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Thioglycollate broth: Principle, Composition, Preparation and Uses

Thioglycollate broth is the enrichment broth most frequently used in diagnostic bacteriology. This broth supports growth of anaerobes, aerobes, microaerophilic, and fastidious microorganisms.  It contains many nutrient factors, including casein, yeast, and beef extracts, and vitamins to enhance the growth of most medically important bacteria. Other nutrient supplements includes; oxidation-reduction indictor (resazurin),  dextrose, vitamin K1, and hemin.

This medium also contains 0.075% agar to prevent convection currents from carrying atmospheric oxygen throughout the broth.  This agar supplement and the presence of reducing agent thioglycolic acid create an anaerobic environment deeper in the tube which allows anaerobic bacteria to grow.

Growth Characteristics of various bacteria in thiolgycollate broth

Growth Characteristics of various bacteria in tholgycollate broth

A. Gram-negative, facultatively anaerobic bacilli (i.e., those that can grow in the presence or absence of oxygen) generally produce diffuse, even growth throughout the broth.

B. Gram positive cocci frequently grow as discrete “puffballs”.

C. Strict aerobic bacteria (i.e., require oxygen for growth), such as Pseudomonas spp., tend to grow toward the surface of the broth,

D. Strictly anaerobic bacteria (i.e., those that cannot grow in the presence of oxygen) grow at the bottom of the broth.

 

Uses of Thioglycollate broth

Composition of Thioglycollate broth

Ingredients per liter of deionized water:*

  1. Pancreatic Digest of Casein: 15 gm
  2. Dextrose: 5.5 gm
  3. Yeast Extract: 5 gm
  4. Sodium Chloride: 2.5 gm
  5. Sodium Thioglycollate: 0.5 gm
  6. L-Cystine: 0.5 gm
  7. Resazurin: 1 mg
  8. Agar 75gm

Final pH 7.1 +/- 0.3 at 25°C.

Principles (primary purpose of ingredients used)

  1. Cystine and casein: They supply carbon and nitrogenous compounds,
  2. Dextrose: It is added as another energy source
  3. Yeast extract or papaic digest of soybean meal are added as growth enhancers.
  4. Sodium chloride: It maintains osmotic equilibrium.
  5. Sodium Thioglycollate: Sodium Thioglycollate is a reducing agent which maintains a low oxygen tension by removing molecular oxygen from the environment i.e., it creates anaerobic conditions when it reduces molecular oxygen to water. Peroxides, which may be lethal to many anaerobic organisms, are not formed under this condition.
  6. Resazurin is an oxidation-reduction indicator that turns pink when increased oxidation has occurred, it is colorless when reduced.
  7. Agar: The addition of a small amount of agar in Thioglycollate Medium aids in the initiation and growth of small inocula and anaerobes by impeding the diffusion of oxygen into the medium. It also retards the dispersion of CO2 and the reducing substance from the microenvironment surrounding the inoculum.

Certain additives can be incorporated into the Thioglycollate medium as desired:

  1. Hemin is incorporated to supply X-factor for the stimulated growth of many fastidious organisms,
  2. Vitamin K because it is a growth requirement for some gram-positive spore-formers and Bacteroides species.
  3. The calcium carbonate chip is added to act as a buffer for the medium and prevents a buildup of toxic acid.

Prepation and Inoculation of Thioglycollate broth

Thioglycollate broth is best prepared from ready to use dehydrated powder, available from most suppliers of culture media.

Reading results in Thioglycollate broth

A: For isolating bacteria  from Blood Culture
Examine daily (up to 14 days) for visible signs of bacterial growth such as turbidity above the red cells layer, colonies growing on the surface of the red cells (cotton balls), hemolysis, gas bubbles, and clots. Remember, not only anaerobes but also other bacteria having diverse oxygen requirements grows in thioglycollate broth. When there is visible signs of bacterial growth, subculture the broth and examine a toulidine blue stained smear for bacteria.

B: For finding oxygen requirements of various bacteria

Results are read after 48 hours incubation of the inoculated test tubes. When oxygen diffuses near the top of the broth a pink band develops (Remember: Oxidation-Reduction indicator Resazurin is pink when oxidized and colorless when reduced). The absence of pink in the rest of the tube indicates the absence of oxygen and a suitable environment for strict anaerobes.  Growth of the organisms in different regions of the test tubes will depend on the oxygen requirement characteristics of those organisms:

  1. Strict aerobes will grow only in the pink band,
  2. Microaerophiles will grow near the bottom of the band where the concentration of oxygen is lower.
  3. Both facultative anaerobes and aerotolerant anaerobes will grow throughout the tube. Facultative anaerobes, grow throughout the media but heavier at the bottom and facultative aerobes  grow thoughout but more at the top. The facultatives grow most densely where oxygen is present.
  4. The aerotolerants grow equally well thoroughout the tube.Usually this test should be read at about 48 hrs although some slower growing microbes may take more incubation time.

References and further readings:

  1. Tille, P. M., & Forbes, B. A. (2014). Bailey & Scott’s diagnostic microbiology (Thirteenth edition.). St. Louis, Missouri: Elsevier.
  2. Cheesbrough M. (2007). District laboratory Practice in Tropical Countries, Part 2. Cambridge University Press.