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OXOID BIOCHEMICAL IDENTIFICATION SYSTEM (O.B.I.S.) CAMPY

Code: ID0800

A rapid colorimetric test for the differentiation of Campylobacter, Helicobacter and Arcobacter species from other Gram-negative organisms.

Intended Use
O.B.I.S. campy is a simple, rapid colorimetric test for the detection of L-alanyl aminopeptidase in Gram-negative organisms. It has been designed for the differentiation of Campylobacter, Helicobacter and Arcobacter species from other Gram-negative organisms and incorporates a Gram-lysis test which will demonstrate Gram status.

Principle of the Test
The O.B.I.S. campy test will differentiate species of Campylobacter, Helicobacter and Arcobacter from all other Gram-negative organisms¹. Unlike other Gram-negative organisms, Campylobacteraceae do not possess the enzyme L-alanyl aminopeptidase (L-ALA). The O.B.I.S. campy test incorporates a rapid test to detect this enzyme and a Gram-lysis reagent to rapidly determine the Gram status. First, the Gram-lysis test (or a Gram stain) must be carried out. This test differentiates between Gram-positive and Gram-negative bacteria². The test is carried out on a glass slide. Sodium hydroxide (0.5M) is used to lyse the cell wall of Gram-negative organisms and release the DNA. The DNA forms a ‘string’ which can be seen when the loop is raised from the surface of the slide. This reaction does not occur with Gram-positive organisms. Once the organism has been identified as Gram-negative, the L-ALA test can be carried out. Each O.B.I.S. campy reaction card has been impregnated with the L-ALA substrate (L-alanyl-7-amino-4-ethylcoumarin) in each of the six reaction zones. An acidic solution of dimethylaminocinnamaldehyde (DMAC) is used as a colour developer. If the substrate has been hydrolysed by the organism, the free 7-amino-4-methylcoumarin will combine with the developer to produce a purple Schiff’s base.

Components of the O.B.I.S. campy Kit (ID0800M)

Each kit contains the following reagents with enough material for 60 tests

Specimens
The test is designed for use from purity plates such as Columbia Blood Agar. Primary isolation media should not be used as the colonies may be too small or too few to carry out an effective test. Pick colonies which have typical Campylobacter morphology from selective Campylobacter isolation media and streak onto Columbia Blood Agar. Incubate in a microaerobic atmosphere for 48 hours, then conduct the O.B.I.S. test.

Test Procedure and Interpretation of Results

Gram-lysis test

1. Take a 10 μl loopful of 0.5M sodium hydroxide solution (white flat-capped bottle) and place onto a clean, glass slide.
2. Using a sterile loop, take a small amount of material from a purity plate.
3. Mix the sample into the NaOH on the slide for up to one minute.
4. At intervals, carefully raise the loop from the mixture to check for the presence of a ‘string’ between the loop and the mixture.
5. Record the result:
• A positive result is characterised by a viscous appearance to the mixture and the presence of the DNA string. This indicates that the organism is Gram-negative.
• A negative result is characterised by the formation of a cell suspension with no string and indicates that the organism is Gram-positive.
• The test may also be carried out using a 0.5M potassium hydroxide (KOH) solution. Alternatively, a traditional Gram stain may be used to ascertain the Gram status of the organism.

L-ALA Test
Once the organism has been identified as Gram-negative, the L-ALA portion of the test can be carried out.

1. Remove one of the Test Cards from the pouch.
2. Using the paddle end of an unused plastic pastette, transfer the equivalent of 5x1 mm diameter colonies from a purity plate to the test area.
3. Spread the sample across the reaction zone (inside a circle) of a Test Card.
4. Dispense one drop of O.B.I.S. campy buffer (white capped dropper bottle) onto each of the inoculated reaction zones.
5. Wait for 30 seconds.
6. Dispense one drop of O.B.I.S. DMAC Developer (purple cap) onto each of the inoculated reaction zones.
7. The appearance of a purple colour around the original colony material within 20 seconds is a positive L-ALA reaction.
   
   A positive reaction indicates the organism is not a Campylobacter, Helicobacter or Arcobacter species. If no colour develops around the original colony material within 20 seconds this is a negative reaction. A negative reaction indicates the organism is a presumptive Campylobacter, Helicobacter or Arcobacter species.

Interpretation Chart

Venn Diagram
The figure 1 illustrates how Campylobacter and Campylobacter-related organisms can be differentiated from other common bacteria using the Gram-lysis and L-ALA tests of O.B.I.S. campy.

figure1: Venn Diagram of NaOH and L-ALA reactions


Gram-positive organisms, other than enterococci, are L-ALA negative.

Quality Control Procedure
The following procedure should be performed each time the kit is used:

Gram-lysis Test

• Positive Control – Use a known Gram-negative (Gram-lysis positive) organism, such as Campylobacter jejuni ATCC^®33291™. Follow the method given in the test procedure.
• Negative Control – Use a known Gram-positive (Gram-lysis negative)
  organism such as Bacillus cereus ATCC^®11778™. Follow the method given in the test procedure.

L-ALA Test

• Positive Control – use a known L-ALA positive organism, such as Pseudomonas aeruginosa ATCC^®27853™. Follow the method given in the test procedure. Ensure that a purple colour forms around the colony material within 20 seconds.
• Negative Control – Use a known L-ALA negative organism such as Campylobacter jejuni ATCC^®33291™. Follow the method given in the test procedure. Ensure that no purple colour forms within 20 seconds.

Performance
In an internal study, 46 Campylobacter species and 6 Arcobacter species were tested. All yielded a Gram-negative, L-ALA negative reaction. 252 other species (non-Campylobacter, Arcobacter or Helicobacter) were tested. Only one organism gave a result similar to Campylobacter, and was due to an atypical Gram-lysis reaction. This resulted in a sensitivity and specificity of 100% and 99.6% respectively³.

Storage and Opening
The O.B.I.S. campy kit must be stored at 2°C to 8°C. Allow the pouches to reach room temperature before use to prevent condensation forming on the Test Cards. Open the pouches by cutting at the notch between the end seal and the clip-lock opening. Remove the number of Test Cards required and reseal the pouch. Use within 60 minutes. If fewer tests are required than the number on the Test Card, cut the card and return the unused portion to the pouch. Do not return used Test Cards to the pouch.

Precipitation will occur after long-term storage of the sodium hydroxide – this does not affect performance. A sterile loop should always be used.

Discard if there is any sign of contamination.

Precautions
This product is for in vitro diagnostic use only.

Do not use the O.B.I.S. campy reagents beyond the stated expiry date.

The DMAC Developer contains a weak acid and will stain the skin. The 0.5M sodium hydroxide solution is corrosive and may cause burns. Do not breathe fumes/vapour. Avoid contact with skin and eyes. In case of contact with eyes, rinse immediately with plenty of water and seek medical advice. After contact with skin, wash immediately with plenty of water and seek medical advice. Wear suitable protective clothing, gloves and eye/face protection.

Used Test Cards and inoculating loops should be disposed of as biohazardous waste. This should be incinerated or autoclaved at 121°C for at least 15 minutes.

Campylobacter are pathogens. A low infective dose can cause gastro-enteritis with potentially serious complications. Take appropriate precautions when handling potentially contaminated samples.

Limitations of the Test
O.B.I.S campy is intended for the detection of L-alanyl aminopeptidase in Gram negative organisms. It can be used for the presumptive identification of Campylobacter and related organisms from pure culture. The reaction with the O.B.I.S. campy L-ALA test is a marker for enzyme activity and atypical strains may occasionally occur. Bacteroides ureolyticus can give the same reactions as Campylobacter species. However, their colonial morphology and anaerobiosis aid differentiation. Over time, the DMAC Developer may give a slightly pink reaction with the campy
buffer even in negatives. However, this is easily differentiated from the clearly purple reaction seen around the colony material in a positive.

Do not use nichrome wire loops to inoculate cards as this material can interfere with the test.

References
1. Hoosain, N. and Lastovica, A. J. (2005) Evaluation of the Oxoid Biochemical Identification System (O.B.I.S.) for the differentiation of Campylobacter and Arcobacter from other Gram-negative organisms. In: Abstracts of CHRO 2005. 13th International Workshop on Campylobacter, Helicobacter and related organisms. Sept 4-8, 2005, Gold Coast, Queensland, Australia. Griffith University.
2. Carlone, G. M., Valadez, M. J. and Pickett, M. J. (1982) Methods for distinguishing Gram-positive from Gram-negative bacteria. J. Clin Micro. 16 (6), 1157-1159.
3. Smith, C. M., Colborne, N. R., Stephens, P .J. and Druggan, P. (2006) A simple and rapid biochemical screening test for the differentiation of Campylobacter spp. from other contaminating micro-organisms. In: Abstracts of Emerging Campylobacter spp. in the food chain, CAMPYCHECK. Feb 8th 2006, Croke Park Conference Centre, Dublin, Ireland.

 ATCC^® is a registered trademark of American Type Culture Collection