ABSTRACT
Slice and mince: - The cleaned tissue is sliced in half with a sterile scalpel and the exposed inner surface is finely minced. The minced surface is then smeared over the entire surface of each selective medium. 20 •21 This is a simple method and requires very little equipment. The isolation rate is approximately 40 to 50% but is decreased by inadequate mincing of the inner surface. 16
Stomacher: - The cleaned tissue is cut into small pieces and placed in a bag with an equal volume of phosphate-buffered saline. The bag is then placed in a Col worth Stomacher (Tekmar Co., Cincinnati, OH) and macerated for a minimum of 5 min. Using a sterile cotton swab, the tissue suspension is spread over the entire surface of each selective medium. 19
The tissue must be macerated for at least 5 min, and some tissues, particularly uterine tissue, may not macerate well. The isolation rate is approximately 40 to 50%. 16 An additional method is to pipette 1 rnl of the tissue suspension onto the solid phase of biphasic medium so that the excess drains into the liquid phase. Usually, a second flask is inoculated with 2 ml of the same tissue suspension. Each flask is observed at 3-, 5-, 7-, and 14-d intervals, and if no growth is observed, each solid phase is reinoculated by tipping the flask. 15 This method is very sensitive, but more prone to contamination than the solid phase methods. The isolation rate is approximately 30 to 40%. 16
Centrifuge: - Centrifugation is effectively used for the isolation of brucellae from milk. Milk is centrifuged at 6000 to 7000 x g for 15 min. Using a sterile cotton swab, cream and sediment are inoculated onto separate plates of selective media. 6
Guinea pig inoculation: - This method is rarely used because of the cost and time required. After injecting a suspension of the sample into several guinea pigs, half of the animals are necropsied at 3 weeks and the other half at 6 weeks. Each animal serum is tested for the presence of antibodies, and the spleen, lymph nodes, and any visible lesions are cultured. 7 •8
Culturing from blood: - Heparinized blood can be directly spread onto agar plates or can be inoculated into trypticase soy broth which is frozen, thawed, and incubated for several days before it is subcultured onto solid medium. The second method is slower but more sensitive. 6 Isolation from blood is recommended only if other specimens cannot be obtained.
