Thioglycollate Broth- Composition, Principle, Preparation, Results, Uses

Thioglycollate broth is a multipurpose, enriched, differential medium used primarily to determine the oxygen requirements of microorganisms. It acts as an enrichment broth which is most frequently used in diagnostic bacteriology. This broth supports the growth of anaerobes, aerobes, microaerophilic, and fastidious microorganisms.

Thioglycollate broth is a complex medium that contains various ingredients to support the growth of different types of bacteria based on their oxygen requirements. The main components of thioglycollate broth are:

• Pancreatic digest of casein or tryptone: This provides carbon and nitrogen sources, as well as amino acids, peptides, and vitamins for bacterial metabolism.
• Yeast extract: This enhances the growth of bacteria by supplying additional nutrients, such as B-complex vitamins and trace elements.
• Dextrose or glucose: This serves as an energy source for bacteria that can ferment carbohydrates.
• Sodium chloride: This maintains the osmotic balance of the medium and prevents the lysis of bacterial cells.
• Sodium thioglycollate: This is a reducing agent that lowers the oxidation-reduction potential of the medium and consumes molecular oxygen from the environment. This creates anaerobic conditions in the lower part of the tube and allows the growth of obligate anaerobes.
• L-cystine: This is another reducing agent that helps to maintain a low oxygen tension in the medium. It also acts as a sulfur source for some bacteria.
• Resazurin: This is a redox indicator that changes color from colorless to pink in the presence of oxygen. It helps to visualize the oxygen gradient in the tube and differentiate between aerobic and anaerobic bacteria.
• Agar: This is added in a low concentration (0.75%) to increase the viscosity of the medium and prevent convection currents that could disturb the oxygen gradient.

The final pH of thioglycollate broth is 7.1 ± 0.2 at 25°C, which is suitable for most bacteria. The medium is sterilized by autoclaving at 121°C for 15 minutes and should be used fresh or boiled and cooled before use.

Thioglycollate broth is a medium that can support the growth and differentiation of various types of microorganisms based on their oxygen requirements. It contains nutrients, vitamins, and sodium thioglycollate, which consumes oxygen and creates a gradient of oxygen concentrations in the broth. It is used for multiple purposes, such as diagnostic bacteriology, sterility testing, and disinfectant evaluation. It may or may not have an oxidation indicator, such as resazurin, to show the presence of oxygen. It does not need special equipment or conditions to cultivate anaerobes.

The principle of thioglycollate broth is based on the ability of sodium thioglycollate to act as a reducing agent and neutralize the toxic effects of oxygen and other oxidizing agents on anaerobic bacteria. Sodium thioglycollate reduces molecular oxygen to water and maintains a low oxygen tension in the medium. This, combined with the diffusion of oxygen from the top of the broth, produces a range of oxygen concentrations in the medium along its depth. The oxygen concentration at a given level is indicated by a redox-sensitive dye such as resazurin that turns pink in the presence of oxygen.

Different bacteria have different oxygen requirements for their growth and metabolism. Some bacteria are obligate aerobes, which need oxygen because they cannot ferment or respire anaerobically. Some bacteria are obligate anaerobes, which are poisoned by oxygen and can only grow in its absence. Some bacteria are facultative anaerobes, which can grow with or without oxygen because they can metabolize energy aerobically or anaerobically. Some bacteria are microaerophiles, which need oxygen but are poisoned by high concentrations of it. Some bacteria are aerotolerant, which do not require oxygen but are not harmed by it either.

Thioglycollate broth allows the differentiation of these bacteria based on their oxygen requirements. Obligate aerobes tend to grow toward the surface of the broth, where the oxygen concentration is highest. Obligate anaerobes tend to grow at the bottom of the broth, where the oxygen concentration is lowest. Facultative anaerobes tend to grow mostly at the top but also throughout the broth, because they can use both aerobic and anaerobic pathways for energy production. Microaerophiles tend to grow in the upper part of the broth but not at the very top, because they need some oxygen but not too much. Aerotolerant organisms tend to grow evenly spread throughout the broth, because they do not depend on oxygen for their growth.

To prepare thioglycollate broth, you will need the following ingredients per liter of distilled water or deionized water:

• Pancreatic digest of casein: 15 g
• Dextrose: 5.5 g
• Yeast extract: 5 g
• Sodium chloride: 2.5 g
• Sodium thioglycollate: 0.5 g
• L-cystine: 0.5 g
• Resazurin: 1 mg
• Agar: 0.75 g

The steps for preparing thioglycollate broth are as follows:

1. Suspend the ingredients in water and heat to boiling to dissolve the medium completely.
2. Distribute the medium into tubes or bottles and sterilize by autoclaving at 121°C for 15 minutes.
3. Cool the medium to 25°C and store in a cool dark place preferably below 25°C.
4. Note: Prepare the medium freshly or boil and cool it just before use.

To use thioglycollate broth for bacterial cultivation and differentiation, you will need to follow these steps:

1. Inoculate the medium with a small amount of the specimen or culture following aseptic technique.
2. Incubate the inoculated tubes or bottles at 35-37°C as soon as possible.
3. Observe the growth patterns after 24 hours and up to 7 days if necessary.

The growth of bacteria in thioglycollate broth can be observed by the presence or absence of turbidity (cloudiness) and the location of growth along the tube. The location of growth reflects the oxygen requirements of the bacteria, as different regions of the tube have different oxygen concentrations. The oxygen concentration is highest at the top of the tube where it is exposed to air, and lowest at the bottom of the tube where it is reduced by sodium thioglycollate.

Based on the growth pattern in thioglycollate broth, bacteria can be classified into five categories:

• Obligate aerobes: These bacteria require oxygen for their metabolism and cannot grow without it. They will only grow at the top of the tube where the oxygen concentration is highest. Examples of obligate aerobes are Pseudomonas aeruginosa and Mycobacterium tuberculosis.
• Obligate anaerobes: These bacteria are killed or inhibited by oxygen and can only grow in its absence. They will only grow at the bottom of the tube where the oxygen concentration is lowest. Examples of obligate anaerobes are Clostridium botulinum and Bacteroides fragilis.
• Facultative anaerobes: These bacteria can grow with or without oxygen, but they prefer oxygen as it allows them to produce more energy (ATP) through aerobic respiration. They will grow throughout the tube, but more densely at the top where the oxygen concentration is higher. Examples of facultative anaerobes are Escherichia coli and Staphylococcus aureus.
• Microaerophiles: These bacteria need oxygen for their metabolism, but they are harmed by high concentrations of oxygen. They will grow in a narrow band near the top of the tube, but not at the very top where the oxygen concentration is too high. Examples of microaerophiles are Helicobacter pylori and Campylobacter jejuni.
• Aerotolerant anaerobes: These bacteria do not use oxygen for their metabolism, but they are not affected by it either. They can grow equally well in any region of the tube, as they rely on fermentation or anaerobic respiration for energy production. Examples of aerotolerant anaerobes are Enterococcus faecalis and Lactobacillus acidophilus.

Thioglycollate broth has various applications in microbiology and medicine. Some of the uses are:

• Isolation of anaerobic bacteria from blood and other specimens. Thioglycollate broth is recommended for the cultivation of aerobic, microaerophilic, and anaerobic microorganisms. It allows the detection of obligate anaerobes, such as Clostridium species, that are often involved in anaerobic infections. A sufficient volume of broth must be used to prevent the blood from clotting and to dilute the blood’s natural bactericidal substances.
• Sterility testing of biological products and materials. Thioglycollate broth is intended for sterility testing with certain biological products that are turbid or otherwise do not lend themselves readily to culturing in Thioglycollate Medium because of its viscosity. It is also recommended for sterility testing of antibiotics, biologicals, and foods and for determining the phenol coefficient and sporicidal effect of disinfectants. Thioglycollate broth can neutralize the toxic effects of mercurial preservatives and peroxides formed in the medium, thereby promoting anaerobiosis, and making the medium suitable to test materials containing heavy metals.
• Differentiation of bacteria based on oxygen requirements. Thioglycollate broth is used to find out the growth characteristics of various bacteria based on their oxygen requirements. It allows the differentiation of obligate aerobes, obligate anaerobes, facultative anaerobes, microaerophiles, and aerotolerant organisms. For example, obligate aerobes grow at the top of the tube where the oxygen concentration is highest, while obligate anaerobes grow at the bottom of the tube where the oxygen concentration is lowest.
• Recruitment of macrophages to the peritoneal cavity. Thioglycollate broth is also used to recruit macrophages to the peritoneal cavity of mice when injected intraperitoneally. It recruits numerous macrophages, but does not activate them. This can be useful for studying the phagocytic and microbicidal activities of peritoneal macrophages.

Thioglycollate broth is a versatile and widely used medium for the cultivation and differentiation of bacteria based on their oxygen requirements. However, it also has some limitations that should be considered when using it for microbiological purposes. Some of the limitations are:

• It is essential that the medium should be freshly prepared or boiled and cooled within four hours of use. This is because storage at lower temperatures increases oxygen absorption, which can affect the anaerobic conditions in the medium. Moreover, thioglycollate media should not be re-heated more than once because toxic oxygen radicals are formed on reheating, which can inhibit the growth of anaerobes.
• It may not support the growth of some fastidious anaerobes that require specific nutrients or growth factors. For example, some species of Clostridium may not grow well in thioglycollate broth because they need complex carbohydrates or amino acids that are not present in the medium. Therefore, other media such as cooked meat medium or brain heart infusion broth may be needed to isolate these organisms.
• It may not detect small numbers of aerobes or anaerobes in mixed cultures. This is because the oxygen gradient in the medium may not be sufficient to separate the different types of bacteria based on their oxygen preferences. Also, some aerobes or anaerobes may grow faster than others and overgrow the medium, masking the presence of less abundant organisms. Therefore, other methods such as streaking on solid media or using selective or differential media may be needed to identify the different bacteria in a sample.
• It may give false-positive results for sterility testing of some products that contain reducing substances. This is because reducing substances such as ascorbic acid, cysteine, glutathione, or sodium metabisulfite can react with the resazurin indicator in the medium and turn it pink, indicating oxidation. Therefore, other methods such as membrane filtration or direct inoculation on solid media may be needed to test the sterility of these products.

These are some of the limitations of thioglycollate broth that should be taken into account when using it for microbiological purposes. However, despite these limitations, thioglycollate broth remains a valuable and convenient medium for the cultivation and differentiation of bacteria based on their oxygen requirements.

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