Organisms this product works with:
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
ID0803M | O.B.I.S. campy Test Cards – one resealable pouch containing 10 cards, each with six reaction zones |
ID0804M | O.B.I.S. campy Buffer – one white capped dropper bottle containing 7 ml of solution |
ID0221M | O.B.I.S. DMAC Developer – one purple capped dropper bottle containing 7 ml of 0.5% w/v dimethylaminocinnamaldehyde in 1M hydrochloric acid |
ID0802M | 0.5M sodium hydroxide – one flat white capped bottle containing 6 ml of 0.5M NaOH (sufficient for 600 Gram-lysis screening tests) |
ID0898 | Paddle pastettes – pack of 60 |
Instruction leaflet |
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
L-ALA Test
Once the organism has been identified as Gram-negative, the L-ALA portion of the test can be carried out.
Interpretation Chart
Organism / typical reactions | NaOH | L-ALA |
Campylobacteraceae | + (i.e. Gram -ve) | - |
Gram-positives | - (i.e. Gram +ve) | n/a |
Gram-negatives other than Campylobacteraceae | + (i.e. Gram -ve) | + |
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
L-ALA Test
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