BIO 261L LAB 6 -ex 24

FTM - Fluid thioglycollate

Fluid thioglycollate medium is a rich liquid medium that supports the growth of both aerobic and anaerobic bacteria. Thioglycollate and cystine in the medium reduce oxygen to water. promotes the growth of anaerobes. The medium also contains the dye resazurin, which is an indicator for the presence of oxygen. Wherever oxygen is present, this dye turns pink or red. Since the oxygen tension is always higher near the surface, the medium will be pink or red at the top and colorless in the middle and bottom. FTM also contains a small amount of agar, which helps to local- ize the organisms and favors anaerobic growth in the bottom of the tube.

differing of enzymes

Most strict anaerobes do not pos- sess catalase or superoxide dismutase. However, some strict anaerobes contain other detoxifying systems to deal with superoxide. Therefore, it is not solely the lack of catalase and superoxide dismutase in these bacteria that is responsible for oxygen sensitivity, but it may involve other factors. Interestingly, aerotolerant anaerobes such as the streptococci possess superoxide dismutase, but they lack catalase.

Obligate (Strict) Anaerobes

Obligate anaerobes cannot tolerate oxygen and must be cultured under conditions in which oxygen is completely eliminated; otherwise, they are harmed or killed by its presence. These organisms carry out fermentation or anaerobic respiration bligate anaerobes are only found among the prokaryotes and in some protozoa. These anaerobes occur in environments such asthe soil, the rumen (stomach) of cattle, and in anaerobic sewage digesters. Clostridium, Methanococcus, and Bacteroides, a bacterium found in the human intestine, are examples of obligate anaerobes.

where the concentration of oxygen is less than that in the atmosphere.

The growth of some bacteria such as the strepto- cocci can be enhanced by cultivation in a candle jar, where the concentration of oxygen is less than that in the atmosphere. Cultures are set up in a jar in which a lighted candle is placed. A lid is placed on the jar and tightened, and the candle is extinguished because the oxygen is partially consumed by combustion. The oxygen concentration decreases and the carbon diox- ide increases to about 3.5% in the jar.

Microaerophiles:

These aerobic bacteria prefer to grow in oxygen concentrationsof 2-10% rather than the 20% found in the atmosphere. The lower concentration of oxygen is necessary for their respiratory metabolism. Their sensitivity to the higher concentrations of oxygen is not, however, completely understood. Helicobacter pylori is a microaerophile that causes stomach ulcers in humans. The oxygen concentration in the stomach is less than the 20% that occurs in the atmosphere.

tryptone glucose yeast extract agar (TGYA)

This solid medium will be used to pre- pare "shake tubes." The medium is not primarily for the cultivation of anaerobes but will be used to deter- mine the oxygen requirements of different bacteria. It will be inoculated in the liquefied state, shaken to mix the organisms throughout the medium, and allowed to solidify. After incubation one determines the oxygen requirements on the basis of where growth occurs in the shake tube: top, middle, or bottom.

The reason for the sensitivity of strict anaerobes to oxygen is not completely understood.

Toxic forms of oxygen such as hydrogen peroxide and superoxide are generated by various chemical mechanisms, and these toxic compounds are abundant in most envi- ronments. Toxic forms of oxygen are highly reactive compounds that can damage biological molecules such as nucleic acids, proteins, and small molecules such as coenzymes.

Facultative Anaerobes

ese bacteria grow very well aerobically but also have the capacity to grow anaerobically if oxygen is not present. Their metabolism is flexible because under aerobic conditions they can carry out respiration to produce energy, but if oxygen is absent they can switch to fermentation that does not require oxygen for energy production. Escherichia coli is a facultative anaerobe.

Aerotolerant Anaerobes

hese anaerobescan tolerate oxygen and even grow in its presence, but they do not require oxygen for energy production. Because they produce their energy strictly by fermentation and not by respiratory means, they are also called obligate fermenters.

obligate (strict) aerobes

hese bacteria must grow in oxygen because their metabolism requires oxygen. They carry out aerobic respiration in which oxygen is utilized as the terminal electron acceptor in the electron transport chain. Examples are Pseudomonas, Micrococcus, and many Bacillus.

Brewer's Anaerobic Agar

his solid medium is excel- lent for culturing anaerobic bacteria in agar plates. Similar to FTM tubes, it contains thioglycollate for reducing ox- ygen and resazurin for indicating the presence of oxygen. For obligate anaerobic growth, it is essential that plates be incubated in an oxygen-free environment. To provide an oxygen-free incubation environment for the petri plates of anaerobic agar, we will use the GasPak anaerobic jar.

GasPak anaerobic jar. Brewer's Anaerobic Agar This solid medium is excel- lent for culturing anaerobic bacteria in agar plates.

hydrogen is generated in the jar, which removes the oxygen by forming water. Palladium pellets cata- lyze the reaction at room temperature. The generation of hydrogen is achieved by adding water to a plastic envelope of chemicals. Note also that CO2 is pro- duced, which is a requirement for the growth of many fastidious bacteria. To make certain that anaerobic conditions actually exist in the jar, an indicator strip of methylene blue becomes colorless in the total absence of oxygen. If the strip is not reduced (decolorized) within 2 hours, the jar has not been sealed properly, or the chemical reaction has failed to occur.

anaerobic respiration

n which inorganic compounds, such as nitrates and sulfate, replace oxygen as the terminal electron acceptor in the electron transport chain

obligate fermenters

produce many food products by fermentation such as cheese, yogurt, and sour cream. Other examples are Enterococcus faecalis found in the human intestinal tract and Streptococcus pyogenes, a pathogen that causes several diseases in humans, such as strep throat and heart and kidney infections.

cultivation of obligate anaerobes

requires specialized conditions that eliminate oxygen and therefore its toxic forms. This can be achieved in an- aerobic incubators or in anaerobic jars that use chemi- cal catalysts to eliminate oxygen. These bacteria can also be cultivated in specialized media that contain chemicals such as thioglycollate, which reacts with oxygen to create anaerobic conditions.

aerobes possess enzymes

systems that will convert the toxic forms of oxygen to less harmful compounds that are not damaging to the cell.

Superoxide dismutase

will act on the superoxide anion and convert it to oxygen and hydrogen peroxide; the latter can be de- graded by catalase.

Peroxidase

will also degrade hydrogen peroxide.

catalase

will degrade hydrogen peroxide into oxygen and water

urinary tract infections

•A urine sample is examined for color, odor, turbidity, pH, mucus, blood or pus. All these might indicate infection. •You will transfer 0.01 ml of a urine sample and then streaked it on a plate. The following week, you counted the colonies and then after multiplying by 100 one can determine the original count.