How to Perform a CAMP Test: Principle and Protocol

The CAMP test is a biochemical procedure used to presumptively identify Streptococcus agalactiae (Group B Streptococcus, GBS) by detecting the synergistic hemolytic activity of its CAMP factor with the beta-hemolysin produced by Staphylococcus aureus. This test is useful for differentiating S. agalactiae from other beta-hemolytic streptococci, particularly when Lancefield grouping is unavailable or as a confirmatory step in a identification scheme. The test relies on the observation of an enhanced zone of hemolysis (arrowhead or bow-tie shaped) at the junction where the two organisms grow in close proximity on sheep blood agar.

At a Glance

Scientific Principle

The CAMP test is named after its discoverers: Christie, Atkins, and Munch-Petersen, who first described the phenomenon in 1944. The test exploits a synergistic hemolytic reaction between two distinct bacterial products:

1. CAMP factor: A diffusible, heat-stable, extracellular protein produced by Streptococcus agalactiae. CAMP factor is not itself hemolytic but binds to the erythrocyte membrane that has been damaged by staphylococcal beta-hemolysin.

2. Beta-hemolysin: A phospholipase C produced by Staphylococcus aureus that hydrolyzes sphingomyelin in the erythrocyte membrane, causing incomplete (beta) hemolysis around the staphylococcal streak.

When these two factors interact, the CAMP factor binds to the sphingomyelinase-damaged erythrocytes, causing complete lysis. This results in a characteristic arrowhead or bow-tie shaped zone of clear (complete) hemolysis at the junction where the two organisms meet on sheep blood agar. The reaction is highly specific for S. agalactiae among beta-hemolytic streptococci, though rare CAMP-positive reactions have been reported in some other streptococcal species (e.g., Streptococcus uberis, Streptococcus porcinus).

The test is performed on sheep blood agar because sheep erythrocytes contain high levels of sphingomyelin, making them susceptible to beta-hemolysin action. Horse or human blood agar may not produce reliable results due to differences in erythrocyte membrane composition.

Materials and Instrumentation

Essential Materials

• Sheep blood agar plates: 5% defibrinated sheep blood in Tryptic Soy Agar base. Plates should be fresh (prepared within 7 days) and stored at 2–8°C. Older plates may have reduced hemolytic activity.
• Inoculating loops: Sterile, disposable 1 µL or 10 µL loops for streaking.
• Sterile swabs or forceps: For handling control organisms if using colony picks.
• Incubator: Capable of maintaining 35–37°C with 5% CO₂ (or a candle jar for CO₂ enrichment).
• Bunsen burner or biosafety cabinet: For aseptic technique.
• Marking pen: For labeling plates.

Organism Requirements

• Test organism: Unknown beta-hemolytic streptococcus to be identified.
• Positive control: Streptococcus agalactiae ATCC 12386 (or other well-characterized strain).
• Negative control: Streptococcus pyogenes ATCC 19615 (Group A Streptococcus, which is CAMP-negative).
• Indicator organism: Staphylococcus aureus ATCC 25923 (known beta-hemolysin producer). This strain consistently produces beta-hemolysin and is recommended for standardized testing.

Quality Control Strains

All control strains should be obtained from a reputable culture collection (e.g., ATCC, NCTC) and maintained according to the supplier's instructions. Working cultures should be subcultured no more than three times from the reference stock to maintain phenotypic stability.

Controls

Proper controls are essential for valid CAMP test interpretation. Without controls, false-positive or false-negative results cannot be distinguished from technical errors.

Positive Control

• Organism: S. agalactiae ATCC 12386
• Expected result: Arrowhead-shaped zone of complete hemolysis at the junction with S. aureus
• Purpose: Confirms that the S. aureus strain is producing beta-hemolysin and that the medium supports the synergistic reaction

Negative Control

• Organism: S. pyogenes ATCC 19615
• Expected result: No enhanced hemolysis at the junction; only the normal beta-hemolysis of S. aureus is visible
• Purpose: Demonstrates that the test system does not produce false-positive reactions with a common beta-hemolytic streptococcus

Medium Control

• Observation: The S. aureus streak alone should show a zone of incomplete (beta) hemolysis around the streak line
• Purpose: Confirms that the sheep blood agar supports beta-hemolysin activity and that the S. aureus strain is viable and producing hemolysin

Reagent Control

• Observation: The test organism streak alone should show its characteristic hemolysis pattern (typically beta-hemolytic for S. agalactiae, but this is not required for the CAMP test)
• Purpose: Ensures the test organism is viable and growing properly on the medium

Conceptual Workflow

Step 1: Preparation

1. Remove sheep blood agar plates from refrigeration and allow them to warm to room temperature (approximately 15–20 minutes). Cold plates may cause condensation that interferes with hemolysis observation.
2. Label the bottom of each plate with the test organism identification, control organisms, date, and your initials.
3. Using a marking pen, draw a line on the bottom of the plate to indicate where the S. aureus streak will be placed.

Step 2: Inoculation of S. aureus Streak

1. Using a sterile inoculating loop, pick a single colony of S. aureus ATCC 25923 from a fresh (18–24 hour) culture.
2. Streak the S. aureus in a single straight line down the center of the plate. The streak should be approximately 2–3 cm long and placed in the middle of the plate.
3. The streak should be heavy enough to produce visible growth but not so heavy that it obscures hemolysis.

Step 3: Inoculation of Test and Control Organisms

1. Using a fresh sterile loop for each organism, streak the test organism perpendicular to the S. aureus line. The test streak should start approximately 2–3 mm away from the S. aureus line and extend outward.
2. For the positive control plate, streak S. agalactiae perpendicular to the S. aureus line.
3. For the negative control plate, streak S. pyogenes perpendicular to the S. aureus line.
4. Multiple test organisms can be streaked on the same plate, but they must be spaced at least 1 cm apart to prevent cross-contamination and to allow clear interpretation.

Step 4: Incubation

1. Place the inoculated plates in an incubator at 35–37°C with 5% CO₂ (or in a candle jar).
2. Incubate for 18–24 hours. Do not exceed 24 hours, as prolonged incubation may cause the hemolysis zones to merge or become difficult to interpret.

Step 5: Reading Results

1. After incubation, examine the plates for the presence of an arrowhead-shaped zone of complete hemolysis at the junction of the test organism and S. aureus streaks.
2. Hold the plate up to a light source or use a colony counter with transmitted light to better visualize hemolysis patterns.
3. Record the result as positive, negative, or equivocal.

Quality Checks

Pre-Analytical Quality Checks

• Verify that sheep blood agar plates are within expiration date and show no signs of contamination, hemolysis, or excessive moisture.
• Confirm that control organisms are viable and pure by examining colony morphology and performing Gram stains as needed.
• Ensure the S. aureus strain has been tested for beta-hemolysin production within the past month. Some strains lose hemolytic activity upon repeated subculture.

Analytical Quality Checks

• Each batch of tests must include both positive and negative controls on separate plates.
• The S. aureus streak must show visible beta-hemolysis (a clear zone around the streak) to confirm hemolysin production.
• Test and control organisms must show adequate growth along their entire streak lines.

Post-Analytical Quality Checks

• Document all results, including control results, before interpreting test results.
• If controls fail (e.g., positive control is negative, negative control is positive), repeat the entire test with fresh reagents and controls.
• Photograph or diagram the hemolysis patterns for permanent records.

Result Interpretation

Positive CAMP Test

A positive CAMP test shows a distinct arrowhead, bow-tie, or wedge-shaped zone of complete (clear) hemolysis at the junction where the test organism streak meets the S. aureus streak. This enhanced hemolysis extends outward from the junction point, creating a characteristic arrowhead pattern pointing toward the test organism.

Interpretation: The test organism is presumptively identified as Streptococcus agalactiae (Group B Streptococcus). Confirmatory testing (e.g., Lancefield grouping, PYR test, or bile esculin test) should be performed for definitive identification.

Negative CAMP Test

A negative CAMP test shows no enhanced hemolysis at the junction. Only the normal beta-hemolysis of S. aureus is visible around the staphylococcal streak, and the test organism shows its characteristic hemolysis pattern (typically beta-hemolysis for most pathogenic streptococci).

Interpretation: The test organism is not S. agalactiae. It may be another beta-hemolytic streptococcus such as S. pyogenes (Group A), S. dysgalactiae (Group C or G), or S. equi (Group C). Further testing is required for identification.

Equivocal Results

Occasionally, a weak or diffuse zone of hemolysis may be observed that does not form a clear arrowhead shape. This may occur due to:

• Weak CAMP factor production by the test organism
• Reduced beta-hemolysin production by S. aureus
• Suboptimal medium conditions
• Overgrowth of one organism

Action: Repeat the test with fresh cultures and controls. If equivocal results persist, consider alternative identification methods such as Lancefield grouping or molecular testing.

Troubleshooting

Limitations

Organism-Specific Limitations

1. False-positive reactions: Some non-GBS streptococci can produce CAMP-like factors. Streptococcus uberis (a bovine pathogen) and Streptococcus porcinus (associated with swine) can give positive CAMP reactions. These organisms are rarely encountered in human clinical specimens but should be considered in veterinary or environmental isolates.

2. False-negative reactions: Rare strains of S. agalactiae may produce weak or undetectable CAMP factor. Some reports suggest that up to 1–2% of GBS isolates may be CAMP-negative. These strains require alternative identification methods.

3. Anaerobic CAMP test: Some anaerobic bacteria (e.g., Propionibacterium acnes, Clostridium perfringens) can produce CAMP-like reactions under anaerobic conditions. This test is specifically for aerobic/facultative anaerobic streptococci.

Technical Limitations

1. Medium dependency: The test requires fresh (≤7 days old) 5% sheep blood agar. Older plates may have reduced sphingomyelin content or altered erythrocyte membrane integrity.

2. Strain variability: Not all S. aureus strains produce adequate beta-hemolysin. The ATCC 25923 strain is recommended for consistent results. Some clinical isolates of S. aureus may produce other hemolysins (alpha, delta) that can interfere with interpretation.

3. CO₂ requirement: The test performs optimally in 5% CO₂. Incubation in ambient air may reduce hemolysin production and CAMP factor activity.

4. Subjective interpretation: The arrowhead shape can be subtle, particularly with weak reactions. Training and experience are required for consistent interpretation.

Scope Limitations

This protocol is designed for educational and research laboratory settings (BSL-1) and is not intended for clinical diagnostic use. Clinical laboratories should follow validated, regulatory-approved procedures for patient specimen testing.

Documentation

Required Records

For each CAMP test performed, document the following:

1. Test identification: Unique identifier for each test organism (e.g., laboratory number, strain designation)
2. Date and time: Date of inoculation and time of reading
3. Medium: Type, lot number, expiration date, and preparation date of sheep blood agar
4. Organisms: Source and passage number of test organism and controls
5. Incubation conditions: Temperature, CO₂ concentration, and duration
6. Results: Positive, negative, or equivocal; include description of hemolysis pattern
7. Control results: Positive and negative control results
8. Technician: Name or initials of person performing the test
9. Comments: Any observations, deviations from protocol, or troubleshooting actions

Example Documentation Entry

Date: 2024-01-15
Test ID: LAB-2024-001
Medium: TSA with 5% sheep blood, Lot #SB1234, Exp: 2024-02-01
Test organism: Streptococcus sp. isolate from throat swab
Positive control: S. agalactiae ATCC 12386
Negative control: S. pyogenes ATCC 19615
Indicator: S. aureus ATCC 25923
Incubation: 35°C, 5% CO₂, 22 hours
Result: Positive – distinct arrowhead hemolysis at junction
Control results: Positive control positive, negative control negative
Technician: J. Smith
Comments: Test organism shows beta-hemolysis; arrowhead pattern consistent with S. agalactiae

Biosafety Considerations

Risk Assessment

The CAMP test as described here uses organisms classified as Biosafety Level 1 (BSL-1) or BSL-2 agents. Streptococcus agalactiae and Staphylococcus aureus are BSL-2 agents in clinical settings but are commonly used in teaching laboratories at BSL-1 with appropriate precautions [1]. The CDC and NIH's Biosafety in Microbiological and Biomedical Laboratories (BMBL) provides the framework for risk assessment [1].

Standard Practices

1. Hand hygiene: Wash hands before and after handling cultures.
2. Personal protective equipment (PPE): Wear a laboratory coat, gloves, and safety glasses.
3. Aseptic technique: Perform all inoculations near a Bunsen burner flame or in a biosafety cabinet.
4. Work surface decontamination: Clean work surfaces with 10% bleach or appropriate disinfectant before and after use.
5. Waste disposal: Dispose of all contaminated materials (plates, loops, gloves) in biohazard waste containers.
6. No eating or drinking: Do not eat, drink, or apply cosmetics in the laboratory.

Decontamination

All cultures and contaminated materials must be autoclaved at 121°C for 30 minutes before disposal [1]. Spills should be covered with absorbent material, flooded with disinfectant, and allowed to sit for 20 minutes before cleanup.

Training

Personnel performing the CAMP test should receive training in:

• Basic microbiological techniques
• Biosafety practices for BSL-1 laboratories
• Proper use of personal protective equipment
• Emergency procedures for spills and exposures

Frequently Asked Questions

Q1: Can I use human blood agar instead of sheep blood agar for the CAMP test?

No, sheep blood agar is specifically required for the CAMP test. Sheep erythrocytes contain high levels of sphingomyelin, which is the substrate for staphylococcal beta-hemolysin. Human erythrocytes have a different membrane composition and may not support the synergistic hemolysis reaction. Horse blood agar may work but produces less consistent results. Always use 5% defibrinated sheep blood agar for standardized CAMP testing.

Q2: Why do I need to incubate the CAMP test in CO₂?

Carbon dioxide (5% CO₂) enhances the production of beta-hemolysin by Staphylococcus aureus and promotes optimal growth of both S. aureus and Streptococcus agalactiae. Incubation in ambient air may result in reduced hemolysin production, leading to false-negative results. If a CO₂ incubator is unavailable, a candle jar (which generates approximately 3–5% CO₂) can be used as an alternative.

Q3: Can I test multiple unknown organisms on the same CAMP plate?

Yes, multiple test organisms can be streaked perpendicular to the same S. aureus line, provided they are spaced at least 1 cm apart. This is an efficient way to screen several isolates simultaneously. However, each plate must still include appropriate controls on separate plates. Be careful to use a fresh sterile loop for each organism to prevent cross-contamination.

Q4: What should I do if my positive control gives a negative CAMP reaction?

A negative positive control indicates a problem with the test system. First, verify that the S. aureus strain is producing beta-hemolysin by examining the hemolysis around the staphylococcal streak. If no hemolysis is visible, the S. aureus strain may have lost its hemolysin production. Subculture from a reference stock or obtain a fresh ATCC culture. If the S. aureus shows hemolysis but the positive control is negative, the sheep blood agar may be too old or improperly stored. Use fresh medium (≤7 days old) and repeat the test.

References and Further Reading

1. Biosafety in Microbiological and Biomedical Laboratories (BMBL), 6th Edition – CDC and NIH. Provides authoritative principles for risk assessment, containment, decontamination, and microbiological laboratory practice. https://www.cdc.gov/labs/bmbl/index.html

2. NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules – National Institutes of Health. Provides institutional and biosafety framework for recombinant and synthetic nucleic acid research. https://osp.od.nih.gov/policies/biosafety-and-biosecurity-policy/nih-guidelines-for-research-involving-recombinant-or-synthetic-nucleic-acid-molecules/

3. NCBI Bookshelf: Molecular Biology and Laboratory Methods – National Center for Biotechnology Information. A searchable collection of authoritative biomedical books and methods references. https://www.ncbi.nlm.nih.gov/books/

Related Articles

• How to Perform a PYR Test: Principle and Protocol
• How to Perform a Gelatin Hydrolysis Test: Principle and Protocol
• How to Perform a Bile Esculin Test: Principle and Protocol
• How to Perform a Phenylalanine Deaminase Test: Principle and Protocol
• How to Perform a Nitrate Reduction Test: Principle and Protocol
• How to Perform a Starch Hydrolysis Test: Principle and Protocol