Fluid Thioglycollate Medium

Thioglycollate broth (Fluid Thioglycollate Medium) is a medium designed to test the aerotolerance of bacteria.  Along with nutrients to support bacterial growth, it contains sodium thioglycollate, thioglycolic acid, L-cystine, methylene blue, and 0.05% agar.  

Sodium thioglycollate, thioglycolic acid, and L-cystine reduce the oxygen to water. Methylene blue is an indicator that is colorless in an anaerobic environment and greenish-blue in the presence of oxygen. The agar helps slow oxygen diffusion and helps maintain the stratification of organisms growing in different layers of the broth.

Oxygen is driven out of the broth by autoclaving, but as the broths sit at room temperature, oxygen begins to diffuse back into the tube. This is evidenced by the small layer of blue-green at the top of the broth. 

Obligate aerobes will only grow in the blue-green oxygen-rich layer.

Obligate anaerobes will only grow in the lower areas of the tube. 

Microaerophiles will grow in a thin layer below the richly-oxygenated layer.

Facultative or aerotolerant anaerobes can grow throughout the medium but will primarily grow in the middle of the tube, between the oxygen-rich and oxygen-free zones.

The Micrococcus luteus on the left only grows in the oxygenated zone; it is an obligate aerobe. The Clostridium sporogenes in the middle only grows in the oxygen-free area; it is an obligate anaerobe. The Escherichia coli on the right grows throughout the tube; it is a facultative anaerobe.