Paper I
Question
Anaerobic culture methods.
Answer
Anaerobic culture methods are laboratory techniques designed to isolate and grow obligate anaerobic bacteria, which are killed or fail to grow in the presence of atmospheric oxygen, by creating and maintaining an oxygen-free (or very low oxygen tension) environment.
Methods:
- Anaerobic jar (McIntosh and Filde’s jar) — inoculated plates are placed in an airtight jar; a gas-generating envelope/kit (chemically produces hydrogen and CO2) combined with a palladium catalyst removes residual oxygen by catalysing its combination with hydrogen to form water; a methylene blue or resazurin indicator strip confirms anaerobiosis (remains colourless if anaerobic conditions are achieved).
- Anaerobic chamber (glove box) — a sealed cabinet continuously flushed with an oxygen-free gas mixture (nitrogen, hydrogen, CO2), allowing inoculation, incubation, and examination of cultures entirely within an anaerobic atmosphere — provides the most reliable and stringent anaerobic environment for routine laboratory use.
- Robertson’s cooked meat (RCM) broth — a reducing liquid medium containing meat particles that consume oxygen and lower the redox potential, supporting growth of anaerobes even without special jar/chamber apparatus; widely used for enrichment.
- Reducing/pre-reduced media — media prepared and stored under anaerobic conditions, or containing reducing agents (e.g., thioglycollate, cysteine) that lower the oxidation-reduction potential, allowing anaerobic growth in ordinary incubators.
- GasPak system — a commercially available, self-contained gas-generating system used within an anaerobic jar, simplifying the process.
Specimen handling: proper anaerobic technique also requires appropriate collection (avoiding air exposure, e.g., needle aspiration rather than swab) and prompt transport in an anaerobic transport medium, since many anaerobes die rapidly on exposure to oxygen.