Microculture in biphasic medium with silicone-coated slides for isolation of mycobacteria

The study reported here, seeking to develop a simple, practical, sensitive, and inexpensive technique for microbial diagnosis of tuberculosis, used a combination of biphasic media and microculture techniques to augment the sensitivity of traditional culture methods. A total of 540 sputum samples (5 mL each) were obtained from 180 patients with suspected tuberculosis in Mexico City. These samples were treated with Hanks reagent, neutralized with 25% HCl, and centrifuged. In each case the resulting residue was combined with liquid media (Sula medium or a phosphate-buffered control solution) and was inoculated into a bottle containing a solid medium (Löwenstein-Jensen-Holm or Middlebrook). A silicone-coated slide appropriate for culture of hydrophobic mycobacteria was inserted in each bottle, and the cultures (examined weekly) were incubated at 37 degrees C until the first macroscopic bacterial growth was detected or for up to eight weeks if none was detected. When such growth was detected, or at the end of eight weeks, each slide was withdrawn from the bottle, sterilized, stained by Kinyoun's method, and examined microscopically. Following 2-4 weeks of incubation, macroscopic bacterial growth was detected in 71 bottles and was confirmed by microscopic examination of the corresponding slides. No macroscopic bacterial growth was found in any of the remaining 469 bottles, but microscopic growth was observed on 77 of the slides examined after eight weeks. The authors conclude that this method represents a noteworthy improvement over standard culture methods in terms of bacterial isolation and suggest that its case, economy, and practicality make it suitable for application in developing countries.