Chapter 7: Aerobe/anaerobe

Clostridium sporogenes aerotolerance in agar deep tube

Obligate anaerobic: throughout, but not surface

Gas pack anaerobic culture chamber

Oxygen must be removed from the air. White packet inside chamber. This packet contains chemicals that,when activated, start a chemical reaction. The chemical reaction releases free hydrogen into the atmosphere of the chamber,which binds with the oxygen in the air. The hydrogen and oxygen reaction is catalyzed by pellets of palladium in a cage underneath the lid,which is not visible in this picture. This reaction continues until all the oxygen from the air is bound with the hydrogen to form water molecules. This reaction makes the oxygen unavailable and therefore not toxic to anaerobes. There is an indicator strip in the gas pack chamber which contains methylene blue. The strip is blue on exposure to oxygen and turns white in an anaerobic atmosphere. The indicator strip provides visual conformation that an anaerobic atmosphere has been created in the jar. There is another way by which we can tell that we have generated an anaerobic atmosphere

Why do you think the Clostridium was cultured on a plate, rather than in a broth tube like the other two species?

The clostridium was cultured on a plate, rather than in a broth tube because it it an obligate anaerobic organism, so it would grow better in an incubated environment under anaerobic conditions (pre-reduced oxygenic) anaerobic jar

Indicator strip in gas pack chamber contains methylene blue

-strip blue on exposure to oxygen and white in anaerobic atmosphere -provides visual confirmation that an anaerobic atmosphere has been created

Materials

1 tube of E. coli broth 1 tube of Pseudomonas aeruginosa broth 1 plate of Clostridium sporogenes, to be passed around and shared 3 prepared agar deep tubes of TSA agar Inoculating needles

Gas pack equation

2H2+O2------2 H2O White packet inside chamber contains chemicals that when activated start a chemical reaction. Releases free hydrogen into the atmosphere of the chamber, which binds with the oxygen in the air. The hydrogen and oxygen reaction is catalyzed by pellets of palladium in a cage underneath the lid. This reaction continues until all the oxygen from the air is bound with the hydrogen to form water molecules. This reaction makes the oxygen unavailable and therefore not toxic to anaerobes.

Pseudomonas aeruginosa aerotolerance in agar deep tube

Aerobic : surface

Tolerance for oxygen in bacteria in free flowing water

Bacteria can be facultative anaerobes because they can use free oxygen available in water and can use dissolved oxygen/manually added or can live without it.

Aerobes

Bacteria require oxygen to live, if oxygen not present they will die.

Tolerance for oxygen in bacteria in the human digestive tract

Bacteria would also be obligate anaerobic in the digestive tract because they do not need oxygen to survive and tissues in this area are poorly aerated. Tissues suppress air flow.

Tolerance for oxygen in bacteria in the mouth and around the teeth

Bacteria would be obligate anaerobic in mouth because they do not need oxygen to survive,and tissues in this area are poorly aerated. Tissues surprise air flow.

Obligate anaerobes

Cannot grow in the presence of oxygen -grow and reproduce with no oxygen

Agar deep tubes

Examine aerotolerance of bacteria -agar first boiled to drive out all the oxygen -agar poured into test tubes and tubes are capped -some oxygen diffuses back in

E. coli aerotolerance in agar deep tube

Facultative: surface and throughout

Facultative anaerobes ex....

Grow either in presence or absence of oxygen -Staphylococcus aureus

Inoculation of an agar deep stab

In this experiment an inoculating needle is used to perform the stab. The needle is held like a pencil. Check to be sure that the needle will reach to the bottom of the tube. Incinerate the needle, dip the needle into the broth or scoop up a bit of colony from the Clostridium plate. remove the cap from the agar deep tube. Hold the needle straight up and down over the tube, and stab all the way down to the bottom of the tube in one continuous motion. Pull it out the same way, making sure the needle does not wiggle. Incubate the three tubes at 37 degrees celcius for 48 hours

Examine the tubes

Look for a fuzzy line that indicates growth of bacteria. Be sure to differentiate the stab line from actual bacterial growth. It helps to hold the tube up towards the ceiling light. Record the results.

Where in tube will the most oxygen be found? The least oxygen?

Most-top of tube Least-bottom of tube

Why might it be advantageous for an organism to be an anaerobe?

The organism can specialize in particular parts of the environment where aerobic organisms cannot live, and thus have no competition for resources.

How might the results of this exercise be different if the agar deep tubes were used promptly after sterilization, cooling, and solidification of the agar?

There would be less oxygen available throughout the agar due to being used immediately after these sterilizing, cooling, and solidifying processes. There is no time for oxygen to diffuse back in. -E. coli: facultative, everywhere except surface -P, aeruginosa: Aerobic, did not grow -C. sporogenes: Obligate, everywhere ex eat surface

How might the results of this exercise be different if the agar deep tubes were stored for several months before using them?

There would be plenty of oxygen throughout the agar due to sitting for several months, allowing time for oxygen to diffuse in. -E. coli: facultative, everywhere including surface -P, aeruginosa: aerobic, everywhere and surface -C. sporogenes: obligate, nowhere