Thioglycollate Broth Composition, Principle, Preparation, Results, Uses

Thioglycolate broth, an enrichment broth that is multipurpose and can be used to determine the oxygen needs of microorganisms, is called a differential medium. It is used most often in diagnostic bacteriology as an enrichment broth. This broth is supportive of the growth and development of microorganisms fastidious, anaerophilic, microaerophilic and aerobes.

Composition of Thioglycollate Broth

Ingredients	Gms/liter
L-cystine	0.50
Sodium chloride	2.50
Glucose	5.50
Yeast extract	5.00
Pancreatic digest of casein	15.0
Sodium thioglycollate	0.5

Final pH (at 25°C): 7.1 ± 0.2

Principle of Thioglycollate Broth

Pancreatic digest of casein and yeast extract provide the growth factors for bacterial multiplication. Clostridium, and other strict anaerobes can grow in this medium under aerobic conditions thanks to sodium thioglycollate and L-cystine. The medium’s toxic effects are neutralized by sodium thioglycollate and mercurial peroxides. This promotes anaerobiosis and makes it suitable for testing heavy metal-containing materials. You can identify aerobic and anaerobic bacteria by growing them in test tubes made of thioglycolate soup.

Obligate aerobes require oxygen because they can’t ferment or breathe anaerobically. They cluster at the top of the tube, where oxygen concentration is the highest. Obligate anaerobes can be poisoned by oxygen so they collect at the bottom of the tube, where oxygen concentration is the lowest. Facultative anaerobes are able to grow with or without oxygen, as they can metabolize energy either anaerobically or aerobically. Because aerobic respiration produces more ATP than fermentation or anaerobic, they tend to cluster at the top.

Because they can’t ferment or breathe anaerobically, microaerophilics need oxygen. They can be poisoned by high levels of oxygen. They tend to gather in the top of the tube but not at the top. Aerotolerant organisms don’t require oxygen because they metabolize energy anaerobically. They are not poisoned like obligate anaerobes. They can be found uniformly distributed throughout the tube.

Preparation and Method of Use of Thioglycollate Broth

1. Take 29g and mix it with 1 liter of distilled.
2. Bring to a boil, and then completely dissolve the medium.
3. Divide into tubes or bottles, and sterilize with an autoclave at 121°C for 15 minutes.
4. Take care to not boil or cool the medium before using.
5. Following aseptic technique, inoculate the medium.
6. After inoculation, the broth should immediately be incubated at 35 to 35degC.

Result Interpretation on Thioglycollate Broth

Organisms	Growth
Candida albicans	Flocculent growth
Clostridium sporogenes	Turbid growth and or colonies
Peptostreptococcus anaerobius	Turbid growth and or colonies
Clostridium perfringens	Turbid growth and or colonies
Bacteroides fragilis	Turbid growth and or colonies
Bacteroides vulgatus	Turbid growth and or colonies
Staphylococcus aureus subsp. Aureus	Turbid growth and or colonies
Pseudomonas aeruginosa	Turbid growth and or colonies
Micrococcus luteus	Turbid growth and or colonies
Streptococcus pneumoniae	Turbid growth and or colonies
Escherichia coli	Turbid growth and or colonies
Salmonella Typhimurium	Turbid growth and or colonies
Salmonella Abony	Turbid growth and or colonies

Uses of Thioglycollate Broth

• For the cultivation of microorganisms that are anaerobic, microaerophilic and aerobic, Thioglycollate broth should be used.
• It allows for the differentiation of obligate and facultative aerobes, anaerobes and anaerobes.
• This medium is used to test for sterility with biological products that are turbide or not able to be culturing in Thioglycollate Medium due its viscosity.
• It is used to test for sterility of biologicals, antibiotics, and food, as well as for determining the phenol and sporicidal effects of disinfectants.
• Thioglycollate medium should be used to remove strict anaerobes from blood when anaerobic infections are suspected.
• Thioglycollate broth can be used to determine the growth characteristics of different bacteria according to their oxygen needs.

Limitations of Thioglycollate Broth

• It is important that the medium be prepared immediately or boiled within four hours.
• Lower temperatures increase oxygen absorption.
• Because toxic oxygen radicles can form on reheating, it is important to not heat thioglycollate media more than once.

Primary purpose of ingredients used

• Casein and cystine: They provide carbon and nitrogenous compounds.
• Dextrose: It can be added to another energy source
• As growth stimulants, yeast extract and papaic digest soybean meal can be added.
• Sodium chloride is a salt that maintains an osmotic equilibrium.
• Sodium Thioglycollate – Sodium Thioglycollate (or sodium thioglycollate) is a reducing agent that maintains a low oxygen tension. It removes molecular oxygen from the surrounding environment, i.e. it creates anaerobic environments when it reduces molecular Oxygen to water. This condition does not allow for the formation of peroxides which can be fatal to anaerobic organisms.
• Resazurin, an indicator of oxidation reduction, turns pink when there has been increased oxidation. It is inert when it is reduced.
• Agar: Thioglycollate medium contains a small amount agar. This aids in the inoculation and growth of small anaerobes and inocula by preventing oxygen from entering the medium. It also slows down the dispersion and removal of CO2 from the microenvironment around the inoculum.

You can add certain additives to the Thioglycollate medium if you wish:

• Hemin is incorporated in order to supply X factor for stimulating the growth of many fastidious organisms.
• Vitamin K is required for certain gram-positive Bacteroides and spore-formers.
• Calcium carbonate chips are added to buffer the medium and prevent toxic acid buildup.

Reading results in Thioglycollate broth

1. Isolation of bacteria  from Blood Culture

Everyday, examine the area for signs of bacterial growth (up to 14 days). These include turbidity above red cells, colonies growing on top of red cells (cotton balls), hemolysis and gas bubbles. Thioglycollate broth can be used to grow anaerobes and other bacteria with different oxygen needs. Subculture the broth if you see signs of bacterial growth. Then, look for bacteria in a toluidine-blue stained smear.

2. For finding oxygen requirements of various bacteria

After 48 hours incubation, the results are read. A pinkish band forms when oxygen diffuses near top of broth. (Remember that Oxidation-Reduction indicator Resazurin can be pink when it is oxidized, and pink when it is reduced). If there is no pink in the rest, it indicates that the environment is not suitable for strict anaerobes. The oxygen requirements of each organism will determine the growth of the organisms in the different parts of the test tubes.

• Aerobes that are strictly controlled will only grow in the pink range.
• The band with the lowest oxygen concentration will see microaerophiles grow.
• Both aerotolerant and facultative anaerobes can grow in the tube. Facultative anaerobes will grow in all media, but they are heavier at the bottom. Similarly, facultative aerobes will grow at the top and throughout the tube. The facultative grows most densely in areas where oxygen is available.
• Aerotolerant microbes grow equally well in all parts of the tube. This test should be done within 48 hours. However, some microbes that are slower growing may require more time.