Reverse CAMP test is a biochemical test that is used for presumptive identification of some pathogenic bacteria mainly Clostridium perfringens and Arcanobacterium haemolyticum. It is also used for identification of Corynebacterium pseudotuberculosis in veterinary microbiology.
Reverse CAMP test is based on the interaction between extracellular products of two different bacteria when they are grown on sheep blood agar plate. This interaction may show synergistic hemolysis or inhibition of hemolysis.
Reverse CAMP test positive reaction for Clostridium perfringens is seen as a distinct bow-tie or reversed arrowhead zone of hemolysis. This is produced near the streak of Streptococcus agalactiae.
CAMP test vs reverse CAMP test is important because both are done on blood agar and both show hemolytic reaction, but the test organism and result interpretation are different.
What is Reverse CAMP Test?
Reverse CAMP test is a test used to detect the interaction of toxin or phospholipase produced by bacteria with hemolytic factor of another organism.
It is mainly used for Clostridium perfringens, Arcanobacterium haemolyticum, and Corynebacterium pseudotuberculosis.
In this test, the suspected organism is streaked perpendicular to the indicator organism on blood agar plate. After incubation, the plate is observed for arrowhead hemolysis, bow-tie hemolysis, or inhibition of hemolysis.
Objective of Reverse CAMP Test
- To identify Clostridium perfringens presumptively and differentiate it from other Clostridium species.
- To detect the production of alpha-toxin (Phospholipase C) by C. perfringens which gives characteristic bow-tie or reversed arrowhead zone of enhanced hemolysis with Streptococcus agalactiae.
- To identify Arcanobacterium haemolyticum and differentiate it from corynebacteria, normal flora and beta hemolytic streptococci like Streptococcus pyogenes.
- To demonstrate phospholipase D production in Arcanobacterium haemolyticum which can inhibit staphylococcal beta-lysin or can show synergistic hemolysis with Streptococcus agalactiae.
- To identify Corynebacterium pseudotuberculosis by showing inhibition of beta hemolysin produced by Staphylococcus aureus.
Principle of Reverse CAMP test
Reverse CAMP test principle is based on the interaction between extracellular hemolytic factors produced by two bacteria on sheep blood agar.
In Clostridium perfringens, alpha toxin or phospholipase C is produced by the test organism. This toxin diffuses in the agar and acts on the red blood cell membrane.
The alpha toxin hydrolyzes sphingomyelin of RBC membrane into ceramide. The RBC becomes sensitized and more susceptible to lysis.
After this, CAMP factor produced by Streptococcus agalactiae binds to the exposed ceramide and destroys the lipid bilayer integrity. This results in complete lysis of red blood cells.
The positive reaction is seen as bow-tie or reversed arrowhead shaped zone of enhanced hemolysis pointing towards S. agalactiae streak.
In Arcanobacterium haemolyticum, phospholipase D may interact with the CAMP factor of Streptococcus agalactiae and produce enhanced hemolysis. It may also inhibit beta hemolysin of Staphylococcus aureus and form an arrow of no hemolysis.
Material Required for Reverse CAMP test
- 5% sheep blood agar plates are used as preferred medium due to high sphingomyelin content in sheep RBC.
- Known CAMP positive Streptococcus agalactiae strain is used to supply CAMP factor.
- Suspected test organism such as Clostridium perfringens or Arcanobacterium haemolyticum is required as pure culture.
- Anaerobic jar or anaerobic chamber is required for growth and toxin production of Clostridium perfringens.
- Incubator is set at 35°C to 37°C for incubation of plate for 24 to 48 hours.
- Inoculating loop, needle or sterile applicator stick is used for streaking organisms in straight perpendicular lines.
Procedure of Reverse CAMP Test (Reverse CAMP Test for Clostridium perfringens)
- Fresh 5% sheep blood agar plate is taken and it is allowed to reach room temperature.
- A known CAMP positive Streptococcus agalactiae strain is streaked in a single straight line across the center of the plate.
- The suspected test organism such as Clostridium perfringens is streaked in a straight line perpendicular to the S. agalactiae streak.
- The streaks should not touch each other. A small gap of about 1 to 2 mm is maintained between them to allow diffusion of factors in agar.
- The plate is placed in anaerobic jar or anaerobic chamber.
- The plate is incubated at 37°C for 24 to 48 hours.
- After incubation, the junction area of both streaks is observed for bow-tie or reversed arrowhead zone of hemolysis.
- For Arcanobacterium haemolyticum, the organism may be streaked perpendicular to Staphylococcus aureus to observe inhibition of beta hemolysin.
Results of Reverse CAMP Test
Positive result for Clostridium perfringens– A distinct bow-tie, arrowhead, or reversed arrow shaped zone of enhanced hemolysis is formed pointing towards the Streptococcus agalactiae streak. This indicates that the test isolate produces alpha toxin which acts synergistically with CAMP factor.
Negative result for Clostridium perfringens– Growth of organism is seen but no enhanced hemolysis zone is formed at the junction area of the two streaks. This helps to differentiate it from other clostridia like Clostridium septicum.
Inhibitory result for Arcanobacterium haemolyticum– The organism inhibits the staphylococcal beta-lysin and an arrow of no hemolysis is seen. This is also called CAMP inhibition test.
Synergistic result for Arcanobacterium haemolyticum– An arrowhead or crescent shaped zone of enhanced hemolysis is produced due to interaction of phospholipase D with CAMP factor.
Arrowhead Hemolysis in Reverse CAMP Test
Arrowhead hemolysis is a triangular or arrow shaped area of enhanced hemolysis formed near the junction of two bacterial streaks.
In reverse CAMP test, it may appear as bow-tie or reversed arrowhead hemolysis. It is commonly seen in positive Clostridium perfringens reaction.
This hemolysis is produced because alpha toxin of C. perfringens and CAMP factor of Streptococcus agalactiae act together on red blood cells.
Reverse CAMP Test for Arcanobacterium haemolyticum
Arcanobacterium haemolyticum is another organism which can give reverse CAMP reaction.
In Arcanobacterium CAMP test, the organism may be tested with Streptococcus agalactiae or Staphylococcus aureus.
With Streptococcus agalactiae, Arcanobacterium haemolyticum may produce arrowhead or crescent shaped zone of enhanced hemolysis.
With Staphylococcus aureus, it produces an inhibitory reaction. The positive result is seen as an arrow of no hemolysis because phospholipase D inhibits staphylococcal beta-lysin.
This test helps to differentiate Arcanobacterium haemolyticum from beta hemolytic streptococci and commensal coryneform bacteria.
Reverse CAMP Positive Bacteria
Reverse CAMP positive bacteria are those bacteria which produce positive reverse CAMP reaction on blood agar due to interaction of their toxin or phospholipase with hemolytic factor of another organism.
Some important reverse CAMP positive bacteria are-
Clostridium perfringens– It gives bow-tie or reversed arrowhead zone of enhanced hemolysis with Streptococcus agalactiae.
Arcanobacterium haemolyticum– It may give synergistic hemolysis with Streptococcus agalactiae or inhibition of beta hemolysis with Staphylococcus aureus.
Corynebacterium pseudotuberculosis– It gives inhibition of beta hemolysin of Staphylococcus aureus and is important in veterinary diagnosis.
CAMP Test vs Reverse CAMP Test
CAMP test and reverse CAMP test are blood agar based tests used for identification of bacteria by hemolytic reaction.
CAMP test is mainly used for identification of Streptococcus agalactiae. In this test, Staphylococcus aureus is used as indicator organism and S. agalactiae gives arrowhead zone of enhanced hemolysis.
Reverse CAMP test is mainly used for identification of Clostridium perfringens and Arcanobacterium haemolyticum. In this test, the test organism produces toxin or phospholipase which reacts with CAMP factor or beta hemolysin of another organism.
In CAMP test, the CAMP factor is produced by the test organism. In reverse CAMP test, CAMP factor is supplied by Streptococcus agalactiae and the toxin is produced by the suspected organism.
Thus, camp vs reverse camp test differs mainly in the organism tested, indicator organism used, and pattern of hemolysis.
Uses of Reverse CAMP test
- It is used for presumptive identification of Clostridium perfringens in clinical laboratory.
- It is used in wound infection and suspected gas gangrene cases.
- It is used for confirmation of Arcanobacterium haemolyticum during pharyngeal screening in adolescent pharyngitis.
- It helps to differentiate Arcanobacterium haemolyticum from commensal diphtheroids and beta hemolytic streptococci.
- It is used in veterinary diagnosis to identify Corynebacterium pseudotuberculosis in caseous lymphadenitis of sheep and goats.
- It is used in food safety laboratory to confirm C. perfringens isolates from suspected food samples or patient fecal matter in food poisoning outbreaks.
- It is used in environmental and epidemiological studies to differentiate toxigenic C. perfringens from other non-toxigenic clostridia.
Advantages of Reverse CAMP test
- High sensitivity- It is highly sensitive and it can identify most of the Clostridium perfringens isolates by giving clear positive reaction.
- Cost effective- It uses standard 5% sheep blood agar which is commonly available in microbiology laboratory.
- Direct proof of toxin production- It gives phenotypic confirmation that the organism is producing toxin like alpha toxin or phospholipase D.
- Clear visual interpretation- The reaction gives distinct bow-tie or reversed arrowhead zone which can be easily recognized on the agar plate.
- Effective differentiation- It helps to differentiate target organisms from similar species like C. perfringens from Clostridium septicum and Arcanobacterium haemolyticum from Streptococcus pyogenes.
Limitations of Reverse CAMP test
- Strict media requirement- The test depends on sheep or bovine blood agar because high sphingomyelin content is needed for proper reaction.
- Risk of false negative- Some Clostridium perfringens strains may produce alpha toxin in low amount and visible synergistic hemolysis is not produced.
- Reagent strain variability– All Streptococcus agalactiae strains do not produce equal CAMP factor. A verified high CAMP producing strain is required.
- Strict incubation atmosphere- For Clostridium perfringens, incubation must be in strict anaerobic condition. If anaerobic condition is not maintained then toxin production is reduced.
- Slower turnaround time- The test needs 24 to 48 hours incubation so it is slower than rapid identification methods like MALDI-TOF.
- Agar quality dependency– If blood agar is very thin or already partially hemolyzed then reaction becomes weak and interpretation becomes difficult.
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.
References
- American Society for Microbiology. (2009). Reverse CAMP test for the identification of Clostridium perfringens. ASM Image Gallery.
- Awad, M. M., Ellemor, D. M., Boyd, R. L., Emmins, J. J., & Rood, J. I. (2001). Synergistic effects of alpha-toxin and perfringolysin O in Clostridium perfringens-mediated gas gangrene. Infection and Immunity, 69(12), 7904–7910.
- Bae, S. Y., Choi, S., Kang, S. J., & Shin, J. H. (2010). A case of Arcanobacterium haemolyticum bacteremia and osteomyelitis diagnosed by 16s rRNA sequencing. Infection and Chemotherapy, 42(4), 241-243.
- Bernheimer, A. W., & Avigad, L. S. (1982). Mechanism of hemolysis by Renalin, a CAMP-like protein from Corynebacterium renale. Infection and Immunity, 36(3), 1253–1256. https://doi.org/10.1128/iai.36.3.1253-1256.1982
- Bernheimer, A. W., Linder, R., & Avigad, L. S. (1979). Nature and mechanism of action of the CAMP protein of group B streptococci. Infection and Immunity, 23(3), 838–844. https://doi.org/10.1128/iai.23.3.838-844.1979
- Camargo, A., Ramírez, J. D., Kiu, R., Hall, L. J., & Muñoz, M. (2024). Unveiling the pathogenic mechanisms of Clostridium perfringens toxins and virulence factors. Emerging Microbes & Infections, 13(1), 2341968. https://doi.org/10.1080/22221751.2024.2341968
- CAMP test. (2025, November 17). In Wikipedia. https://en.wikipedia.org/wiki/CAMP_test
- Centers for Disease Control and Prevention. (1971). Details – Public Health Image Library (PHIL).
- Consonni, A., Briozzo, E., Giubbi, C., Tonolo, S., Luzzaro, F., & Mauri, C. (2026). Insights into Arcanobacterium haemolyticum: A narrative review of an emerging pathogen revisited. Pathogens, 15(3), 335. https://doi.org/10.3390/pathogens15030335
- Gase, K., Ferretti, J. J., Primeaux, C., & McShan, W. M. (1999). Identification, cloning, and expression of the CAMP factor gene (cfa) of Group A Streptococci. Infection and Immunity, 67(9), 4725–4731. https://doi.org/10.1128/iai.67.9.4725-4731.1999
- Hansen, M. V., & Elliott, L. P. (1980). New presumptive identification test for Clostridium perfringens: Reverse CAMP test. Journal of Clinical Microbiology, 12(4), 617-619. https://doi.org/10.1128/jcm.12.4.617-619.1980
- Hanson, A. (2006). CAMP test protocols. American Society for Microbiology.
- Ilhan, Z. (2013). Detection of Corynebacterium pseudotuberculosis from sheep lymph nodes by PCR. Revue Méd Vét, 164(2), 60-66.
- Kang, H., Park, G., Kim, H., & Chang, K. (2016). Haemolytic differential identification of Arcanobacterium haemolyticum isolated from a patient with diabetic foot ulcers. JMM Case Reports, 3(1), e005016. https://doi.org/10.1099/jmmcr.0.005016
- Kiu, R., & Hall, L. J. (2018). An update on the human and animal enteric pathogen Clostridium perfringens. Emerging Microbes & Infections, 7(1), 141. https://doi.org/10.1038/s41426-018-0144-8
- Microbiology pictures. (2015). The CAMP test and the reverse CAMP test. www.microbiologyinpictures.com
- Microbiology pictures. (2015). The reverse CAMP test with Corynebacterium pseudotuberculosis and Staphylococcus aureus on blood agar. www.microbiologyinpictures.com
- Mubarak, A., & Moussa, I. M. (2022). Sero-diagnosis of Corynebacterium pseudotuberculosis affecting camels using recombinant phospholipase D (rPLD) and anti-camels IgY-conjugate. Journal of King Saud University – Science, 34(8), 102344. https://doi.org/10.1016/j.jksus.2022.102344
- Niyomdecha, N., Phakamas, W., & Nana, A. (2016). Modified human blood agar as substitute for sheep blood agar in laboratories of developing countries. Journal of Pure and Applied Microbiology, 10(3), 1773-1779. https://doi.org/10.22207/JPAM.10.3.11
- Pokhrel, P. (2015). Reverse CAMP test for the identification of Clostridium perfringens. Microbiology Notes.
- Rao, S. P. N. (2009). CAMP TEST. Microrao.
- Raza, A., Mahmood, S., Khan, A., Goshi, S., Khalid, R., Ijaz, M., Shaheen, M., Noor, A., Bano, S., Asghar, S., Afzal, T., & Akbar, Z. (2024). Mechanisms of action of toxins released by Clostridium perfringens. BioScientific Review, 6(1), 16-33. https://doi.org/10.32350/bsr.61.i
- Sakurai, J., & Oda, M. (2004). Clostridium perfringens alpha-toxin: Characterization and mode of action. Journal of Biochemistry, 136(5), 569-574. https://doi.org/10.1093/jb/mvh161
- Sapkota, A. (2022). CAMP test- principle, procedure, types, results, uses, limitations. Microbe Notes.
- Syame, S. M., Abuelhag, H. A., Kandil, M. M., Gaber, E. S., & Ebessy, E. A. (2021). Bacteriological, molecular and histopathological evaluation of four vaccines’ protective role against caseous lymphadenitis in sheep. International Journal of Health Sciences, 6(S6), 9941–9954. https://doi.org/10.53730/ijhs.v6nS6.12561
- The reverse CAMP test: Analytical mechanisms, biochemical principles, and clinical utility in diagnostic microbiology. (n.d.).
- Vu, M. L. D., & Rajnik, M. (2024). Arcanobacterium haemolyticum infections. In StatPearls. StatPearls Publishing.
- What is the difference between CAMP and reverse CAMP test. (n.d.). Difference Between.
- Yeh, E., Pinsky, B. A., Banaei, N., & Baron, E. J. (2009). Hair sheep blood, citrated or defibrinated, fulfills all requirements of blood agar for diagnostic microbiology laboratory tests. PLoS ONE, 4(7), e6141. https://doi.org/10.1371/journal.pone.0006141
- Zwart, M. (n.d.). Reverse CAMP test Clostridium perfringens – Microbiology [Video]. YouTube.