinogenic substrate, in response to associated health concerns. The use of chromogenic rather than fluorogenic substrates eliminates the
need for UV detection, providing both safety and ease of use.
O.B.I.S. albicans uses Test Cards impregnated with p-nitrophenyl-N-acetyl-ß-D-galactosaminide (pNP-NAGal) and L-prolinyl 7-amido-4-methylcoumarin (PRO-AMC). Following addition of the O.B.I.S. NaOH Developer the presence of ß-galactosaminidase is indicated by the formation of a yellow colour. The addition of O.B.I.S. DMAC Developer indicates the presence of L-proline aminopeptidase by the formation of a magenta colour. Absence of either enzyme (indicated by no colour change) confirms that the culture is not C. albicans.

COMPONENTS OF THE O.B.I.S albicans KIT (ID700M)

Each O.B.I.S mono kit contains the following reagents with enough material for 60 tests:

ID0703M Test Cards: 1 Pouch containing 10 cards and a moisture absorbent sachet. There are 6 test reaction areas on each card, providing a total of 60 tests. Each Test Card is impregnated with pNP-NAGal and PRO-AMC.
ID0701M
O.B.I.S. Rehydration Solution – 1 dropper bottle containing 7 ml of 0.1% Tween 80® solution.
ID0702M O.B.I.S. NaOH Developer – 1 dropper bottle containing 7 ml of 0.1M sodium hydroxide.
ID0221M
O.B.I.S. DMAC Developer – 1 dropper bottle containing 7 ml of 0.5% w/v dimethylaminocinnamaldehyde dissolved in 1M hydrochloric acid.
ID0090M O.B.I.S. Sleeves – 1 pouch containing 30 plastic reaction sleeves.
ID0898 60 Plastic Paddle Pastettes.
Instruction leaflet.

MATERIALS REQUIRED BUT NOT INCLUDED
37ºC incubator

PRECAUTIONS
This product is for in vitro diagnostic use.
Do not use the O.B.I.S. albicans Test Cards or reagents beyond the expiry date.
Specimen material may contain pathogenic organisms, take appropriate precautions when handling.
The O.B.I.S. NaOH Developer is a weak base and the O.B.I.S. DMAC Developer contains an acid. Avoid direct contact by wearing suitable personal protective equipment. If the material comes into contact with the skin, mucous membranes or eyes, immediately rinse the affected area with plenty of water.
Used Test Cards and paddle pastettes must be disposed of as bio-hazardous waste and incinerated or autoclaved for 15 minutes at 121°C.

STORAGE AND OPENING
The O.B.I.S. mono kit must be stored at 2- 8°C. Allow the pouches to reach room temperature before use to prevent the formation of condensation on the test cards.
Open the pouches by cutting at the notch between the end seal and the clip-lock opening.
Once opened, remove the number of Test Cards required for testing within the next 60 minutes and reseal the pouch immediately.
If fewer tests are required, cut the Test Card and return the unused portions to the pouch. Do not return used portions to the pouch as they will be contaminated.
When stored as directed, reagents will retain their activity until the expiry date shown on the box.

QUALITY CONTROL PROCEDURE
Each day the kit is used the following control procedures should
be performed:
1. Positive Control – Candida albicans ATCC® 10231. Follow the method given in the test procedure. Ensure that a yellow colour forms immediately after addition of O.B.I.S. NaOH Developer (ID0702) AND a magenta colour forms within 10 seconds after addition of O.B.I.S. DMAC Developer (ID0221).
2. Negative Control – Candida kefyr ATCC® 8555 or Saccharomyces cerevisiae ATCC® 9763. Follow the method given in the test procedure. Ensure that no yellow colour forms after addition of O.B.I.S. NaOH Developer (ID0702M) AND no magenta colour forms within 10 seconds after addition of O.B.I.S. DMAC Developer (ID0221).
Do not use the reagents if reactions with control organisms are incorrect.

SPECIMENS
For details of specimen collection and processing, standardmethods or procedures should be consulted.
When identifying Candida albicans: Fresh primary or secondary cultures grown on Sabouraud Dextrose Agar (SDA) (CM0041) for 24 to 48 hours give best results. Cultures older than 96 hours should not be used as enzymic activity may be impaired. In the case of insufficient growth, a subculture (passage) should be performed.

TEST PROCEDURE
1. Using the paddle end of an unused plastic Paddle Pastette, transfer approximately three to five 1 mm colonies, or equivalent, onto the circular test area, ensuring the material is smeared thinly and evenly within the circular test area.
2. Moisten the inoculated test area with 1 drop of O.B.I.S. Rehydration Solution (ID0701 – white cap).
3. Place the card into a plastic Reaction Sleeve ensuring the test area is covered and incubate at 37°C for 60 minutes.
4. After incubation, remove the Test Card from the plastic Reaction Sleeve and dispense 1 drop of O.B.I.S. NaOH Developer (ID0702 – yellow cap) onto the inoculated test area. Development of a yellow colour on and around the smear of inoculum indicates presence of ß-galactosaminidase.
Record the result on the Test Card before progressing to step 5.
No colour change indicates absence of this enzyme, i.e. the organism is not Candida albicans. If there is no colour development no further steps are required.
5. Dispense 1 drop of O.B.I.S. DMAC Developer (ID0221 – purple cap) onto the test area. Development of a magenta colour on and around the smear of inoculum within 10 seconds indicates presence of L-proline aminopeptidase. No colour change after 10 seconds indicates absence of this enzyme, i.e. the organism is not Candida albicans. Record the result on the Test Card.

READING AND INTERPRETATION OF RESULTS
Positive Result: A positive result is indicated by the development of a yellow colour in the inoculated portion of the test area following addition of O.B.I.S. NaOH Developer (ID0702) AND the development of a magenta colour in the inoculated portion of the test area within the 10 second period following addition of O.B.I.S. DMAC Developer (ID0221).
Negative Result: A negative result is indicated by lack of colour development in the inoßculated portion of the test area within the 10 second period
following addition of either the O.B.I.S. NaOH Developer (ID0702) or the O.B.I.S. DMAC Developer (ID0221).
Following addition of O.B.I.S. NaOH Developer (ID0702), it is important that the result is recorded immediately, before the addition of the O.B.I.S. DMAC Developer (ID0221), as the reactions are non-reversible.
The inoculum itself may appear as a straw-yellow coloured smear. To aid interpretation of positive and negative results after addition of the O.B.I.S. NaOH Developer (ID0702), refer to the positive and negative control reactions for comparison.
Following addition of the O.B.I.S. DMAC Developer (ID0221), a pale yellow background will be apparent. This should not be confused with the yellow colouration observed for positive reactions after addition of the O.B.I.S. NaOH Developer (ID0702).
Some yeast species express L-proline-aminopeptidase, but not ß-galactosaminidase. If a negative result is recorded following addition of O.B.I.S. NaOH Developer (ID0702), there is no need to add the O.B.I.S. DMAC Developer (ID0221). Presumptive identification of Candida albicans is only achieved by a positive result for the presence of both enzymes.

LIMITATIONS OF THE TEST
O.B.I.S. albicans is intended for the presumptive identification of Candida albicans from pure culture.
Less commonly encountered isolates of Candida dubliniensis (a recently described species, closely related to Candida albicans) are also positive for this test.
For best results only use colonies grown Sabouraud Dextrose Agar (CM0041). Other media may interfere with enzymic activity or cause false positive cross-reactions3.
Continuous passaging may affect enzyme expression in the target organism. Therefore, it is preferable to use cultures that have not been passaged more than three times.
Only Paddle Pastettes should be used for the transfer of material from the culture media to the Test Card. Use of microbiological loops for this purpose may result in weak positive reactions.
The reactions with O.B.I.S. albicans are markers for enzyme activity and atypical strains may occasionally occur.
Incubation beyond 10 seconds following addition of the DMAC Developer (ID0221) may produce non-specific colour reactions. Therefore, it is important that the test is read as directed.

TRIAL RESULTS
Results from organisms grown on Sabouraud Dextrose Agar (SDA). Isolates were incubated at 30°C for 48 hours.

Comparison of O.B.I.S. albicans with Germ Tube

 
Germ Tube
 
+
-
O.B.I.S. albicans
+
83
0
Sensitivity 100%
-
0
133
Specificity 100%

 

REFERENCES
1. Willinger, B., Manafi, M. and Rotter, M. L. Comparison of rapid methods using fluorogenic-chromogenic assays for detecting Candida albicans. Lett. Appl. Microbiol. 1994; 18: 47–9.
2. Heelan, J. S., Siliezar, D. and Coon, K. Comparison of rapid testing methods for enzyme production with the germ tube method for presumptive identification of Candida albicans. J. Clin. Microbiol. 1996; 34(11): 2847–9.
3. Data on file at Oxoid.
4. Bascomb, S. and Manafi, M. Use of enzyme tests in the characterisation and identification of aerobic and facultatively anaerobic Gram-positive cocci. Clin. Microbiol. Reviews 1988; 11: 318–340.

 
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