INTRODUCTION

Much of the information found in this chapter is adapted from the literature of the manufacturers of the kit systems described. Where possible, manufacturers details will be listed.

The brand used in practicals will vary from time to time, and so details of exact requirements/procedures may not be the same. These commercial kits vary in the method used to extract the antigenic material from the bacterial cell walls. Always check the instructions supplied with the kit.

LANCEFIELD GROUPING OF STREPTOCOCCI BY LATEX CO-AGGLUTINATION KITS

These kits are used for Lancefield grouping of beta-haemolytic Streptococcci. There are a number available on the market. In some kits, the bacteria are first treated with an extraction enzyme, which removes the Lancefield group specific material from the cell walls of the bacteria, leaving it in solution. Drops of the extract are then reacted with individual reagents containing latex particles coated with antibodies to Lancefield groups A,B,C,D,F and G antigens respectively. Agglutination is seen in the well corresponding to the extracted antigen. In other kits, colonies can be tested directly (check specific instructions).

PROCEDURE

1. Add 400 μl of extraction enzyme to a small sterile glass tube. Emulsify several colonies in the solution. If working from a broth, add a drop of broth to the enzyme solution.
2. Incubate at 37oC in a heating block for 10-15 minutes.
3. Mix the latex reagents well.
4. Place a drop of each reagent (A,B,C,D,F and G) in a separate well on the card supplied.
5. Add a drop of the extract to each well and mix with toothpicks. Use a clean toothpick for each well.
6. Rock the card gently until agglutination appears in one well. Observe for 2 minutes.
7. If agglutination does not occur, the strain may be a beta-haemolytic Streptococcus other than groups A,B,C,D,F and G or there may not have been enough inoculum in the extract. Repeat with a stronger extract. If agglutination occurs in more than one well, the isolate is not pure. Re-isolate the organism and repeat.
8. Dispose of test card into the infectious waste containers.

POSITIVE Agglutination in one well

NEGATIVE No agglutination in any well