IMMUNOSEROLOGY
Exercise 1: Preparation of Bacterial Antigens 1.
Bacterial Antigens of Medical Importance
1. O or Somatic Antigen
2. H or Flagellar Antigen
3. K or Vi or Capsular Antigen
DEFINITION OF TERMS
 Derived from the German Ohne Hauch
 Refers to the cell body alone excluding
the flagella and capsule
 Important in the classification of enteric
Gram (-) bacilli into major groups
 Some enteric bacteria possess the O Ag
without the flagellar H Ag (e.g. Klebsiella
Somatic
pneumoniae, Shigella spp.)
Ag (O
Ag)  Relatively stable toward heat, alcohol and
acids
 Preparation: by heating or treating the
cells with alcohol or acids
 O Agglutination: fine granular precipitate
 Results from the reaction
between nonflagellated cells
and anti-O serum
 Derived from Hauch
 Relatively labile toward heat, alcohol and
acids
Flagellar  More specific than O antigen
Ag (H  Of more value into separating bacteria
Ag) into specific strains or types
 Preparation: by adding equal volume of
formol to an 18-24 hour broth culture
Capsula  Occur in most bacteria but play a
r Ag (K predominant role in only a few:
or Vi Ag)  Streptococcus pneumoniae
 Bacillus anthracis
PART I. PREPARATION OF H ANTIGEN
Materials
 Cultures of S. typhi inoculated on TSA
 Pasteur pipette
 10 mL pipette
 BaSO4 Standard
 0.6% formalinized saline
 Glass beads
 10 mL thioglycolate broth
 Appropriately labelled test tubes
PROCEDURE PROCEDURE
Add glass beads and 8mL of 0.6% formalinized 1. Add glass beads to a bottle containing cultures of S.
saline to a bottle containing cultures of S. typhi typhi inoculated on trypticase soy agar.
inoculated on trypticase soy agar (TSA). 2. Add absolute alcohol instead of 0.6 % formalinized
saline mixture.
2. Scrape off the growth on the surface of the agar 3. Gently rock the bottle to scrape off the growth on the
by gently rocking the bottle. surface of the agar.
4. Harvest the suspension using a pipette and transfer it to
3. Using a pipette harvest the suspension and
a sterile test tube.
transfer to a sterile test tube
5. Place the test tube with the bacterial suspension in a
4. Transfer 1.0 mL of the bacterial suspension to
water bath for 60 minutes at 60 C. Be sure to fully
another test tube.
immerse the bacterial suspension.
5. Gradually add 0.6% formalinized saline until 6. Centrifuge the suspension at 1,500 RPM for 2 minutes.
turbidity matches the standard No. 4 BaSO4
solution. Equivalent to 1.2 billion organisms per
7. Decant and discard the supernatant.
mL.
8. Add 8 mL of 0.6% formalinized saline to the
6. Allow the suspension to incubate at room precipitate.
temperature for 48 hours. 9. Transfer 1 mL of the bacterial suspension to another
7. Test for sterility by aseptically transferring 1 drop test tube.
of antigen to 10 mL of thioglycollate broth. 10. Gradually add 0.6% formalinized saline until the turbidity
Incubate at 37 C for 24 hours. of the solution matches the BaSO4 solution.
8. Observe for turbidity. Sterile antigens will not 11. Allow the suspension to incubate at RT for 48 hours.
exhibit growth.
12. Test for sterility by aseptically transferring 1 drop of
antigen to 10 mL of thioglycollate broth.
13. Incubate at 37 C for 24 hours.
14. Observe for turbidity. Sterile antigens will not exhibit
growth.
PART II. PREPARATION OF O ANTIGEN
Materials
 Cultures of S. typhi inoculated on TSA
 Pasteur pipette
 10 mL pipette
 BaSO4 Standard
 0.6% formalinized saline
 Glass beads
 10 mL thioglycolate broth
 Appropriately labelled test tubes
 Centrifuge
 Water bath
 Absolute Alcohol