Tube Agglutination

Material

Pipettes

Test tube

Grease pencil

Pack for tube

Serum

Agglutination tube

Antigen 50% water bath

Normal saline

PROCEDURE

1. Set ten test tube on a rack
2. A volume of normal saline should be added to tube one and 1 volume each tube from tube 2 to tube ten.
3. Dilute tube one in five dilution with serum
4. Dilute tube 1 volume serum in ten dilution tube three one in twenty dilution proceed to the final tube this will yield the following result 1 in 5, 1 in 10, 1 in 20, 1 in 40, 1 in 80, 1 in 160, 1 in 320, 1 in 640, 1 in 1280, 1 in 2560.

Begin from the 1 in 2560 that is the highest dilution and transfer 0.5ml from each tube to the corresponding agglutination tube rack.

Add 0.5ml of antigen to each tube, the addition of the serum dilute in further again. At this point the first dilution yield 1 in 10 dilution white these second dilution yield 1 in 20 dilution and so on to the final dilution.

Proceed control solution on this tube add 0.5ml of saline now set agglutination rack in a water bath, the water bath should adjusted until 1/3 tube covering is achieved, after these examine the test to determine the agglutination on the experiment.

Slide test agglutination: in other to achieve quick agglutination of colonies from an agar media, the slide agglutination is recommended. On these test, the colonies is suspended in a tube with known volume of saline and known volume of serum and mixed.

The colonies serve as an antigen. But it should be noted that only O agglutination can be carried out with the method because solid media are not good for formation of flagella, if it should be carried out false negative result is bound to occur with H slide agglutination mean why confirmation is advisable with this technique of slide agglutination.

PROCEDURE

Place a drop of saline on a slide follow by a drop of serum deserved for testing. Used wire loop transfer colonies subjected for examination to the slide and mixed, make sure that homogenous smooth suspension is obtained. Auto agglutination will take place, but in case of no auto agglutination has taken place, them mixed the serum with the homogenous suspension and observed for agglutination to take place.

Remember that agglutination only take place in the present of electrolyte so it should be born in mind that 0.9% of sodium chloride should be added as diluents, and also immunoglobulin’s are destroy by excess heat, in this case temperature above 50ºC must not be used. Antigen flagella agglutinate more rapidly than that of somatic antigen the reading may be taken after 2 hours incubation at 50ºC.

But with refrigeration at 4ºC, somatic antigen can best be incubated at 37ºC for 2 hours over night, then read and record.

And it should be noted that when incubating, water level of the water bath should be adjusted to 1/3 in other for the water to cover the tube, when the water cover the tube it will permit convection of currents in the tube and mixing, therefore raised agglutination reaction in the tube.

Carrier particle agglutination: when particles phase is absent in the transformation of the either antigen or antibody, and then it is necessary to stick either the antigen or antibody to a carrier particle such as latex beads erythrocytes or a coagulated complex protein of staphylococcus aureus.

Latex agglutination: when antibodies to microorganism are attracted to the latex substance the reagent found hear are used to see homologous antigen in the body specimen i.e fluids these agglutination process has been developed for examination of haemophilus, pneumococcus and meningococcus in the CSF antigen, urine, and serum and for examination of streptococci via serological identification of bacteria.

Haemagglutination: when tannic acids are treated with erythrocyte it sticks treponoma pallidum extracts and haemaglutination test and are used to see syphilis antibodies. But when hepatitis B infection involved erythrocyte that is covered with homologous antiserum will see Austrelia antigen (HBsAg) in the serum of the patient.

Coagulation: staphylococcus aureus is composed excess of protein A, so when these protein react with IgG molecules, the antibody surrounding staphylococcus areus agglutinated in the presence of the homologous antigen, and these reaction can be used for quick checking of the antigen in the body fluids fluid examination and differentiation of micro organism.

For example phadebact test which is used for examination of Nesseria gonorrhea.

Precipitation: On this screening solution, antigen reaction with specific antibody to form precipitate, I.e. lancefield streptococcus grouping.