Objectives: Aerotolerance Culturing Organisms with Various Oxygen Requirements Exercises 2-6, 2-7, 2-8

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1

List five groupings of microorganisms according to their oxygen tolerance. Know the characteristics of each group.

1. Obligate aerobes- require Oxygen to survive (ML, CV)

2. Facultative anaerobes- do not require oxygen
but can use it (SA, SM)

3. Obligate anaerobes- oxygen is toxic (CS)

4. Aerotolerant anaerobes- oxygen not toxic but
can't use it (EF)

5. Microaerophiles- High oxygen level is toxic but
need a little oxygen

2

Understand the characteristics of capnophiles as they relate to aerotolerance

sub category of microaerophiles they love carbon dioxide but need a small amount of oxygen

3

Understand how each of the methods used create environments that are anaerobic and/or have low oxygen tension/increased carbon dioxide concentration.

Stab cultures heated or boiled drive oxygen out.

Old tubes will need to be reheated to remove oxygen that may have gotten into it.

Water, phyglycolaid will reduce any oxygen

Resizure- indicator will turn pink to indicate how much air has defused

Gas pack- white indicator button = no oxygen, blue= contaminated. Methylene blue turns blue in presence of oxygen. releases carbondioxide capanophiles enviroment, sorbic acid, vitamen C removes the oxygen.

4

Accurately appraise the redox potential of a GasPak anaerobic system and tubes of Fluid Thioglycollate Medium. Identify the redox potential indicators.

Gas Pak - is a method used in production of an anaerobic environment. It is used to culture bacteria which die or fail to grow in presence of oxygen (anaerobes). Redox indicator is a paper indicator strip saturated with methylene blue that changes color to blue in the presence of oxygen and is colorless when reduced. Pak creates an anaerobic, microaerophilic, or CO2 enriched conditions.

Fluid Thiglycollate Medium - used primarily to determine the oxygen requirements of microorganisms. Sodium thioglycolate in the medium consumes oxygen and permits the growth of obligate anaerobes.[1] This, combined with the diffusion of oxygen from the top of the broth produces a range of oxygen concentrations in the media along its depth. The oxygen concentration at a given level is indicated by a redox sensitive dye like resazurine that turns pink in the presence of oxygen.

5

Know the purpose of catalase (peroxidase) and superoxide dismutase in bacterial metabolism and how these relate to aerotolerance.

catalase breaks hydrogen peroxide down into oxygen and water.
Superoxide dismutase is an enzyme that break down super oxides (O2-) into either hydrogen peroxide or molecular oxygen (O2).

these enzymes remove oxygen free radicals

6

Understand why anaerobes are inhibited/killed by oxygen

they lack catalase or superoxide dismutase enzymes to remove oxygen

7

Understand why tubed media are boiled for at least 10 minutes prior to inoculation or should be used soon after initial preparation. (agar shake cultures, stab cultures, Fluid Thioglycollate medium)

to remove air and oxygen

8

Name at least one bacterial genus that tolerates oxygen but does not produce catalase

Streptococcus

Organism's test to see if it has catalase is to pour peroxide on it an will break down the hydrogen peroxide by bubbling

9

Understand why it is important to time how long it takes for the candle in a candle jar to go out

It is measuring how long it takes for the oxygen to be removed from the jar. Heat is also accumulating in the jar. The candle goes out and the jar cools when oxygen depleted.

10

Understand why adding a small amount of agar to the Thioglycollate Medium would be beneficial. (Small amount would be enough to slightly thicken the medium but not solidify it.)

thickened agar slows diffusion of air into medium

11

Understand the meaning of the term “fastidious” as it relates to bacterial culturing

picky as to requirements such as nutrients, temperature, PH, salinity, oxygen

12

Understand why water (moisture) accumulates in the candle jar after the lid has been tightened down

Palladium catalyzes a reaction between the hydrogen and free oxygen in the jar to produce water. Removal of free oxygen produces anaerobic conditions in the jar within approximately an hour, as evidenced by a white indicator strip and moisture on the inside of the jar.