Thioglycollate Broth: Introduction, Composition, Preparation, Principle, Test Procedure, Result Interpretation, Uses and Keynotes

achaurasiya

5 years ago

Thioglycollate Broth: Introduction, Composition, Preparation, Principle and Interpretation, Uses and Keynotes

Introduction of Thioglycollate Broth

Thioglycollate broth is recommended for sterility testing of biologicals and for the cultivation of anaerobes, aerobes, and microaerophiles from pharmaceutical as well as clinical samples.

Composition of Thioglycollate Broth

Ingredients Gms / Litre

Tryptone: 15.0
Yeast extract: 5.0
Dextrose (Glucose): 5.5
Sodium chloride(NaCl):2.5
L-Cystine: 0.5
Sodium thioglycollate: 0.5
Resazurin sodium: 0.001
Agar: 0.75
Final pH ( at 25°C): 7.1±0.2

Preparation of Thioglycollate Broth

Suspend 2.975 grams in 100 ml distilled water or deionized water.
Heat to boiling to dissolve the medium completely.
Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
Cool to 25°C and pour into the suitable test tubes or bottles.
Store in a cool dark place preferably below 25°C. Note: If more than the upper one-third (1/3) of the thioglycollate broth has acquired a pink-purple color, it may be restored once by heating in a water bath or in free-flowing steam until the pink-purple color clears.

Principle of Thioglycollate Broth

Dextrose, tryptone, yeast extract, L-cystine provide the growth factors necessary for organism multiplication while Sodium chloride allows for adjustments in osmotic pressure. L-cystine and sodium thioglycollate allows anaerobic bacteria to grow in this medium even under aerobic conditions. A low level of agar in the medium favors the growth of aerobes as well as anaerobes, even if sodium thioglycollate is deleted from the medium. Sodium thioglycollate act as a reducing agent and neutralizes the toxic effects of mercurial preservatives and peroxides formed in the medium, thereby enhancing anaerobiosis, and making the medium suitable to test materials containing heavy metals. Resazurin is a redox indicator that changes to red if there is an increase in the oxygen level. The small number of agar assists in maintaining a low redox potential for stabilizing the medium. And hence this environment of medium supports the growth of anaerobes, aerobes, microaerophilic, and fastidious microorganisms.

Test Requirements

Thioglycollate Broth
Test sample (clinical /pharmaceuticals/ test organisms)
Incubator
Bunsen burner and inoculating loop (optional-depending on the nature of the sample to be tested.)
Control Strain ( obligate aerobe,Pseudomonas aeruginosa ATCC 9027
and obligate anaerobe, Clostridium sporogenes ATCC 19404)

Test procedure ( specimen/organism inoculation)

Take out the broth from storage.
Inoculate the sample in it.
Incubate the medium at 30-35°C for up to 72 hours.
Examine for turbidity.

Result Interpretation of Thioglycollate Broth

No turbidity: No growth

Turbidity: Growth ( Presence of organisms)

Obligate aerobe: Growth at top of the broth

Obligate anaerobe: Growth at the bottom of thioglycollate broth

Growth in the thioglycollate broth depends on the nature of organisms requiring oxygen-

Obligate aerobes: Growth at top of the broth due to the highest oxygen concentration since they need oxygen and they cannot ferment or respire anaerobically.

Obligate anaerobes: Growth at the bottom of the tube where the oxygen concentration is lowest since the oxygen is poisonous to the organisms.

Facultative anaerobe: Growth in the whole broth due to being able to grow with or without oxygen since they can metabolize energy aerobically or anaerobically but mainly gather at the top because aerobic respiration generates more ATP than either fermentation or anaerobic respiration.

Microaerophilic organisms: Growth at the upper part of the thioglycollate broth since they need oxygen because they cannot ferment or respire anaerobically. However, high concentrations of oxygen may be fatal.

Aerotolerant organisms: Uniform growth in the whole broth since they do not require oxygen as they metabolize energy anaerobically. Unlike obligate anaerobes, though, they are not deadly by oxygen.

Colony morphology of different organisms are as follows-

Luxuriant growth under aerobic conditions

Luxuriant growth under anaerobic conditions

Clostridium sporogenes
Clostridium perfringens
Bacteroides fragilis

Note: Candida albicans shows flocculent growth.

Uses of Thioglycollate Broth

Thioglycollate broth is the choice of medium for the cultivation of aerobic, microaerophilic, and anaerobic microorganisms.
It also permits the differentiation of obligate aerobes, obligate anaerobes, facultative anaerobes, microaerophiles, and aerotolerant microorganisms.
Thioglycollate broth is the recommended medium for sterility testing of antibiotics, biologicals, and foods and for determining the phenol coefficient and sporicidal effect of disinfectants.
It is also routinely used to check the sterility of stored blood in blood banks.

Limitations of Thioglycollate Broth

Proper anaerobic conditions must be maintained for the optimal recovery of microorganisms.
It is necessary that the thioglycollate broth should be freshly prepared or boiled and cooled within four hours of use.
Low-temperature storage increases oxygen absorption.
Thioglycolate broth should not be re-heated more than once because toxic oxygen radicles are formed on reheating.

Keynotes on Thioglycollate Broth

The prepared thioglycolate broth should be a light yellow colored clear solution.
This medium supports the growth of a wide variety of microorganisms that may be either anaerobe, aerobes, microaerophilic, or even fastidious organisms, or all of them.
It is used to find out the growth characteristics of various bacteria based on their oxygen requirements.
Brewer formulated Fluid Thioglycollate Medium for rapid cultivation of aerobes as well as anaerobes including microaerophiles by including a reducing agent and small amount of agar.