These include bubo aspirates (in bubonic plague), sputum (in pneumonic plague), and blood and cerebrospinal fluid (CSF) (in septicemic plague).
Bubo aspirates is collected by injecting 1 mL of sterile saline into the bubo with a 20-G needle. The fluid is aspirated there-after by withdrawing several times. The bubo aspirate smears are stained with Gram, Wright, Wayson, or Giemsa stain for demonstration of the typical bipolar (safety pin) morphology of Y. pestis. Blood and CSF of patients who are septicemic can also be stained to reveal the bacteria
Isolation of Y. pestis by culture confirms the diagnosis of plague. The organism can be isolated from blood, CSF, sputum, and bubo aspirates, depending on the clinical presentation, whether it is bubonic, pneumonic, or septicemic. Y. pestis is slow grow-ing, but it does not require any special growth media. Clinical specimens are inoculated on blood or nutrient agar and on specialized selective media for isolation of Y. pestis. Selective medium, such as blood agar with sodium azide is used for selective isolation of Y. pestisfrom sputum and bubo aspirates containing numerous other bacteria.
Dark-brown colonies on blood agar and pinpoint colonies on MacConkey agar are the characteristic features of colonies of Y. pestis. The identifying features of Y. pestis colonies are summarized.
Y. pestis can be isolated from bubo aspirate or sputum by inoculat-ing in guinea pigs or white rats. The bubo aspirate is injected sub-cutaneously into the animal. Y. pestis causes death of the animal within 2–5 days. Postmortem of the dead animal shows necrosis and edema at the site of inoculation. Draining lymph nodes are enlarged, congested and show grayish white patches. Gram- and methylene blue-stained smears of lymph nodes, spleen, and heart blood reveal typical bipolar stained Gram-negative bacilli.
Sputum collected from suspected cases of pneumonic plague is inoculated by applying them over the shaven skin or to the nasal mucosa of a guinea pig. The bacteria present in the specimen penetrate the skin through minute abrasions. Bacilli are demonstrated in the sputum and in the blood from the heart of the guinea pig, which dies 2–3 days after inoculation.
Serological tests usually supplement diagnosis of plague. Enzyme-linked immunosorbent assay (ELISA) and indirect hemagglutination (IHA) tests are employed to detect specific antibodies in acute and convalescent sera. A fourfold or greater difference in antibody titer between acute and convalescent sera collected 10 days apart, demonstrated by ELISA or IHA, suggests plague infection. A single positive ELISA or IHA test in a patient who has not received plague vaccine nor has had previous plague is also presumptive of infection.