Vibrio cholerae Serotyping: Introduction, Principle, Test Requirements, Test Procedure, Result- Interpretation and Keynotes

Introduction of Vibrio cholerae Serotyping

Vibrio Serotyping helps to differentiate serotypes of Vibrio. Serotypes refer to separate groups within a species of microorganisms that all share a similar property. More specifically, each serotype has the same number of antigens on its surfaces. For example, V.cholerae O1 serovar Inaba, V. cholerae O1 serovar Ogawa, V. cholerae O1 serovar Hikojima. Serotypes are differentiated on the basis of agglutination tests and used to help with the identification of Salmonella, Shigella, Vibrio cholerae, Haemophilus influenzae, etc. Serotyping of Shigella is performed on the basis of these bacteria having the somatic O antigen. There are many different serotypes that can be identified using the type-specific monovalent antisera. Denka Seiken co Ltd antisera available for use in the laboratory are as follows-

• V. cholerae V. O1 antiserum
• V. cholerae O139 antiserum

Principle of Vibrio Serotyping

Vibrio Serotyping works on the principle of agglutination. When a particulate antigen (agglutinogen) combines with its antibody (agglutinin) in the presence of electrolytes at a suitable temperature and pH, the particles are clumped or agglutinated. Agglutination is the aggregation of already insoluble particles or cells into larger clumps. Interaction between the antibody (Ab) and particulate antigen results in visible clumping called agglutination.

Test Requirements for Vibrio cholerae Serotyping

Vibrio serotyping needs the following requirements-

• Test organism ( pure colony)
• Antisera (serogroups)
• Sterile, clean, and grease-free glass slides
• Normal saline
• Inoculating loop/ Sterile mixing sticks
• Bunsen burner
• Gloves
• Waste bin
• Control strains

Test Procedure of Vibrio cholerae Serotyping

 (Slide agglutination of live organisms)

1. Suspend a certain amount of bacterial growth (3-5 times the amount of a matched head) in 0.5 ml physiological saline and use antigenic suspension.
2. Place a drop of antiserum and physiological saline (30 ul) as control onto a cleaned glass slide partitioned into several parts with a glass pencil.
3. Place an antigenic suspension onto the serum and physiological saline on the sliding glass.
4. Mix the reagents tilting the glass slide back and forth for 1 minute and the agglutination pattern is observed. Agglutination is grossly observed with light through the slide including fluorescent light. It should be first confirmed that no agglutination is found in the reaction with antigenic suspension and physiological saline. Only strong agglutination observed within 1 minute in the reaction with each serum should be regarded as positive. Delayed or weak agglutination is regarded as negative.

Result and Interpretation of Vibrio cholerae Serotyping

Agglutination : Positive

No agglutination: Negative

Refer to the table below for the actual judgment.

Quality Control (QC): It is recommended that quality control should be performed with at least one organism to demonstrate a positive reaction and at least one organism to demonstrate a negative reaction. Do not use the product if the reactions with the control organisms are incorrect. Check for signs of deterioration. Do not use reagents if they are contaminated or cloudy.

Keynotes on Vibrio Serotyping

1. Salmonella, Shigella, Vibrio cholerae, and Haemophilus influenzae are the most common bacteria in that serotyping is performed.
2. Serotyping works on the principle of agglutination.
3. Only do agglutination on pure colonies, which biochemically are the suspected organism. If the specimen consists of multiple strains, the serotype may not be correctly identified.
4. Nutrient agar is best for the agglutination tests.
5. Serotyping is recommended from non-selective media such as MHA, Blood agar, or TSI.
6. In the case of Shigella, taking growth from the KIA agar for routine slide agglutination test heating is not required.

Further Readings

1. https://assets.publishing.service.gov.uk/government/uploads/system/uploads/attachment_data/file/762019/TP_3i4.pdf
2. https://www.frontiersin.org/articles/10.3389/fmicb.2019.02554/full
3. https://sfamjournals.onlinelibrary.wiley.com/doi/10.1111/lam.12690
4. https://link.springer.com/chapter/10.1007/978-3-642-69943-6_18
5. http://swcorp.co.kr/pdf/bacteriology/42_Vibrio%20cholerae%20Insert%20121809.pdf
6. https://www.trios.cz/wp-content/uploads/sites/149/2016/08/Bacterial-Typing-Antisera-Handbook.pdf
7. https://jcm.asm.org/content/jcm/19/2/181.full.pdf
8. https://www.who.int/water_sanitation_health/dwq/admicrob6.pdf
9. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC96140/