Avoid contact with skin and mucosa. On disposal,

flush with large quantities of water.

Sample Collection and Storage

1. No special preparation of patient is required prior to

sample collection by approved techniques. Do not use

hemolyzed serum samples.

2. Clean and dry glassware free from detergents must be

used for sample collection.

3. Do not heat/inactivate the serum.

4. Though freshly collected serum is preferred, samples

can be stored at 2 to 8°C, for 24 hours or frozen for 8

days should a delay in testing occur.

Materials Provided with the Kit

Stained Brucel-A/Brucel-M antigen suspensions.

Additional Material Required

Slide Test Method

Stop watch, positive control, isotonic saline, and glass

slide with clear/white background, appropriate pipettes/

micropipettes, mixing sticks and a high intensity direct

light source.

Quantitative Method

Timer, test tubes (12mm × 75mm), test tube rack, appropriate pipettes/micropipettes, isotonic saline/0.25%

phenol saline, incubator (37°C).

Procedure

Bring all reagents to room temperature.

Shake and mix the Bruce antigen suspensions well

before dispensing.

The procedure for Brucel-A and Brucel-M is identical.

Slide Test Method

Qualitative Method

1. Place one drop of positive control onto the reaction

circle of glass slide.

2. Place 80 µL of saline onto the next reaction circle of

the glass slide.

3. Place 80 µL of patient serum to be tested onto the next

reaction circle.

4. Add one drop of the appropriate Brucel antigen

suspensions in each of the above circles (containing

positive control, saline, and the patient serum to be tested).

5. Mix contents of each circle uniformly over the entire

circle with separate mixing sticks.

6. Gently rock the slide back and forth, observe for

agglutination macroscopically, at one minute against

white background.

636 Concise Book of Medical Laboratory Technology: Methods and Interpretations Semiquantitative Method

1. Using a pipette place 80 µL, 40 µL, 20 µL, 10 µL, and

5 µL of patient serum to be tested on 5 different circles

on the glass slide. The corresponding titers obtained

will be 1:20, 1:40, 1:80, 1:160, and 1:320 respectively.

2. Place one drop of appropriate Brucel antigen

suspension to each circle.

3. Mix contents of each circle uniformly over the entire

circle with separate mixing sticks.

4. Gently rock the slide back and forth, observe for

agglutination macroscopically at one minute against

a white background.

Tube Test Method

1. Take 8 test tubes and label them 1 to 8.

2. Pipette 1.9 mL of isotonic saline or preferably 0.25%

phenol saline to tube No. 1.

3. To each of the remaining tubes (2–7), add 1.0 mL of

isotonic saline or preferably 0.25% phenol saline.

4. To the tube No. 1, add 0.1 mL of serum sample to be

tested. Mix well.

5. Transfer 1.0 mL of the diluted serum from tube No. 1

to tube No. 2 and mix well.

6. Transfer 1.0 mL of the diluted serum from tube

No. 2 to tube No. 3 and mix well. Continue this serial

dilution till tube No. 7.

7. Discard 1.0 mL of the diluted serum from tube No. 7.

8. Pipette 1.0 mL of isotonic saline in tube No. 8, which

serves as a negative control.

9. To all the tubes add 1 drop of appropriate Brucel

antigen suspensions and mix well.

10. Cover the tubes and incubate at 37°C for 24 hours.

11. Observe for agglutination macroscopically in each

tube of the dilution series.

Interpretation of Results

Slide Test Method

Qualitative Method

Agglutination is a positive test result and indicates the

presence of specific antibodies to Brucella in the patient

serum.

No agglutination is a negative test result and indicates

absence of specific antibodies to Brucella in the patient

serum.

Semiquantitative Method

Agglutination is a positive test result. The titer of patient

serum corresponds to the visible agglutination in the test

circle with the minimum amount of serum sample.

Tube Test Method

The titer of patient serum is the reciprocal of the last dilution

of the serum sample that gives a granular agglutination.

In negative reaction, the appearance of the suspension

remains unchanged, which shows a typical swirl when the

tube is flicked.

Remarks

1. Turbid and contaminated serum should not be used

for testing.