Faculty of Medicine and Health Sciences Microbiology Lab Experiment 13 Culture of Anaerobic Bacteria and Respiratory Enzymes (Second Semester 2015-2016)

Culture of the obligate anaerobes: Obligate anaerobic bacteria are bacteria that grow only without oxygen and, in fact, oxygen inhibits or kills them. They obtain energy by anaerobic respiration or fermentation. A variety of obligate anaerobic bacteria, which are usually normal flora of the body, may cause various opportunistic infections. Accordingly, most anaerobic infections are endogenous ones An example of anaerobic opportunistic pathogen: Bacteroides fragilis Note: anaerobic infections are characterized by a foully smell. This is due to tissue fermentation by the infecting anaerobic bacterial pathogen . In addition, most anaerobic infections are mixed infections that include facultative anaerobic bacteria that would consume O2 . This will encourage the growth of the anaerobic bacterium at the infection site.

Classification of bacteria based on Oxygen requirement: Based on Oxygen requirement, bacteria may be classified as: Obligate aerobes: they require O2 for growth. They use O2 as a final electron acceptor in aerobic respiration. Obligate anaerobes : they do not need or use O2 . In fact, O2 is a toxic substance for them, which either kills or inhibits their growth. Facultative anaerobes : these are bacteria that can switch between aerobic and anaerobic types of metabolism. Under anaerobic conditions, they generate energy mainly by fermentation . In the presence of O2 they switch to aerobic respiration. Aerotolerant anaerobes: these are bacteria that are exclusively anaerobic (fermentative). However, they are insensitive (not killed) to the presence of O2. They live by fermentation alone whether or not O2 is present in their environment Microaerophilic: these bacteria need O2 in low concentration

Utilization or exposure to O2 results in the generation of highly reactive and toxic molecules known as free radicals such as superoxide (O2.-) and Hydrogen peroxide (H2O2). Unless the cell is able to neutralize these molecules, free radicals oxidize various cellular molecules to inactivate them resulting in cell death. In aerobic bacteria, facultative anaerobic bacteria, aerotolerant and microaerophilic , the lethal accumulation of free radicals is prevented by superoxide dismutase and catalase (or peroxidase). Obligate anaerobes lack such enzymes and therefore undergo lethal oxidations by various free radicals when they are exposed to O2, and thus they may get killed or have their growth inhibited.

Superoxide Dismutase, Catalase and Peroxidase: These are the enzymes that detoxify oxygen radicals that are generated by cells in case of O2 utilization or incase the presence of O2. The distribution of these enzymes in cells determines their ability to survive in the presence of O2.

Common Methods Used In culturing obligate Anaerobes in the lab include: Candle Jar: Any a sealable, non-flammable jar large enough to hold petri dishes can be used as a candle jar. After placing the cultured plates in the jar, and lit candle inside, then the jar is closed firmly. The candle flame will consume most of the oxygen in the jar and will produce an elevated level of carbon dioxide. Then the jar is placed in the incubator. Candle jar is ideal for the growth of microaerophilic organisms.

GasPak Jar: The GasPak is a commercially-produced disposable hydrogen and carbon dioxide generator envelope. When water is added, hydrogen and carbon dioxide are produced. In the presence of palladium catalyst, the hydrogen then combines with oxygen to form water, thus creating an anaerobic atmosphere within the jar. The cultured plates are placed in a GasPak jar, then water is added to the GasPak and the lid of the jar is sealed. After that, the jar is placed in an incubator Methylene blue impregnated paper may be used an indicator for the generation of an anaerobic atmosphere within the jar. In the absence of O2, Methylene blue becomes colorless.

Anaerobic Incubators: Example: Carbon dioxide incubators: Carbon dioxide incubators are frequently used to culture anaerobes. After the cultures are placed in the incubator, the air is evacuated and replaced by carbon dioxide gas and other gases such as Nitrogen

Preparing Broth for Culturing Anaerobic bacteria: Water contains soluble O2, which must be removed when a broth is intended to be used for culturing anaerobic bacteria. It is possible to remove soluble from water by using a reducing chemical agent that reduces O2 to H2O such as Sodium Thioglycolate . Example: Thioglycolate broth.

Respiratory Enzymes: 1-Catalase Test: Catalase is the enzyme that breaks hydrogen peroxide (H2O2) into H2O and O2. H2O2 is a potent oxidizing agent that can react with many cellular components. Because of this, any cell that uses O2 or can live in the presence of O2 must have a way to get rid of H2O2. One of those ways is to have the catalase enzyme Procedure: Place a small amount of growth from your culture onto a clean slide. Add a few drops of H2O2 ( 3%) onto the smear. If needed, mix with a toothpick. A positive result is the rapid evolution of O2 as evidenced by bubbling.

A negative result is no bubbles or only a few scattered bubbles. A positive result is the rapid evolution of O2 as evidenced by bubbling Examples: Staphylococci are catalase positive Streptococci re catalase negative Notes: I- Be very careful not to scrape up any of the blood agar. Red blood cells contain the catalase enzyme. Any contaminating with the blood agar while taking bacteria from the blood agar plate could give a false positive result. II- DO NOT use a metal loop or needle with H2O2; it will give a false positive and degrade the metal.

2- The Oxidase Test: Procedure: The oxidase test identifies bacteria that produce the enzyme Cytochrome oxidase.  Cytochrome oxidase participates in the electron transport chain by transferring electrons from a donor molecule to oxygen.  The oxidase reagent contains a chromogenic reducing agent, which is a compound that changes color when it becomes oxidized.  The chromogenic reducing agent is N,N,N′,N′-tetramethyl-p- phenylenediamine ((TMPD)). If the tested bacterium produces Cytochrome oxidase, the oxidase reagent will turn blue or purple within 15 seconds. Procedure: With a sterile swab, obtain a small amount of bacteria from an agar slant or plate. Place one drop of reagent onto the culture on the swab. Positive reactions turn the bacteria violet to purple immediately or within 10 to 30seconds. Delayed reactions should be ignored.