Reverse CAMP Test: Principle, Procedure, Results and Uses

The reverse CAMP test is a phenotypic microbiological test used mainly for the presumptive identification of Clostridium perfringens. In the classical test, C. perfringens is streaked near a CAMP-positive strain of Streptococcus agalactiae on blood agar. Interaction between the bacterial extracellular factors produces a characteristic zone of enhanced hemolysis, often described as a bow-tie or arrowhead-shaped reaction.

Used mainly for: Presumptive identification of Clostridium perfringens and demonstration of characteristic hemolytic interactions.

Quick facts

What is Reverse CAMP Test?

Reverse CAMP test is a biochemical test used for the presumptive identification of Clostridium perfringens. It is based on the synergistic hemolytic reaction produced between Clostridium perfringens and group B Streptococcus agalactiae on blood agar.

In this test, the suspected organism and Streptococcus agalactiae are streaked at right angles to each other on a blood agar plate. A positive reverse CAMP reaction is seen by an arrow-shaped or bow-tie zone of enhanced hemolysis near the junction of the two organisms. Thus, this reaction can be used for rapid presumptive identification of C. perfringens.

Objectives of Reverse CAMP Test

• To presumptively identify Clostridium perfringens by detecting its characteristic enhanced hemolytic reaction with group B Streptococcus agalactiae.
• To differentiate C. perfringens from other related Clostridium species based on the positive reverse CAMP reaction.

Principle of the Reverse CAMP Test

The Reverse CAMP test is based on the synergistic hemolysis produced by the alpha toxin of Clostridium perfringens and the CAMP factor produced by Streptococcus agalactiae. The alpha toxin of C. perfringens is a phospholipase C which acts on the membrane phospholipids of erythrocytes. This makes the erythrocytes more susceptible to the hemolytic action of CAMP factor.

When C. perfringens and S. agalactiae are streaked at right angles to each other on blood agar, their extracellular products diffuse through the medium. At the area where these products interact, increased destruction of red blood cells takes place. This produces a characteristic arrow-shaped or bow-tie zone of enhanced hemolysis near the junction of the two bacterial streaks.

Synergistic Hemolysis in Clostridium perfringens

C. perfringens produces alpha toxin (phospholipase C) which has hemolytic activity. S. agalactiae produces CAMP factor. When these two factors meet in the blood agar, their combined action produces stronger hemolysis than that produced by either factor alone. This is referred to as synergistic hemolysis and forms the basis of a positive reverse CAMP reaction.

Materials Required for the Reverse CAMP Test

The following are the materials required for performing the Reverse CAMP test–

1. Sheep blood agar plate containing about 5% sheep blood.
2. Pure culture of the suspected Clostridium perfringens.
3. CAMP-positive culture of Streptococcus agalactiae (group B Streptococcus).
4. Sterile inoculating loop or inoculating needle.
5. Anaerobic jar or anaerobic incubation system for maintaining anaerobic condition.
6. Incubator maintained at about 35–37°C. The plate is incubated anaerobically to allow the growth of C. perfringens.

Reverse CAMP Test Procedure

The following are the steps for performing the Reverse CAMP test–

Plate Inoculation

1. Take a fresh sheep blood agar plate.
2. A CAMP-positive culture of Streptococcus agalactiae is streaked in a straight line along the center of the blood agar plate.
3. The suspected culture of Clostridium perfringens is streaked at right angle to the streak of S. agalactiae.
4. The two streaks should not touch each other. A small gap of about 1–2 mm is maintained between the streaks.
5. This arrangement allows the extracellular products produced by both organisms to diffuse and interact in the agar.

Incubation and Plate Examination

1. The inoculated blood agar plate is incubated anaerobically at 37°C for about 18–24 hours.
2. After incubation, the area between the perpendicular streaks is examined for enhanced hemolysis.
3. A positive reaction is indicated by the formation of a characteristic arrowhead or bow-tie shaped zone of enhanced hemolysis at the interaction zone between C. perfringens and S. agalactiae.
4. Absence of this characteristic synergistic hemolysis is considered as a negative reverse CAMP reaction.

Reverse CAMP Test Results

The Reverse CAMP test is interpreted by observing the hemolysis formed at the interaction zone between Clostridium perfringens and Streptococcus agalactiae. The characteristic enhanced hemolysis indicates a positive reaction.

Positive Reverse CAMP Test

A positive Reverse CAMP test shows an arrowhead-shaped or bow-tie zone of enhanced hemolysis near the junction of the two bacterial streaks. This enhanced hemolysis is produced due to the synergistic reaction between C. perfringens and group B S. agalactiae. It is considered as a characteristic positive reaction of C. perfringens.

Negative Reverse CAMP Test

In a negative reaction, the characteristic enhanced hemolysis is absent at the interaction zone. No arrowhead or bow-tie shaped enhancement is formed between the two streaks. Several other Clostridium species may show a negative Reverse CAMP reaction, while most clinical isolates of C. perfringens are positive.

Reverse CAMP Test Quick Interpretation

Reverse CAMP Positive Organisms

The following are the important organisms that show a positive Reverse CAMP reaction–

• Clostridium perfringens – It is the classical Reverse CAMP-positive organism. It produces an arrowhead or bow-tie shaped zone of enhanced hemolysis when grown near Streptococcus agalactiae. Most isolates of C. perfringens show this characteristic reaction.
• Arcanobacterium haemolyticum – It can also show a positive Reverse CAMP reaction with Streptococcus agalactiae. The hemolysis produced by S. agalactiae is enhanced near the growth of A. haemolyticum. This reaction can be used as one of the tests for its identification.

Organism-Specific Reverse-CAMP-Type Reactions

CAMP Test vs Reverse CAMP Test

CAMP test and Reverse CAMP test are blood agar based tests which are used for presumptive identification of bacteria by observing characteristic hemolytic interaction between two organisms. The major differences between CAMP test and Reverse CAMP test are as follows-

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Common Interpretation Errors

The following are some of the common errors during interpretation of the Reverse CAMP test–

• Normal hemolysis around the bacterial growth should not be considered as a positive reaction. A positive Reverse CAMP test requires a clear zone of enhanced hemolysis at the interaction area between Clostridium perfringens and Streptococcus agalactiae.
• The result should not be interpreted only from the direction of the arrowhead. The positive reaction may appear as an arrow-shaped or bow-tie zone of enhanced hemolysis, depending on the arrangement of the two streaks. The enhanced interaction zone is more important for interpretation.
• A negative Reverse CAMP test does not completely exclude C. perfringens. About 96–97% of the tested C. perfringens isolates were positive in earlier evaluations, therefore occasional isolates can show a negative reaction.
• A positive result should not be considered as final identification of C. perfringens. Reverse CAMP test is a presumptive identification test, and the result should be considered together with other characteristics or confirmatory identification methods.
• Hemolysis away from the junction of the two organisms should not be confused with synergistic hemolysis. The characteristic reaction is examined where the extracellular products of C. perfringens and group B S. agalactiae interact on the blood agar plate.

Uses of the Reverse CAMP Test

The following are some of the important uses of the Reverse CAMP test–

• It is used for the rapid presumptive identification of Clostridium perfringens isolated from clinical and other specimens.
• It is used to differentiate C. perfringens from other related Clostridium species based on its characteristic positive reverse CAMP reaction.
• It can be used as an additional phenotypic test for identification of suspected C. perfringens colonies after their isolation in the laboratory. The positive organism produces a characteristic zone of enhanced hemolysis with Streptococcus agalactiae.
• It is also used to demonstrate the synergistic hemolysis produced by C. perfringens and the CAMP factor of S. agalactiae on blood agar.

Advantages of Reverse CAMP test

The following are some of the important advantages of the Reverse CAMP test–

• It is a useful test for the presumptive identification of Clostridium perfringens from clinical isolates.
• The test has good sensitivity. About 96–97% of C. perfringens isolates were reported to produce a positive Reverse CAMP reaction in earlier studies.
• The positive reaction is generally easy to observe. A characteristic arrow-shaped or bow-tie zone of enhanced hemolysis is formed at the interaction area.
• It helps in differentiation of C. perfringens from several other Clostridium species which show a negative Reverse CAMP reaction.
• The test is based on growth and hemolytic reaction on blood agar and does not require complex molecular detection methods. It can therefore be used as a practical phenotypic test during routine identification of suspected C. perfringens.

Limitations of the Reverse CAMP Test

The following are some of the important limitations of the Reverse CAMP test–

• The Reverse CAMP test provides only presumptive identification of Clostridium perfringens. Other identification tests may be required for definite confirmation of the organism.
• Not all strains of C. perfringens give a positive reaction. Studies have reported about 96–97% of the tested isolates as Reverse CAMP positive, therefore a small number of strains can give a negative reaction.
• The test requires a suitable CAMP-positive culture of Streptococcus agalactiae and sheep blood agar. Improper growth or arrangement of the bacterial streaks can affect development of the characteristic hemolytic zone.
• Anaerobic incubation is required for proper growth of C. perfringens. Thus, an anaerobic culture system is necessary while performing the test.
• Reverse CAMP reaction is not completely restricted to C. perfringens. Some other bacteria such as Arcanobacterium haemolyticum can also enhance hemolysis with S. agalactiae. Therefore, the reaction should be interpreted together with colony morphology, Gram staining and other identification tests.

FAQs on Reverse CAMP Test

What is reverse CAMP test?

Reverse CAMP test is a biochemical test used for presumptive identification of Clostridium perfringens, Arcanobacterium haemolyticum, and some other reverse CAMP positive bacteria.

What is reverse CAMP test principle?

It is based on interaction between hemolytic toxin or phospholipase of test organism and hemolytic factor produced by indicator organism on sheep blood agar.

Which bacteria are reverse CAMP positive?

Important reverse CAMP positive bacteria are Clostridium perfringens, Arcanobacterium haemolyticum, and Corynebacterium pseudotuberculosis.

What is reverse CAMP test positive result?

Positive result is seen as bow-tie or reversed arrowhead hemolysis in Clostridium perfringens. In Arcanobacterium haemolyticum, it may show arrow of no hemolysis with Staphylococcus aureus.

What is the difference between CAMP test and reverse CAMP test?

In CAMP test, the test organism usually produces CAMP factor. In reverse CAMP test, the suspected organism produces toxin or phospholipase which reacts with CAMP factor or beta hemolysin of another organism.

Why is reverse CAMP test used for Clostridium perfringens?

It is used because Clostridium perfringens produces alpha toxin which acts with CAMP factor of Streptococcus agalactiae and gives characteristic bow-tie or reversed arrowhead hemolysis.

What is CAMP inhibition test?

CAMP inhibition test is a form of reverse CAMP reaction where the test organism inhibits beta hemolysin of Staphylococcus aureus and produces an arrow of no hemolysis.

What is arrowhead zone of hemolysis?

Arrowhead zone of hemolysis is a triangular zone of enhanced clearing on blood agar formed due to synergistic action of bacterial hemolytic factors.

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