1. Bacterial Agglutination Abad, Mary Raina Angeli Z. Advincula, Janina Karla D.C. Al Dhaheri, Sara Ali Anacan, Keight Arren R.

2. Objectives

3. perform bacterial agglutination test explain the principle of serological identification of bacteria interpret the result based on agglutination differentiate serological identification of bacteria from culture identification

4. Materials and Methods

5. Materials glass slides 70% alcohol saline solution pasteur pipets heat-killed E.coli E.coli anti serum

6. Methods 1. Wash slides thoroughly with soap and water. Rinse with 70% ethanol and air dry. 2. Mark two circles at the opposite ends of the slide 3. Using a Pasteur pipets, place a drop of saline solution on one end and anti-serum on the other. 4. Add a drop of heat-killed E.coli on each circle. Rock gently back and forth for two seconds.

7. Test Procedure

8. Put 1 drop of saline solution into a circle of a E.coli latex test kit agglutination slide. Using another dropper/pippetor, dispense 1 drop of the bacterial suspension in the same circle in the slide close to the E.coli latex drop. Mix the latex reagent and the bacterial suspension with a clean mixing stick and rock the slide gently two or three times. Examine for agglutination within a maximum of 2 minutes. LATEX KIT + BACTERIAL SUSPENSION

9. LATEX KIT + COLONY SUSPENSION Using an inoculating loop, remove a colony from the selected agar plate and emulsify the colony in the drop of saline to produce a heavy smooth suspension. Suspensions should only be made from colonies with morphologies resembling E. coli. Mix with an inoculating loop. Rock the slide gently up to 2 minutes and observe for autoagglutination or clumping.

10. DESIRED RESULT Reagent Control Saline soln. + E. coli latexAgglutination Positive Control Positive control + E. coli latexNo agglutination Serologic Test1. Bacterial suspension + E. coli latex 2. Bacterial colony + E. coli latexAgglutination

11. Definition of Terms

12. Agglutination - the clumping of particles. The word agglutination comes from the Latin agglutinare, meaning "to glue to."Latin Serology ­– a science that studies bloodserum and immune responses that are found in it. O antigen – carbohydrate antigen within the wall of microorganism H antigen – flagellar components that also act as an antigen

13. Principle behind Bacterial Agglutination

14. Bacterial agglutination is a highly specific identification of microorganisms that can be obtained by serological techniques. In vitro, antigens and antibodies combine and produce reactions that are visibly measured. The chemical compositions of antigens differ; therefore, each antigen will react only with a complimentary antibody that is unique to that antigen.

15. In gram-negative bacilli, the carbohydrate antigens within the wall of the organism are called “O” antigens. O from German “Ohne” = body Each species has a different array of O antigens that can detected in serological tests.

16. In like manner, those bacilli that are motile also contain characteristic flagellar protein components called “H” antigens. H from German “hauch” = motility

17. Antigen-antibody reaction can be demonstrated in several ways: ▪ Latex agglutination- are coated with polyvalent antisera against a wide range of antigens, when mixed with the suspension of microorganism latex particles rapidly agglutinate to form visible clumps ▪ Coagglutination – the aggregation of particulate antigens combined with agglutinins with more than one specificity. ▪ Enzyme-linked assays. – any enzyme immuno assay using an enzyme-labeled immunoreactant and an immunosorbent These tests depend on linking antibody to a particle or enzyme in order for a positive reaction to be observed.

18. A simple test, which detects O and H antigens of gram-negative enteric bacilli (usually Salmonella and Shigella species and Escherichia coli), is the Latex agglutination test.

19. When the unknown organism isolated in culture is mixed with an antiserum that contains antibodies specific for its antigenic makeup, agglutination (clumping) of the bacteria occurs.

21. If the antiserum does not contain specific antibodies, no clumping is seen.

22. A control test in which saline is substituted for the antiserum must always be included to be certain that the organism does not clump in the absence of the antibodies

23. ✔ NOTE: In latex agglutination testing, the antibody has been conjugated to a latex particle in order to enhance the visibility of the agglutination reaction. Latex bead Homospecific antibody