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 nutritive factors, including casein, yeast, beef extracts, and vitamins to enhance the growth of most medically important bacteria. Other nutrient supplement includes; oxidation-reduction indicator (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 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
- When anaerobic infection is suspected, thioglycollate medium is recommended to isolate strict anaerobes from blood. Sufficient volume of broth must be used to prevent the blood from clotting and to dilute out the blood’s natural bactericidal substances. The blood should be diluted atleast 1 in 10 with broth (SPS, anticoagulant, is inhibitory to anaerobic strpetocci, so is not added in thioglycollate broth)
- Thioglycollate broth is used to find out growth characteristics of various bacteria based on their oxygen requirements.
Composition of Thioglycollate broth
Ingredients per liter of deionized water:*
- Pancreatic Digest of Casein: 15 gm
- Dextrose: 5.5 gm
- Yeast Extract: 5 gm
- Sodium Chloride: 2.5 gm
- Sodium Thioglycollate: 0.5 gm
- L-Cystine: 0.5 gm
- Resazurin: 1 mg
- Agar 0.75 gm
Final pH 7.1 +/- 0.3 at 25°C.
Principles (primary purpose of ingredients used)
- Cystine and casein: They supply carbon and nitrogenous compounds,
- Dextrose: It is added as another energy source
- Yeast extract or papaic digest of soybean meal are added as growth enhancers.
- Sodium chloride: It maintains osmotic equilibrium.
- 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.
- Resazurin is an oxidation-reduction indicator that turns pink when increased oxidation has occurred, it is colorless when reduced.
- 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:
- Hemin is incorporated to supply X-factor for the stimulated growth of many fastidious organisms,
- Vitamin K because it is a growth requirement for some gram-positive spore-formers and Bacteroides species.
- The calcium carbonate chip is added to act as a buffer for the medium and prevents a buildup of toxic acid.
Preparation and Inoculation of Thioglycollate broth
Thioglycollate broth is best prepared from ready to use dehydrated powder, available from most suppliers of culture media.
- The medium is usually used at a concentration of 29.5g in every litre distilled water (concentration may vary depending on manufacturer).
- Prepare as instructed by the manufacturer. Dispense the well mixed medium in 50 ml amounts in bottles fitted with screw-caps that have a central hole and rubber liner. Sterilize by autoclaving (with caps loosened) at 121°C for 15 minutes.
- When cool, tighten the bottle caps. Cover each bottle top with a foil cap or other protective covering (previously soaked in 70% v/v ethanol).
- Label the bottles. Date the medium and give it a batch number. Store in a cool dark place.Shelf-life: Up to 2 years providing there is no change in the volume or apperance of the medium to suggest oxidation or contamination. Important: if at the time of use, more than a narrow band at the surface of the medium appears pink, this indicates oxidation and the broth should not be used. It should be reheated by placing the bottle in a container of boiling water for 15 minutes (with bottle cap loosened) to drive off the dissolved oxygen.
- Inoculate the medium following aseptic technique.
- Written inoculation instructions should be issued with each bottle of medium.
- Once inoculated, the broth should be incubated at 35-35°C as soon as possible.
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 are visible signs of bacterial growth, subculture the broth and examine a toluidine blue stained smear for 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:
- Strict aerobes will grow only in the pink band,
- Microaerophiles will grow near the bottom of the band where the concentration of oxygen is lower.
- 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 facultative grow most densely where oxygen is present.
- 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:
- Tille, P. M., & Forbes, B. A. (2014). Bailey & Scott’s diagnostic microbiology (Thirteenth edition.). St. Louis, Missouri: Elsevier.
- Cheesbrough M. (2007). District laboratory Practice in Tropical Countries, Part 2. Cambridge University Press.