2. Composition of HiMedia’s Fluid Thioglycollate Medium (MAP009):

The formulation of Fluid Thioglycollate Medium (FTM) is meticulously designed to support the growth of a broad spectrum of microorganisms, encompassing aerobic, anaerobic, and microaerophilic species. Key components and their functional roles include:

• Sodium thioglycollate: Serves as a potent reducing agent, lowering the oxidation-reduction potential of the medium and establishing conditions favorable for anaerobic microbial growth.
• L-cystine: Provides sulfur-containing amino acids essential for the proliferation of fastidious anaerobic organisms.
• Glucose: Functions as a readily metabolizable energy source, supporting microbial growth and metabolic activity throughout the incubation period.
• Yeast extract and tryptone: Supply essential nitrogen, vitamins, trace elements, and other growth factors necessary for diverse microbial requirements.
• Resazurin: When added to 1000 mL of freshly prepared medium, resazurin functions as a redox indicator and undergoes a color change from colorless to purplish pink upon exposure to oxygen. This visual change enables confirmation of oxygen penetration and assessment of anaerobic conditions within the medium. The medium should be dispensed into suitable containers that provide an appropriate surface-to-depth ratio, such that no more than the upper half of the medium exhibits a color change indicative of oxygen uptake at the end of the incubation period.
• Sodium chloride: Maintains osmotic balance, ensuring cellular integrity and optimal growth conditions.
• Agar: A lower concentration of agar is used to limit oxygen diffusion within the medium, thereby helping to maintain anaerobic conditions.

This carefully balanced formulation enables Fluid Thioglycollate Medium (FTM) to support the growth of microorganisms with differing oxygen requirements within a single medium. By providing a reduced environment along with adequate nutritional components, FTM allows the reliable recovery of strict aerobes, facultative anaerobes, and obligate anaerobes, making it an essential medium for sterility testing, environmental monitoring, and quality control in regulated pharmaceutical and biopharmaceutical laboratories.

When stored at 2–30 °C in a sterile, airtight container, the medium retains its functional integrity. A pink coloration in more than the upper one-third of the medium indicates oxygen penetration. In such cases, the medium may be restored once by heating the container in a water bath or under free-flowing steam until the pink color is eliminated. The medium should then be rapidly cooled, ensuring that non-sterile air is not introduced, to maintain its suitability for microbiological use.

FTM, when incubated at 20–25 °C, may be used in place of Soybean Casein Digest Medium (SCDM), provided it has been suitably validated through growth promotion testing for aerobic bacteria, anaerobic bacteria, and fungi. This approach is particularly applicable for products containing mercurial preservatives that are not amenable to testing by the membrane filtration method.

3. Types of Fluid Thioglycollate Medium

Fluid Thioglycollate Medium (FTM) is available in multiple formats to accommodate diverse laboratory requirements, ranging from routine microbiological testing to regulated sterility assurance programs.

3.1 Dehydrated Powder:

Dehydrated Fluid Thioglycollate Medium (FTM) is supplied in non-sterile containers and is available in both powder and granulated forms. In addition to the conventional formulation containing animal-derived peptones, FTM is also offered in chemically defined, animal-free formulations such as HiVeg media, which utilizes vegetable-based tryptone, and HiCynth media, thereby eliminating the risk of BSE/TSE associated with animal-derived peptones. This format is suitable for laboratories that prefer in-house preparation of fresh media, offering flexibility in batch sizes while maintaining control over sterilization and handling procedures

3.2 Ready-to-Use Sterile Broth:

Ready-to-use FTM is supplied as pre-prepared, sterile liquid broth, minimizing variability associated with manual media preparation. This format reduces the risk of contamination from handling and is particularly advantageous for pharmaceutical and biopharmaceutical laboratories operating under GMP conditions. Ready-to-use media facilitate standardized execution of sterility testing protocols and support regulatory compliance. HiMedia’s ready-to-use broth is available in volumes ranging from 5 mL to 1000 mL and is supplied in different bottle configurations, including screw-cap and crimp-pull closures, to meet diverse user requirements.

3.3 Supplemented or Enriched Variants:

Certain FTM formulations are enriched with additional nutrients, such as vitamins, hemin, or growth factors, to support the proliferation of extremely fastidious anaerobic microorganisms. These variants are commonly employed in clinical microbiology, research laboratories, and studies involving strict anaerobes, where enhanced growth conditions are required.

• HiMedia FTM/ HM Peptone B (M380) is recommended by the USDA Animal and Plant Health Inspection Service (APHIS) for the detection of viable bacteria in live veterinary vaccines, supporting reliable microbial recovery.
• HS medium (M245), contains sodium hydrosulphite instead of sodium thioglycollate, is designed to establish and maintain both oxidized and reduced conditions, thereby enhancing the recovery of a broad spectrum of microorganisms, including anaerobes.
• Thioglycollate Medium w/K Agar (M430) is formulated to support the growth of aerobic, microaerophilic and anaerobic bacteria, with agar aiding in the establishment of oxygen gradients essential for sterility and microbial limit testing.
• Formulations containing 0.5% soya lecithin and 4% polysorbate (polysorbate 80) demonstrate enhanced efficiency in the sanitization assessment of container and equipment surfaces and are suitable for applications involving water-miscible cosmetics and related products, ensuring effective neutralization of disinfectants and preservatives.

Example: HiMedia Laboratories provides FTM in dehydrated powder and sterile ready-to-use formats. These media are fully compatible with USP <71> sterility testing requirements and are supplied with batch-specific documentation, including Certificates of Analysis, Technical Data Sheets and Safety Data Sheet (SDS) to support regulatory compliance and laboratory traceability.

4. Applications of Fluid Thioglycollate Medium:

4.1 Sterility Testing of Pharmaceutical Products:

Fluid Thioglycollate Medium (FTM) is a cornerstone in sterility testing as prescribed by USP <71>, complementing Soybean Casein Digest Medium (SCDM) to ensure comprehensive microbial detection across aerobic and anaerobic spectra. FTM’s reducing environment, achieved through sodium thioglycollate and L-cystine, creates an oxygen gradient that supports obligate anaerobes in the lower strata while maintaining facultative and microaerophilic growth near the surface. This stratification is critical for detecting contaminants that may evade growth in conventional aerobic media, thereby minimizing the risk of false-negative sterility results.

In practical applications, FTM is compatible with both direct inoculation and membrane filtration methods. Direct inoculation allows for bulk product testing, whereas membrane filtration concentrates microorganisms from large-volume sterile preparations. Method suitability studies are mandatory when testing formulations containing bacteriostatic or preservative agents, as these can inhibit microbial proliferation and obscure contamination detection. Such validations ensure that FTM reliably demonstrates growth promotion capability and maintains sensitivity, fulfilling stringent regulatory expectations for pharmaceutical sterility assurance.

4.2 Microbial Quality Control:

In the context of pharmaceutical and biopharmaceutical manufacturing, FTM serves as a critical medium for microbial quality control, particularly for the detection of anaerobic contaminants. The medium’s nutrient-rich formulation, incorporating tryptone, yeast extract, and glucose, provides a robust environment for fastidious microorganisms that may survive manufacturing processes. Growth promotion tests using standard anaerobic and facultative strains, such as Clostridium sporogenes, validate the medium’s ability to detect low-level contaminants that could compromise product safety or stability.

Pseudomonas aeruginosa and Staphylococcus aureus are typically incubated at 30–35 °C. FTM, when incubated at 20–25 °C, may be used as an alternative to Soybean Casein Digest Medium (SCDM), provided it has been validated according to growth promotion testing for aerobic bacteria, anaerobic bacteria, and fungi. Growth promotion testing should be performed using reference organisms including Staphylococcus aureus, Phocaeicola vulgatus, Bacillus subtilis,, Pseudomonas aeruginosa, Clostridium sporogenes, Bacteroides fragilis, Vulgatus Candida albicans, and Aspergillus brasiliensis.

By supporting sensitive detection of microbial proliferation in raw materials, intermediates, and finished products, FTM plays a pivotal role in ensuring compliance with GMP and pharmacopoeia standards. Its use enables manufacturers to proactively identify contamination events, assess process hygiene, and verify the sterility of products prior to release, thereby mitigating potential health risks to patients.

4.3 Environmental Monitoring:

Fluid Thioglycollate Medium (FTM) plays a critical role in environmental monitoring programs within cleanrooms, isolators, and other controlled manufacturing environments. Inoculation of surface, air, and critical equipment samples into FTM enables the recovery of anaerobic and facultative anaerobic microorganisms that may not be detected using conventional aerobic monitoring media. The medium’s low oxidation–reduction potential is particularly effective for isolating microorganisms residing in oxygen-limited niches, including floor drains, air handling systems, and high-efficiency particulate air (HEPA) filter outlets.

Routine environmental monitoring using FTM supports a comprehensive microbial surveillance strategy that aligns with Good Manufacturing Practice (GMP) requirements and regulatory expectations. This approach facilitates early identification of microbial contamination, enables timely corrective and preventive actions, and provides documented evidence of sustained aseptic control across manufacturing operations, thereby reinforcing overall product quality and integrity.

4.4 Research Applications:

The ability of Fluid Thioglycollate Medium (FTM) to sustain the growth of both obligate and facultative anaerobic microorganisms has made it an indispensable tool in advanced microbiological and biomedical research. The medium is extensively used in studies involving anaerobic pathogens, gut microbiota, and fastidious organisms that require reduced oxygen tension for survival and proliferation. Its carefully optimized composition, including reducing agents and nutrient-rich components, allows researchers to investigate microbial metabolic pathways, interspecies interactions, and physiological adaptations under oxygen-limited conditions. These studies provide valuable insights into mechanisms of pathogenicity, host–microbe interactions, probiotic functionality, and the broader dynamics of microbial ecology.

In addition to its role in fundamental research, FTM is frequently employed in experimental systems for anaerobic fermentation, biofilm formation, and the evaluation of antimicrobial compounds under low oxidation–reduction potential conditions. The medium enables reproducible assessment of microbial behavior in environments that closely mimic in vivo anaerobic niches. Collectively, these research applications highlight the versatility of FTM beyond routine quality control testing and emphasize its contribution to scientific advancement in microbiology, pharmacology, biotechnology, and translational biomedical research.

5. Advantages of Using Fluid Thioglycollate Medium:

Fluid Thioglycollate Medium provides enhanced recovery of stressed and sub-lethally injured microorganisms that may result from exposure to heat, disinfectants, preservatives, or manufacturing stresses. The reducing environment and nutrient-rich composition of FTM support microbial repair mechanisms, enabling damaged cells to recover and proliferate. This characteristic is particularly important in sterility testing, where microorganisms may be present in a compromised physiological state and would otherwise remain undetected in less supportive media.

FTM allows visual assessment of oxygen exposure and redox conditions through the inclusion of resazurin as an oxidation–reduction indicator. The color change from colorless to pink in the presence of oxygen provides immediate, non-invasive feedback on the medium’s suitability for anaerobic growth. This feature aids laboratory personnel in identifying oxidation-related issues, such as improper storage or excessive exposure to air, thereby improving test reliability and media quality control.

The liquid nature of FTM facilitates uniform contact between the test sample and the growth medium, enhancing the probability of microbial recovery, particularly when low bioburden levels are expected. Unlike solid media, which may limit microbial diffusion, liquid FTM enables microorganisms to disperse freely and access nutrients efficiently. This characteristic improves sensitivity in sterility testing and environmental monitoring applications, where contaminant levels are often minimal.

FTM can support extended incubation periods without significant nutrient depletion or loss of reducing capacity. However, according to USP guidelines, the medium should not be used beyond the storage period for which it has been validated. The medium maintains its physicochemical stability throughout the 14-day incubation period, ensuring consistent growth conditions that support the recovery and detection of slow-growing anaerobic microorganisms. This sustained stability is essential for compliance with USP <71> and other internationally recognized sterility testing requirements

6. HiMedia FTM:

HiMedia Laboratories provides Fluid Thioglycollate Medium (FTM) specifically developed for USP <71> sterility testing and pharmaceutical quality control applications. The medium is available in multiple validated formats, including dehydrated powder for in-house preparation, sterile ready-to-use broth, CULT Sterile Prewetted Swab and SCDM Media Tube designed to support aseptic workflows and minimize operator-related variability. These format options allow laboratories to select configurations aligned with their sterility testing volume, infrastructure, and regulatory expectations.

Each batch of HiMedia FTM is accompanied by comprehensive regulatory documentation, including batch-specific Certificates of Analysis (CoA), Technical Data Sheets (TDS), and Safety Data Sheets (SDS). Additionally, stability studies for each batch are available to ensure consistent performance and compliance with regulatory requirements.These documents provide detailed information on sterility status, growth promotion testing results using pharmacopoeial challenge organisms, pH range, redox characteristics, and other critical physicochemical parameters. Such documentation facilitates traceability, audit readiness, and compliance with GMP and pharmacopoeial requirements during regulatory inspections.

HiMedia FTM is validated for use in both direct inoculation and membrane filtration sterility testing methods, ensuring compatibility with a wide range of pharmaceutical dosage forms. The medium supports reliable recovery of aerobic, anaerobic, and microaerophilic microorganisms, including slow-growing and stressed organisms that may be present in sterile products. This broad recovery capability enhances detection sensitivity and strengthens overall sterility assurance programs.

From a quality assurance perspective, HiMedia manufactures FTM under strictly controlled conditions in compliance with established quality management systems. Raw materials are qualified, manufacturing processes are standardized, and finished products undergo rigorous in-process and final quality control testing. This systematic approach ensures batch-to-batch consistency, reproducible performance, and sustained compliance with global regulatory standards, reinforcing HiMedia’s role as a trusted supplier of sterility testing media for regulated industries.

Acknowledgement: The authors express their sincere gratitude to Dr. Rahul Warke (Director, Microbiology Department) and Dr. Girish Mahajan (Senior Vice President, Microbiology Department) for their exceptional scientific guidance, critical insights, and continuous support throughout the development of this work. The authors also thank Ms. Vrutti Mistry (Scientific Writer) for her support in drafting the manuscript.

Reference:

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3. US Pharmacopeia (USP). <71> STERILITY TESTS. In: United States Pharmacopeia (USP). ; 2022.
4. European Pharmacopoeia (Ph. Eur.). Chapter 2.6.1 Sterility Testing in Pharmaceutical Manufacturing. In: European Pharmacopoeia (Ph. Eur.)-12th Edition. ; 2025
5. THE MINISTRY OF HEALTH, LABOUR AND WELFARE. 4.06. Sterility Test Chapter. In: THE JAPANESE PHARMACOPOEIA-.; 2016.