Haemophilus Serotyping: Introduction, Principle, Requirements, Test Procedure, Result Interpretation and Keynotes

Haemophilus Serotyping: Introduction, Principle, Requirements, Test Procedure, Result Interpretation and Keynotes

Introduction of Haemophilus Serotyping

Haemophilus Serotyping helps to differentiate serotypes of Haemophilus influenzae. 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, encapsulated strains of Haemophilus influenzae may be differentiated into 6 serotypes (a to f), of which Haemophilus influenzae serotype b (Hib) is the principal cause of invasive diseases. 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.

Remel™ Agglutinating Sera,  for Haemophilus influenzae, is available for use in the laboratory, are as follows-

  1. Haemophilus influenzae type a Agglutinating Sera
  2. Haemophilus influenzae type b Agglutinating Sera
  3. Haemophilus influenzae type c Agglutinating Sera
  4. Haemophilus influenzae type d Agglutinating Sera
  5. Haemophilus influenzae type e Agglutinating Sera
  6. Haemophilus influenzae type f Agglutinating Sera

Note: These agglutinating sera are intended for use in slide agglutination tests to identify serologically the type antigen of pathogenic strains of H. influenzae (types a to F) for epidemiological and diagnostic purposes.

Principle of Haemophilus Serotyping

Haemophilus 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. In brief, the serological test is based on the fact that antibodies in serum, produced in response to exposure to bacterial antigens, will agglutinate with bacteria carrying homologous antigens.

Test Requirements for Haemophilus Serotyping

Haemophilus 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
  • Timer
  •  Light source over dark background
  • Control strains

Test Procedure of Haemophilus 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 (30µl) 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 slide 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 on 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.


Resul and Interpretation of Haemophilus Serotyping

Agglutination : Positive

No agglutination: Negative

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.

Limitations of Haemophilus Serotyping


  1. Cross‑reactions have been reported to occur with organisms of other species and thus species confirmation is mandatory using morphological, cultural, and biochemical methods.
  2. The optimal concentration of the antigen should be sufficient in the specimen otherwise a negative result will be obtained.
  3. Antisera provide only serological identification and hence full identification of an organism must only be made in conjunction with biochemical assaying.


Keynotes on Haemophilus Serotyping

  1. Salmonella, Shigella, Vibrio cholerae, and Haemophilus influenzae are the most common bacteria in those 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 as MHA, Blood agar, or TSI in the case of enteric Gram-negative bacteria while for H. influenzae chocolate agar is preferred.
  6. In the case of Shigella, taking growth from the KIA agar for routine slide agglutination test heating is not required.
  7. Haemophilus influenzae agglutinating Sera are produced in rabbits and are preserved with 0.5% phenol.
  8. The antisera should be stored at 2 -8°C under which condition they will retain their potency at least until the date shown on the bottle label.

Further Readings on Haemophilus Serotyping

  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://www.cdc.gov/vaccines/pubs/pinkbook/hib.html
  5. https://www.who.int/immunization/monitoring_surveillance/burden/vpd/WHO_SurveillanceVaccinePreventable_05_HaemophilusInfluenzae_BW_R2.pdf
  6. https://bmcinfectdis.biomedcentral.com/articles/10.1186/1471-2334-9-45
  7. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3358072/
  8. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3322004/
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