The "lytic phenomenon" between Staphylococcus aureus and Streptococcus agalactiae (group B), the basis for the CAMP test, was first observed on cultures of milk being tested for hemolytic Streptococcus during an outbreak of scarlet fever. Colonies of Streptococcus were only surrounded by zones of complete hemolysis when they were growing in close proximity to colonies of b-hemolytic Staphylococcus (3). Further studies showed that this enhanced hemolysis was a result of the production of a heat stable, filterable agent produced by only Streptococcus agalactiae (group B). Nongroup B Streptococcus failed to exhibit this enhanced hemolysis when grown near colonies of b-hemolytic Staphylococcus (3).
Munch-Petersen used this phenomenon to develop a test to identify Streptococcus agalactiae (group B), a major cause of mastitis in milk samples. He observed that "when two streptococcus colonies grow 5 mm from the b-producing staphylococcus and 5 to 6 mm from each other, the lysed area may become half moon shaped" (9).
Murray, in his paper published in 1952, was the first to refer to this test as the CAMP test, named after the first researchers (Christie, Atkins, Munch-Petersen) to observe this reaction. Murray inoculated the test organism (Streptococcus) in a straight line at right angles to the b-hemolytic Staphylococcus aureus. At the time of Murray's paper the CAMP test was the standard test for the identification of Lancefield group B (S. agalactiae) in milk as a means to screen for mastitis infections in cows (10).
Darling developed a standardized method of inoculating and incubating the CAMP test (see protocol section). The CAMP test was found to be effective for the "prompt and reliable" identification of Streptococcus agalactiae (group B). This test replaced other tests used to identify Streptococcus agalactiae (group B) in the clinical lab as results could be observed in as little as 18 hours and required few manipulations (4). The reliable CAMP test was found to rarely give false positives with other Streptococcus (11).
The CAMP test is used to identify Streptococcus agalactiae (group B) (CAMP positive) and to differentiate it from Streptococcus pyogenes (group A) (CAMP negative) and nongroup B Streptococcus (CAMP negative) (4).
The b-lysin produced by b-hemolytic Staphylococcus aureus acts synergistically with the CAMP factor produced by both b-hemolytic and nonhemolytic Streptococcus agalactiae (group B). This synergistic reaction results in an enhanced and very visible zone of hemolysis in the region between the two cultures. The synergistic zone is not observed in group A, C, and G Streptococcus (8).
The CAMP test is performed on standard sheep blood agar plates, trypticase soy agar +5% sheep blood. Atlas protocol Blood Agar Plates-recipe
Staphylococcus aureus is inoculated onto a sheep blood agar plate by making a narrow streak down the center of the plate with a loop or the edge of a needle (4). A strain of S. aureus known to produce a high level of b-toxin should be used (4, 8). The test organism (suspect group B Streptococcus) is streaked in a straight-line inoculum at right angles to the S. aureus. The Streptococcus streak should be within 2 mm of, but not touching, the S. aureus streak (Fig. 1) (4). The plates are incubated at 35°C for 24 hours. Incubation in ambient air is recommended to reduce the number of false positives (8).
FIG. 1. Streaking pattern for the CAMP test.
A positive result is indicated by an "arrowhead"-shaped enhanced zone of b-hemolysis. (See Atlas Protocol Blood Agar Plates-interpretation of b-hemolysis) in the area between the two cultures with the "arrow point" toward the S. aureus streak. No enhanced zone of b-hemolysis is observed in a CAMP negative reaction (8) (Fig. 2).
FIG. 2. CAMP test for the identification of Streptococcus agalactiae (group B). (A) Streptococcus (group B) shows a positive CAMP reaction. (B) Streptococcus pyogenes (group A) shows a negative reaction when inoculated at a right angle to (C) Staphylococcus aureus.
Other applications of the CAMP test.
CAMP test for the identification of Listeria monocytogenes
A version of the CAMP test was first used by Groves to identify pathogenic Listeria monocytogenes. He found that most of the pathogenic L. monocytogenes he tested were also CAMP positive (5). To perform the CAMP test for the identification of L. monocytogenes, L. monocytogenes is streaked at a right angle to the streak of b-hemolytic Staphylococcus aureus on a sheep blood agar plate. It is recommended that the plate be incubated in ambient air at 35°C for 24 hours (8). A positive CAMP reaction is indicated by a weaker enhanced zone of b hemolysis and a smaller less obvious rectangular zone of hemolysis (5) (Fig. 3). Listeria monocytogenes is a biosafety level 2 organism and should be handled with the necessary precautions (2) (see safety section). Atlas Images CAMP Test for the identification of L. monocytogenes
FIG. 3. CAMP test for the identification of Listeria monocytogenes. (A) Listeria monocytogenes shows a positive CAMP reaction when streaked at a right angle to (B) Staphylococcus aureus.
Reverse CAMP test for the identification of Clostridium perfringens
Gubash first reported a synergistic effect between Streptococcus agalactiae (group B) and hemolytic Clostridium perfringens. The synergistic relationship was studied to determine if using Clostridium perfringens in place of Staphylococcus aureus would decrease the number of false positives observed using the traditional CAMP test. An arrowhead-shaped zone of hemolysis was observed when the two organisms grew at right angles on the sheep blood agar plate (6).
Hansen used the synergistic relationship between these two microbes to develop a test, known as the reverse CAMP test, using Streptococcus agalactiae (group B) for the identification of Clostridium perfringens. The reverse CAMP is performed by streaking Clostridium perfringens down the center of a sheep blood agar plate (in place of S. aureus in the traditional CAMP). Streptococcus agalactiae (group B) is streaked at a right angle to Clostridium perfringens so that the streaks are not touching but are separated by 1 to 2 mm. Plates are incubated anaerobically to allow for the growth of anaerobic Clostridium perfringens. A positive reverse CAMP results in an arrow-shaped zone of hemolysis between the streaks of reverse CAMP-positive Clostridium perfringens and Streptococcus agalactiae (group B) (Fig. 4) (7, 12).
FIG. 4. The reverse CAMP test for the identification of Clostridium perfringens. (A) Streptococcus agalactiae (group B) is streaked at a right angle to (B) reverse CAMP-positive Clostridium perfringens.The reverse CAMP test can also be done by streaking Streptococcus agalactiae (group B) down the center of a sheep blood agar plate. The test organism, Clostridium sp., is streaked at a right angle to and within 1 to 2 mm of the Streptococcus agalactiae (group B) inoculum. Plates are incubated anaerobically to allow for the growth of anaerobic Clostridium perfringens. A positive reverse CAMP result, shown by Clostridium perfringens, is a "bow tie" or reversed arrow zone of enhanced hemolysis at the junction of the two cultures (Fig. 5) (1, 8). Atlas Images CAMP Test Atlas Images- Reverse Camp test for the Identification of Clostridium perfringens
FIG. 5. The reverse CAMP test for the identification of Clostridium perfringens. (A) Reverse CAMP-positive Clostridium perfringens and (B) reverse CAMP-negative Clostridium septicum streaked at right angles to (C) Streptococcus agalactiae (group B).
The ASM advocates that students must successfully demonstrate the ability to explain and practice safe laboratory techniques. For more information, read the laboratory safety section of the ASM Curriculum Recommendations: Introductory Course in Microbiology and the Guidelines for Biosafety in Teaching Laboratories.
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This section is to evolve as feedback on the protocol is discussed at ASMCUE. Please contact the project manager for further information.
1. Buchanan, A. G. 1982. Clinical laboratory evaluation of a reverse CAMP test for presumptive identification of Clostridium perfringens. J. Clin. Microbiol. 14:761–762.
2. Centers for Disease Control and Prevention. 1999. Biosafety in microbiology and biomedical laboratories, 4th ed. U.S. Government Printing Office, Washington, D.C.
3. Christie, N. E., N. E. Atkins, and E. Munch-Petersen. 1944. A note on a lytic phenomenon shown by group B Streptococcus. Aust. J. Exp. Biol. Med. Sci. 22:193–195.
4. Darling, J. F. 1975. Standardization and evaluation of CAMP reaction for the prompt, presumptive identification of Streptococcus agalactiae (Lancefield group B) in clinical material. J. Clin. Microbiol. 1:171–174.
5. Groves, R. D., and H. J. Welshimer. 1977. Separation of pathogenic from apathogenic Listeria monocytogenes by three in vitro reactions. J. Clin. Microbiol. 5:559–563.
6. Gubash, S. 1978. Synergistic hemolysis phenomenon shown by an alpha-toxin producing Clostridium perfringens and streptococcal CAMP factor in presumptive streptococcal grouping. J. Clin. Microbiol. 6:480–488.
7. Hansen, M. V., and L. F. Elliott. 1980. New presumptive test for Clostridium perfringens: reverse CAMP test. J. Clin. Microbiol. 12:617–619.
8. MacFaddin, J. F. 2000. Biochemical tests for identification of medical bacteria, 3rd ed. Lippincott, Williams, and Wilkins, Philadelphia, Pa.
9. Munch-Petersen, E., and R. Christie. 1947. The effect of the interaction of Staphylococcus b toxin and group B streptococcus substance on red blood corpuscles and its use as a test for the identification of Streptococcus agalactiae. J. Pathol. Bacteriol. 59:367–371.
10. Murphy, J. M., O. M. Stuart, and F. I. Reed. 1952. An evaluation of the CAMP test for the identification of Streptococcus agalactiae in routine mastitis testing. Cornell Vet. 42:133–147.
11. Wilkinson, H .W. 1977. CAMP-disk test for presumptive identification of group B streptococci. J. Clin. Microbiol. 6:42–45.
12. Winn, W., S. Allen, W. Janda, E. Koneman, G. Procop, P. Schreckenberger, and G. Woods. 2006. Color atlas and textbook of diagnostic microbiology, 6th ed. Lippincott Williams and Wilkins, Philadelphia, Pa.
This resource was peer-reviewed at ASM Conference for Undergraduate Educators 2006.
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