JAWAHARLAL NEHRU TECHNOLOGICAL UNIVERSITY HYDERABAD B.Pharmacy III Year PHARMACEUTICAL MICROBIOLOGY Previous Question Paper (November/December- 2020 - R17)

Anaerobes are microorganisms that grow in the absence of free oxygen. Cultivation requires specialized techniques to remove oxygen from the environment and maintain a strictly anaerobic atmosphere.

The primary methods for cultivating anaerobes include:

• Anaerobic Jars (GasPak System): This is the most common laboratory method. The culture plates are placed in an airtight jar along with a chemical sachet containing sodium borohydride and sodium bicarbonate. Upon adding water, the sachet releases hydrogen and carbon dioxide. The hydrogen reacts with oxygen in the presence of a palladium catalyst to form water, effectively removing oxygen. A methylene blue or resazurin indicator strip is used to confirm the absence of oxygen (turning colorless when anaerobic conditions are achieved).
• Anaerobic Chambers (Glove Boxes): These are specialized, enclosed workstations filled with an inert gas mixture (usually 85% nitrogen, 10% hydrogen, and 5% carbon dioxide). Researchers manipulate cultures through attached rubber gloves. This method allows for the incubation, inspection, and subculturing of strict anaerobes without exposure to atmospheric oxygen.
• Reducing Media: Cultivation in liquid media requires the addition of reducing agents to lower the oxidation-reduction potential. Common additives include sodium thioglycollate, L-cysteine, or ascorbic acid. These chemicals react with dissolved oxygen in the medium. Fluid Thioglycollate Medium (FTM) is a standard medium that supports the growth of both aerobic and anaerobic bacteria.
• Deep Agar Shake Cultures: This technique involves inoculating the organism into molten agar medium, which is then allowed to solidify. The depth of the agar tube creates an oxygen gradient; the bottom of the tube remains anaerobic, allowing for the growth of obligate anaerobes, while the top remains aerobic.
• Candle Jar Method: Although primarily used for microaerophiles, a candle jar can be used to reduce oxygen tension. A lit candle is placed in a sealed jar; as the candle burns, it consumes most of the oxygen and replaces it with carbon dioxide, though this does not create a strictly anaerobic environment.

Successful cultivation of anaerobes also necessitates the use of pre-reduced media and the rapid transfer of samples to the anaerobic environment to prevent oxygen toxicity, which can damage or kill sensitive anaerobic cells.