Lab diagnosis of bacterial infection.
Specimen:

Blood culture in acute stage of the dis­ease is the most definite method of diagnosis. Urine, sputum and breast milk may yield positive culture as the organisms are intermittently excreted in these specimens. Biopsied lymph node or bone marrow aspirate culture is done in chronic cases. Serology is useful in diagnosing acute infection. Multiple samples of blood should be collected for culture and serological testing.

Culture and isolation:

(1) Blood cultures: Because of the systemic na­ture of infection, blood cultures are first tried.

(2) When the disease has reached an advance stage, bone marrow aspirate or liver biopsy may be necessary to obtain adequate samples of culture.

Since the organisms may be scanty, at least 10 cc blood (or biopsied material) is cultured in a bottle of liver infusion or glucose serum broth of Trypticase Soy Broth and incubated at 37°C under 5-10% CO2. B. abortus, but not other species, must be incubated under 5-10% CO2 environment.

Sub-cultures are made on liver infusion agar or Trypticase Soy agar every 3-5 days. As growth may often be delayed, culture is retained for six weeks. Brucellae grow faster when the blood culture is transferred to agar medium (e.g. biphasic cultures as in Castaneda’s method).

(1) Castaneda’s method of blood culture provides both liquid (liver infusion broth) and solid media (3% nutrient agar slope) in one bottle. Its use reduces chances of contamination and risk of infection to laboratory personnel.

Since the bottle contains both liquid and solid media, the broth flows over the surface of agar slant when the bottle is tilted. This results in automatic sub-culture. The bottle is kept in the incubator in upright position. In positive cases, colonies develop on the slant. Blood culture comes positive in 30-50% of cases. B. melitensis is more readily cultured than B. abortus.

2. Trypticase Soy broth:

Specimen of blood is inoculated in Trypticase Soy broth and then sub­cultures on serum dextrose agar every 3-5 days.

3. BACTEC is a rapid and automated diagnostic system for culture in which results are obtained in 5-6 days.

Identification:

Isolated organism is identified by:

1. Requirement of CO2 for growth.

2. Production of urease and H2S.

3. Sensitivity to the dyes basic fuchsin, thionin.

4. Agglutination with mono-specific sera, and

5. lysis by bacteriophage.

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