VMC 311: Systematic Veterinary Bacteriology and Mycology (2+1)

TUBE AGGLUTINATION TEST FOR BRUCELLA

• The tube agglutination tests are more accurate than the plate test, but require relatively long incubation time.

Materials

• Dryer’s agglutination tube.
  • Test serum and positive serum
  • Brucella abortus plain antigen
• Normal saline

Procedure

• Set up six agglutination (Dryer’s) tubes in a row and add 0.8 ml of saline to tube No. 1 and 0.5 ml to the other five tubes.
• Add 0.2 ml of test serum to tube No. 1. Mix well and then transfer 0.5 ml to tube No. 2. Repeat two fold dilutions upto tube No.6 and then discard 0.5 ml (dilution 1:5, 1:10 and so on in each tube).
• Add 0.5 ml of plain antigen to each tube and mix well (final dilution of serum resulted will be 1:10, 1:20 and so on).
• Control tubes are also set in the test.
  • Positive control with 0.5 ml of known positive serum and 0.5 ml plain antigen.
  • Negative control with 0.5 ml saline and 0.5 ml plain antigen.
  • 50 % control with 0.75 ml saline and 0.25 ml plain antigen.
• All tubes are then incubated at 37°C for 18-20 h before the results are read

Interpretation

• Mat formation will be observed at the bottom of positive tubes, while a pellet or button will be seen in negative tubes.
• The highest serum dilution showing 50% agglutination (ie 50% clearing) or more is taken as the end point or titre in the serum.
• A titre of 1:40 or above indicates an infected animal.

Uses

• In this test, antiserum is serially diluted and mixed with constant amount and concentration of antigen.
• It is commonly used for diagnosis of diseases like Salmonellosis, Brucellosis, Tularemia, Campylobacteriosis etc. (Click here for visual)