NIH Thioglycollate Broth

Intended Use

NIH Thioglycollate Broth and Sterility Test Broth (USP Alternative Thioglycollate Medium) may be used for sterility testing instead of FTM.
Fluid Thioglycollate Medium and NIH Thioglycollate Broth/Sterility Test Broth meet United States Pharmacopeia (USP)performance specifications.

Summary and Explanation

NIH Thioglycollate Broth and Sterility Test Broth, which are the USP Alternative Thioglycollate Medium, are Fluid Thioglycollate Medium without the agar or indicator components. They are used for the same sterility test procedures except that anaerobic incubation is recommended rather than aerobic incubation. They also meet the requirements of the USP growth promotion test.14
Thioglycollate Medium without Indicator (135C) is the medium of choice for diagnostic work because the lack of indicator avoids possible toxicity to organisms.11 This medium supports a minimal inoculum with early visibility of growth.
When used as an enrichment broth to support plated media, thioglycollate media are often supplemented with hemin and vitamin K1.16 Fluid Thioglycollate Medium, Enriched is BBL™ Fluid Thioglycollate Medium supplemented with vitamin K1 and hemin. Enriched Thioglycollate Medium is BBL Thioglycollate Medium without Indicator-135C supplemented with vitamin K1 and hemin. Enriched broth media are recommended for use in the isolation and cultivation of fastidious or slow growing, obligately anaerobic microorganisms present in clinical materials.17,18 They are also recommended for the isolation and cultivation of a wide variety of aerobic and facultatively anaerobic microorganisms. Enriched Thioglycollate Medium is prepared with an anaerobic head space and is provided in screw-capped tubes in accordance with CDC recommendations.17 Vitamin K1 and hemin have been shown to be required by certain anaerobes for growth.19,20 The addition of calcium carbonate enhances the maintenance of stock cultures by neutralizing acids produced during growth.16
The Enriched Thioglycollate Medium (Broth) recommended by the CLSI for inoculum preparation for susceptibility tests of anaerobes consists of Enriched Thioglycollate Medium (Thioglycollate Medium without Indicator [135] with 1 μg/mL of Vitamin K1 and 5 μg/mL of hemin) supplemented with 1 mg/mL of sodium bicarbonate or a marble chip to neutralize acids produced during growth of the test organisms.21

Principles of the Procedure

Dextrose, peptone, L-cystine and yeast extract provide the growth factors necessary for bacterial replication. Sodium chloride provides essential ions. Sodium thioglycollate is a reducing
agent that prevents the accumulation of peroxides which are lethal to some microorganisms. The L-cystine is also a reducing agent, since it contains sulfhydryl groups which inactivate heavy metal compounds and maintain a low redox potential, thereby supporting anaerobiosis. Methylene blue is  an indicator of the level of oxidation/reduction in the medium; increased oxidation raises the Eh, causing the methylene blue indicator to become green. Resazurin is an oxidation-reduction indicator, being pink when oxidized and colorless when
reduced. The small amount of agar assists in the maintenance of a low redox potential by stabilizing the medium against convection currents, thereby maintaining anaerobiosis in the
lower depths of the medium. The USP lists 5.5g/L of dextrose in the formulations for Fluid Thioglycollate Medium and Alternative Thioglycollate Medium; some of the following
formulations include the anhydrous form of dextrose (5.0g/L). 

Vitamin K1 is a growth requirement for some strains of Prevotella melaninogenica18 and is reported to enhance the growth of some strains of Bacteroides species and grampositive
nonsporeformers.22 Hemin is the source of the X factor, which stimulates the growth of many microorganisms. Calcium carbonate neutralizes acids produced during growth, which helps to maintain the viability of fastidious organisms; e.g., pneumococci, gram-negative cocci, Clostridium perfringens and other acid-sensitive bacteria. Dipotassium phosphate is a buffering agent.

User Quality Control

NOTE: Differences in the Identity Specifications and Cultural Response testing for media offered as both Difco™ and BBL™ brands may reflect differences in the development and testing of media for industrial and clinical applications, per the referenced publications.
NIH Thioglycollate Broth
Dehydrated Appearance: Light tan, free-flowing, homogeneous.
Solution:                       2.9% solution, soluble in purified water upon boiling.
                                    When hot, solution is light amber, clear to very
                                    slightly opalescent, may have a slight precipitate.
Prepared Appearance:     Light amber, clear to very slightly opalescent,
                                    may have a slight precipitate.
Reaction of 2.9%
Solution at 25°C:            pH 7.1 ± 0.2
Cultural Response
NIH Thioglycollate Broth
Prepare the medium per label directions. Inoculate duplicate tubes and incubate at 30-35°C for 18-48 hours (up to 72 hours, if necessary) under anaerobic conditions (tight caps).

Mercurial Neutralization Test – To perform, add 1% Merthiolate™ to medium, inoculate (103 CFU) with Staphylococcus aureus ATCC 6538P and Streptococcus pyogenes ATCC 19615, and incubate at 30-35°C for 18-48 hours. Recovery of organisms indicates that Merthiolate has been neutralized.
*Merthiolate is a trademark of Eli Lilly and Company.

Formula

NIH Thioglycollate Broth
Approximate Formula* Per Liter
Casitone.................................................................... 15.0 g
Yeast Extract................................................................ 5.0 g
Dextrose...................................................................... 5.5 g
Sodium Chloride........................................................... 2.5 g
L-Cystine..................................................................... 0.5 g
Sodium Thioglycollate.................................................... 0.5 g

Directions for Preparation from Dehydrated Product

1. Suspend the powder in 1 L of purified water:
   NIH Thioglycollate Broth – 29 g;Mix thoroughly.
2. Heat with frequent agitation and boil for 1 minute to completely dissolve the powder.
3. Follow label directions specific for each medium.
4. Autoclave at 121°C for 15 minutes, or as directed on the label.
5. Store fluid thioglycollate media at 15-30°C. If more than 30% of the medium is pink prior to use, reheat once (100°C) to drive off absorbed oxygen.
6. Test samples of the finished product for performance using stable, typical control cultures.

Precautions

Do not reheat the media more than once; continued reheating gives rise to toxicity.

Procedure

Follow the procedures outlined in the references and, where applicable, in product package inserts.

Expected Results

After incubation, growth is evidenced by the presence of turbidity compared to an uninoculated control. Strict aerobes tend to grow in a thin layer at the surface of the broth; obligate anaerobes will grow only in that portion of the broth below the upper oxidized layer.

Limitation of the Procedure16

Anaerobes can be overgrown by more rapidly growing facultative organisms. Examine and Gram stain broth if plating medium reveals no growth. Never rely on broth cultures exclusively for isolation of anaerobes. Some anaerobes may be inhibited by metabolic products or acids produced from more rapidly growing facultative anaerobes.

*Store at 2-8°C.

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