Anaerobic Jar

This experiment illustrates the oxygen requirements of an organism.

For this exercise, we use the GasPak anaerobic system by Becton Dickinson. This system consists of a polycarbonate jar, a lid with a gasket to prevent air flow, a strip infused with methylene blue, and a pouch containing sodium borohydride, sodium bicarbonate, citric acid, and a palladium catalyst. Please note that your campus may use a different indicator or a different type of CO2 generator pouch, but the principle of the experiment and the results are the same.

When water is added to the pouch (if necessary), the sodium borohydride, sodium bicarbonate, and citric acid react to form hydrogen and carbon dioxide.  The palladium catalyzes a reaction between the hydrogen and the oxygen within the jar; this reaction creates water, which forms as condensation on the inside of the jar. Methylene blue is blue in the presence of oxygen but is colorless in an anaerobic environment. When the oxygen is converted to water and condensation forms on the side of the jar, the indicator strip will turn from blue to white.

Materials:

2 - Tryptic Soy Agar (TSA) platesper group

1 - Culture each of <i>Escherichia coli</i>, <i>Micrococcus luteus</i>, and <i>Clostridium 

     sporogenes </i> (selection may vary among campuses)

1 - Anaerobic jar, pouch, and indicator per class

Protocol:

• Spot streak each TSA plate with your 3 organisms.

• Tape each plate closed; do not tape the plates together.

• Place one plate in the 37°C incubator.

• Place one plate in the anaerobic jar.  Your instructor will start the reaction and seal the jar.  The entire jar will then be incubated at 37°C.  

Anaerobes should grow in the anaerobic jar while obligate aerobes should not. Obligate aerobes and facultative anaerobes will grow outside the jar while obligate anaerobes will not. On which plate(s) would microaerophiles grow?