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

What is Thioglycollate Broth?

• Thioglycollate broth is a widely used enrichment broth in diagnostic bacteriology. It serves as a versatile medium that supports the growth of various types of microorganisms, including anaerobes, aerobes, microaerophilic, and fastidious bacteria. The composition of thioglycollate broth includes several nutritive factors such as casein, yeast, beef extracts, and vitamins, which promote the growth of medically significant bacteria.
• To enhance its functionality, thioglycollate broth contains additional nutrient supplements. These include an oxidation-reduction indicator called resazurin, dextrose, vitamin K1, and hemin. The presence of these supplements allows for differential analysis and helps in determining the oxygen requirements of microorganisms.
• One key feature of thioglycollate broth is the addition of 0.075% agar, which serves to prevent convection currents from carrying atmospheric oxygen throughout the broth. This agar supplement, combined with the reducing agent thioglycolic acid, creates an anaerobic environment deeper in the tube. This anaerobic environment facilitates the growth of anaerobic bacteria.
• Thioglycolate broth is widely used as an enrichment medium in diagnostic bacteriology. It is commonly employed to determine the oxygen requirements of microorganisms and to cultivate anaerobes, microaerophiles, and aerobes. It serves as a general-purpose medium and is recommended for sterility testing of biological products.
• One of the advantages of thioglycollate broth is that it does not require an anaerobic container or special sealing for the cultivation of anaerobes. The presence of sodium thioglycolate in the medium helps consume oxygen, creating a suitable environment for the growth of obligate anaerobes.
• In summary, thioglycollate broth is a multipurpose, enriched, and differential medium used in diagnostic bacteriology. It supports the growth of various types of microorganisms and is particularly valuable for cultivating anaerobes. Its composition, including nutritive factors and additional supplements, enhances the growth of medically significant bacteria and facilitates the determination of oxygen requirements.

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

The principle of Thioglycollate Broth is based on the differential oxygen requirements of various types of bacteria. The components of the medium play specific roles in creating an environment suitable for different types of bacteria to grow.

The growth factors necessary for bacterial multiplication are provided by dextrose, pancreatic digest of casein, yeast extract, and L-cystine. These nutrients support the overall growth of bacteria in the medium.

L-cystine and sodium thioglycollate are crucial components that allow strict anaerobes, including Clostridium species, to grow even under aerobic conditions. Sodium thioglycollate acts as a reducing agent, neutralizing the toxic effects of mercurial preservatives and peroxides that may be present in the medium. By doing so, it promotes an anaerobic environment, making the medium suitable for testing materials containing heavy metals.

When bacteria are grown in test tubes of thioglycollate broth, the distribution of bacteria within the tube can indicate their oxygen requirements and metabolic capabilities:

1. Obligate aerobes gather at the top of the tube where the oxygen concentration is highest. They require oxygen for their metabolism and cannot ferment or respire anaerobically.
2. Obligate anaerobes are poisoned by oxygen, so they gather at the bottom of the tube where the oxygen concentration is lowest. They cannot survive in the presence of oxygen and rely on anaerobic respiration or fermentation for their energy needs.
3. Facultative anaerobes can grow with or without oxygen because they have the ability to metabolize energy aerobically or anaerobically. However, aerobic respiration generates more ATP for them, so they mostly gather at the top of the tube.
4. Microaerophilic organisms require oxygen for their metabolism, but they are sensitive to high concentrations of oxygen. As a result, they gather in the upper part of the test tube, just below the surface, but not at the very top.
5. Aerotolerant organisms do not require oxygen as they can metabolize energy anaerobically. Unlike obligate anaerobes, they are not harmed by the presence of oxygen. These organisms can be found evenly spread throughout the test tube, as they are not affected by oxygen concentration.

Preparation of Thioglycollate Broth

The preparation and method of use of Thioglycollate Broth involve the following steps:

1. Weigh and suspend: Measure 29 grams of Thioglycollate Broth powder and suspend it in 1 liter of distilled water. Ensure that the powder is completely dissolved.
2. Boiling and dissolution: Heat the suspension to boiling, allowing the medium to come to a boil. Continue boiling until the medium is completely dissolved.
3. Distribution and sterilization: After dissolution, distribute the medium into tubes or bottles as required. Make sure to use appropriate containers that can withstand autoclaving. Sterilize the tubes or bottles containing the medium by autoclaving at 121°C for 15 minutes. This process ensures the elimination of any contaminating microorganisms.

Note: It is recommended to prepare the medium freshly before use. If not possible, boil and cool the medium just before use to maintain its sterility.

4. Inoculation: Following aseptic techniques, inoculate the Thioglycollate Broth with the desired microorganisms. This step involves introducing a small sample of the microorganism into the medium using a sterile loop, needle, or other appropriate methods.
5. Incubation: Once inoculated, it is crucial to incubate the Thioglycollate Broth at 35-37°C as soon as possible. The incubation temperature supports the growth of a wide range of microorganisms. Place the tubes or bottles in an incubator set to the specified temperature and allow the cultures to grow.

By following these steps, Thioglycollate Broth can be properly prepared and utilized for various applications. It is essential to maintain aseptic conditions during preparation and ensure timely incubation of the inoculated medium to obtain accurate and reliable results.

Method of Use of Thioglycollate Broth

The preparation and method of use of Thioglycollate Broth involve specific steps to ensure proper handling and incubation of the culture bottle. Here is a breakdown of the process:

1. Label the blood culture bottle: Before starting, label the ready-to-use blood culture bottle appropriately for identification purposes.
2. Remove the cap: Take off the aluminum foil cap from the culture bottle.
3. Disinfect the rubber stopper: Disinfect the exposed part of the rubber stopper using an appropriate disinfectant. This step helps maintain the sterility of the medium.
4. Draw the patient’s blood: Using a sterile or disposable needle and syringe, collect the patient’s blood according to the recommended procedure for specimen collection.
5. Transfer the blood sample: Immediately after drawing the blood, transfer the blood sample into the culture bottle. Puncture the rubber stopper with the needle and inject the blood into the broth.
6. Venting (optional): If required, use a sterile venting needle with a filter (e.g., LA038) for venting the culture bottle. This step allows the release of gases while maintaining sterility. Place an alcohol swab over the rubber stopper and insert the venting needle through it. Make sure to insert and withdraw the needle in a straight line. Note that venting is not necessary for anaerobic cultures.
7. Mix the contents: After venting (if applicable), discard the venting needle. Gently invert the bottle 2-3 times to mix the blood sample with the Thioglycollate Broth. This step ensures proper distribution of the blood within the medium.
8. Incubation: Place the culture bottle in an upright position, preferably in a biological safety cabinet or appropriate incubator. For most routine cultures, incubate the bottle at 35-37°C for 24-72 hours. Some specific cultures may require extended incubation for up to seven days. Follow the recommended incubation time for the target organisms or diagnostic purposes.

Growth characteristics of various bacteria in thioglycollate broth

In Thioglycollate Broth, the growth characteristics of various bacteria can provide valuable information about their oxygen requirements. Here are the growth characteristics typically observed:

A. Gram-negative, facultatively anaerobic bacilli: These bacteria have the ability to grow in both the presence and absence of oxygen. In Thioglycollate Broth, they generally produce diffuse and even growth throughout the medium. This means that their growth is distributed throughout the broth, rather than concentrated in specific regions.

B. Gram-positive cocci: Gram-positive cocci, such as certain Staphylococcus and Streptococcus species, often exhibit a characteristic growth pattern resembling discrete “puffballs.” These bacteria form compact colonies or clusters that are visibly separated within the medium.

C. Strictly aerobic bacteria: Strict aerobic bacteria, including species like Pseudomonas, have a requirement for oxygen to grow. In Thioglycollate Broth, they tend to grow toward the surface of the broth, where the oxygen concentration is highest. Their growth is more concentrated at the top portion of the medium.

D. Strictly anaerobic bacteria: Strictly anaerobic bacteria cannot grow in the presence of oxygen and thrive in oxygen-depleted environments. In Thioglycollate Broth, these bacteria exhibit growth at the bottom of the broth, away from the oxygen-rich surface. Their growth is concentrated in the lower portion of the medium.

By observing these distinct growth characteristics, it is possible to gain insights into the oxygen requirements and metabolic capabilities of different bacteria. This information can aid in the identification and classification of bacterial species and contribute to diagnostic and research purposes.

Result Interpretation on Thioglycollate Broth

The interpretation of results in Thioglycollate Broth can provide valuable information about the oxygen requirements and growth characteristics of different bacteria. Here is how to interpret the results:

1. No turbidity: If there is no turbidity (cloudiness) observed in the broth, it indicates no growth of organisms in the medium.
2. Turbidity: The presence of turbidity indicates the growth of organisms in the Thioglycollate Broth.

To determine the oxygen requirements of bacteria:

A. For isolating bacteria from Blood Culture: Examine the broth daily for up to 14 days. Look for visible signs of bacterial growth such as turbidity above the layer of red cells, colonies growing on the surface of the red cells (resembling cotton balls), hemolysis, gas bubbles, and clots. Subculture the broth when there are visible signs of bacterial growth and examine a toluidine blue stained smear for bacteria.

B. For finding the oxygen requirements of various bacteria: Results are read after 48 hours of incubation of the inoculated test tubes. The following observations can be made:

• Pink band: When oxygen diffuses near the top of the broth, a pink band develops. This indicates the presence of oxygen in that region. The oxidation-reduction indicator, resazurin, turns pink when oxidized and colorless when reduced.
• Absence of pink: The absence of pink in the rest of the tube indicates the absence of oxygen, creating a suitable environment for strict anaerobes.
• Growth distribution: The growth of organisms in different regions of the test tubes depends on their oxygen requirements.
  • Strict aerobes: They will grow only in the pink band where oxygen is present.
  • Microaerophiles: They will grow near the bottom of the pink band, where the oxygen concentration is lower.
  • Facultative anaerobes and facultative aerobes: They will grow throughout the tube. Facultative anaerobes will have more growth at the bottom, while facultative aerobes will have more growth at the top. The density of growth depends on the presence of oxygen.
  • Aerotolerant organisms: They will grow equally well throughout the tube, as they are not affected by the presence of oxygen.

The interpretation of the test should be done around 48 hours of incubation, although slower-growing microbes may require more incubation time to show growth.

By analyzing the growth patterns and oxygen requirements of bacteria in Thioglycollate Broth, valuable information can be obtained for identification, isolation, and understanding the characteristics of different microorganisms.

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

Quality Control

Quality control measures are essential to ensure the reliability and consistency of Thioglycollate Broth. The following information outlines the quality control parameters and cultural responses used for testing the medium:

1. Appearance: The Thioglycollate Broth should be sterile and appear as a clear solution in a glass bottle.
2. Color: The medium should have a light yellow color, indicating a clear solution.
3. Quantity of Medium: The recommended quantity of medium is 70ml in a glass bottle for adult use.
4. Reaction: The pH of the Thioglycollate Broth should fall within the range of 6.90 to 7.30, ensuring the appropriate acidity or alkalinity.
5. Sterility test: The Thioglycollate Broth must pass the sterility test, indicating the absence of any contaminating microorganisms.
6. Cultural response: The cultural characteristics of the medium are observed after incubation at 35-37°C for 24-72 hours. This allows for the evaluation of growth patterns and microbial responses.
7. Limitations: One limitation mentioned is the need to maintain proper anaerobic conditions for optimal recovery of organisms. This indicates that the medium’s performance may be affected if strict anaerobic conditions are not maintained.
8. Organism Inoculum: Specific strains of microorganisms are used for quality control testing. The provided CFU (colony-forming unit) range indicates the expected growth response under anaerobic conditions for each organism.
9. Growth under anaerobic conditions: The listed microorganisms, such as Clostridium sporogenes, Clostridium perfringens, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Salmonella Abony, Bacteroides fragilis, and Bacteroides vulgatus, should exhibit luxuriant growth (abundant growth) when incubated under anaerobic conditions.
10. Growth under aerobic conditions: The cultural response of microorganisms under aerobic conditions is not explicitly mentioned in the provided information.

These quality control measures ensure that Thioglycollate Broth is of satisfactory quality, suitable for microbiological testing, and capable of supporting the growth of specific microorganisms under appropriate conditions.

Uses of Thioglycollate Broth

Thioglycollate Broth has several important uses in microbiology and diagnostic procedures. Here are some key applications:

• Cultivation of microorganisms: Thioglycollate broth is recommended for the cultivation of aerobic, microaerophilic, and anaerobic microorganisms. It provides a versatile medium that supports the growth of a wide range of bacteria with diverse oxygen requirements.
• Differentiation of bacteria based on oxygen requirements: Thioglycollate broth allows for the differentiation of various types of bacteria according to their oxygen requirements. It enables the identification of obligate aerobes, obligate anaerobes, facultative anaerobes, microaerophiles, and aerotolerant organisms.
• Sterility testing: Thioglycollate broth is commonly used for sterility testing of certain biological products that may be turbid or have high viscosity. It provides a suitable medium for the detection of microorganisms that may be present in these products.
• Determination of phenol coefficient and sporicidal effect: Thioglycollate broth is recommended for determining the phenol coefficient, which measures the effectiveness of disinfectants against specific microorganisms. It is also used to assess the sporicidal effect of disinfectants, evaluating their ability to kill spore-forming bacteria.
• Isolation of strict anaerobes: In cases where anaerobic infections are suspected, Thioglycollate Broth is commonly used to isolate strict anaerobic bacteria from blood samples. It provides an environment conducive to the growth of these bacteria, aiding in their isolation and identification.
• Assessment of bacterial growth characteristics: Thioglycollate broth is widely used to study the growth characteristics of different bacteria based on their oxygen requirements. By observing the growth patterns and distribution of bacteria within the medium, valuable information about their metabolic capabilities can be obtained.

Overall, Thioglycollate Broth serves as a versatile and valuable medium in various microbiological applications, ranging from cultivation and differentiation of bacteria to sterility testing and assessing the efficacy of disinfectants.

Limitations of Thioglycollate Broth

Thioglycollate Broth, despite its versatility and wide range of applications, has certain limitations that should be considered. Here are some important limitations:

• Freshness of the medium: Thioglycollate Broth should be freshly prepared or boiled and cooled within four hours of use. The medium can deteriorate over time, leading to changes in its composition and potentially affecting the growth and viability of microorganisms.
• Storage temperature: Storing Thioglycollate Broth at lower temperatures can lead to increased absorption of oxygen from the environment. This can potentially affect the oxygen concentration within the medium and impact the growth characteristics of certain bacteria.
• Limited re-heating: It is not recommended to reheat Thioglycollate Broth more than once. Upon reheating, toxic oxygen radicals can be formed, which may affect the integrity of the medium and the growth of microorganisms.

These limitations highlight the importance of using freshly prepared or properly stored Thioglycollate Broth to ensure accurate and reliable results. Adhering to the recommended guidelines for preparation, storage, and use of the medium is crucial to minimize any potential drawbacks and obtain the best outcomes in microbiological procedures.

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.

Keynotes on Thioglycollate Broth

Keynotes on Thioglycollate Broth highlight important aspects of the medium:

1. Appearance: The prepared Thioglycollate Broth should have a light yellow color and appear as a clear solution.
2. Versatile growth support: Thioglycollate Broth is a medium that supports the growth of a wide variety of microorganisms. It can accommodate the growth of anaerobes, aerobes, microaerophilic organisms, as well as fastidious organisms.
3. Oxygen requirement assessment: Thioglycollate Broth is commonly used to determine the growth characteristics of bacteria based on their oxygen requirements. The medium helps in differentiating between obligate aerobes, obligate anaerobes, facultative anaerobes, microaerophiles, and aerotolerant organisms.
4. Brewer’s formulation: Fluid Thioglycollate Medium, developed by Brewer, is a modified version of Thioglycollate Broth. It includes a reducing agent and a small amount of agar, which promotes the rapid cultivation of both aerobic and anaerobic bacteria, including microaerophiles.

These keynotes emphasize the versatility of Thioglycollate Broth in supporting the growth of diverse microorganisms and its utility in assessing bacterial oxygen requirements. The mention of Brewer’s formulation highlights an alternative formulation that optimizes the medium for the cultivation of both aerobic and anaerobic bacteria.

FAQ

References

1. https://www.himedialabs.com/media/TD/LQ007A.pdf
2. https://microbiologie-clinique.com/thioglycolate-broth.html
3. https://microbeonline.com/thioglycollate-broth/#Reading_results_in_Thioglycollate_broth
4. https://www.sigmaaldrich.com/IN/en/product/sial/70157
5. https://www.austincc.edu/microbugz/fluid_thioglycollate_medium.php
6. https://universe84a.com/thioglycollate-broth-introduction/
7. https://www.fishersci.com/shop/products/thioglycollate-10-pk-1/NC0174636