THIOGLYCOLLATE BROTH: RATIONALE, PREPARATION AND USES - BIOLOGY - 2025

The thioglycollate broth is a medium enriched fluid consistency. It is known by the acronym FTM for its acronym in English Fluid Thioglycollate Medium. It was created by Brewer and modified in 1944 by Vera, who added casein peptone to it.

This medium has a low oxidation -reduction potential, therefore it is not recommended for the development of strict aerobic bacteria, but it is ideal for recovering facultative aerobic, strict anaerobic and undemanding microaerophilic bacteria.

Thioglycollate broth without indicator. Shows vaginal discharge. Source: Bobjgalindo

The high performance observed with this medium in the isolation and recovery of a wide variety of microorganisms, has made it accepted by the United States Pharmacopeia (USP), by the Official Association of Agricultural Chemists (AOAC) and by the European Pharmacopoeia (EP).

These organizations recommend it for sterility control testing of pharmaceutical products and for enrichment of various types of samples.

The cado thioglycolate is composed of yeast extract, pancreatic digest of casein, anhydrous dextrose, L-cystine, sodium chloride, sodium thioglycollate, resazurin and agar in small quantity.

There are several versions of this medium, among them are: thioglycollate broth with indicator, thioglycollate broth without indicator, thioglycollate broth with indicator enriched with hemin and vitamin K _1, and thioglycollate broth with calcium carbonate.

Notably, the thioglycollate broth variant enriched with hemin and vitamin K serves to enhance the growth of fastidious anaerobes, and the thioglycollate broth variant containing calcium carbonate is useful to counteract the acids produced during microbial growth.

Basis

Thioglycollate broth is considered a non-selective enrichment medium as it allows the growth of most non-fastidious bacteria. The nutritional requirements are provided by the yeast extract, the pancreatic digest and glucose.

On the other hand, this medium, despite being a broth, contains a small amount of agar; This means that it has a low oxidation-reduction potential, due to the fact that it slows the entry of oxygen, in such a way that the oxygen decreases as it goes deeper into the tube.

That is why this medium is ideal for the development of facultative aerobic, microaerophilic and strict anaerobic bacteria, the latter 2 without the need to incubate under these conditions. The same medium regulates the amount of oxygen within the medium, being absent at the bottom of the tube and in sufficient quantity on the surface.

Likewise, thioglycollate and L-cystine act as reducing agents, helping to prevent the accumulation of substances that are harmful to bacterial development, such as peroxide. In addition, these compounds contain sulfhydryl groups (-SH-), neutralizing the inhibitory effects of mercury derivatives, arsenicals, among other heavy metals.

For its part, resazurin is an indicator of oxide-reduction. This substance is colorless when reduced and pink when oxidized. There are indicator and non-indicator thioglycollate broth variants. Its use will depend on the type of sample and the preference of the laboratory.

Meanwhile, sodium chloride maintains the osmotic balance of the thioglycollate broth and the use of glucose in anhydrous form prevents excess moisture in the dehydrated medium.

Preparation

Thioglycollate Broth with Indicator

Weigh out 29.75 g of the dehydrated medium and dissolve in 1 liter of distilled water. The mixture is allowed to stand for approximately 5 minutes. Bring to a heat source and stir frequently until completely dissolved.

Pour the medium into test tubes and autoclave at 121 ° C for 15 minutes. Allow to cool before use. Check the insert of the commercial house for its conservation. Some recommend storing at room temperature in a dark place, and others in a refrigerator protected from light.

The pH of the prepared medium is 7.1 ± 0.2.

The color of the dehydrated medium is light beige and the prepared medium is light amber with some opalescence.

Thioglycollate broth with indicator enriched with hemin and vitamin K

There are commercial media that already contain hemin and vitamin K ₁, especially for the cultivation of anaerobes.

If you do not have the enriched medium for anaerobes, you can prepare the basic thioglycollate broth. For this, 10 mg of hemin hydrochloride and 1 mg of vitamin K _{1 are added} for each liter of medium. However, if blood or serum is added to the thioglycollate broth, the addition of hemin or vitamin K is not necessary.

Thioglycollate broth with calcium carbonate

It comes commercially and is prepared following the instructions on the insert.

Thioglycollate broth without indicator

This has the same composition of the basic thioglycollate, but does not contain resazurin.

Weigh 30 g of the dehydrated medium and dissolve in a liter of distilled water. The rest of the preparation is the same as that described in the thioglycollate broth with indicator.

Use

Thioglycollate broth is useful for the enrichment of clinical samples, especially those from sterile sites. It is also useful for non-clinical samples, such as cosmetics, drugs, etc.

For the inoculation of liquid samples (such as CSF, synovial fluid, among others), the samples are first centrifuged and then 2 drops of the sediment are taken and placed in the thioglycollate broth. Incubate at 35 ° C for 24 hours. If in this time there is no growth (turbidity), it is incubated up to a maximum of 7 days.

If the sample is taken with a swab, the culture media are first inoculated in plates and finally the swab is introduced into the broth, the protruding portion is split and the tube is capped, leaving the swab inside. Incubate at 35 ° C for 24 hours, 7 days maximum.

For solid samples, homogenize in physiological saline solution (SSF) and then inoculate the thioglycollate broth with 2 drops of the suspension.

It can sometimes be used as a transport medium for samples where the presence of stringent anaerobes is suspected or as a backup enrichment broth.

The thioglycollate broth variant with calcium carbonate is used for the maintenance of control strains for a longer time, because it has the ability to neutralize the acids produced by the use of glucose; These acids are toxic to certain bacteria.

Growth in the thioglycollate broth will be observed by the turbidity of the medium. It is recommended to perform a Gram stain and subsequently subculture in non-selective and selective media, depending on the type of sample and the microorganisms suspected.

QA

For sterility control it is recommended to incubate one or two broths without inoculation. The expected result is a clear broth, with no color change, although it is normal to see a slight pink color on the surface of the tube.

For quality control, between 10 - 100 CFU of certified control strains should be inoculated, such as Staphylococcus aureus ATCC 6538, Micrococcus luteus ATCC 9341, Bacillus subtilis ATCC 6633, Pseudomonas aeruginosa ATCC 9027, Clostridium sporogenes ATCC 19404, Clostridium 11437 sporogenes Bacteroides vulgatus ATCC 8482.

Incubate at 30-35 ° C in aerobiosis for 24 hours up to a maximum of 3 days, since these microorganisms are fast growing.

Good development is expected in all cases, except for Micrococcus luteus and Bacillus subtilis, where there may be moderate development.

For quality control of thioglycollate broth enriched with hemin and vitamin K ₁, the control strains Bacteroides vulgatus ATCC 8482, Clostridium perfringens ATCC 13124 and Bacteroides fragilis ATCC 25285 can be used. The expected result is satisfactory growth.

recommendations

-Occasionally it can be observed that the surface of the thioglycollate broth with indicator turns pink; This is due to oxidation of the medium. If the pink color covers 30% or more of the total broth, it can be heated in a water bath for 5 minutes, cooled again and used.

This will remove the absorbed oxygen, returning the medium to its original color. This procedure can only be done once.

-To improve the growth of aerobic bacteria, incubate with the lid slightly loose. However, it is preferable to use for this purpose a brain heart infusion broth or trypticase soy broth for the proper development of strict aerobes.

-The freezing of the medium or overheating should be avoided, as both conditions damage the medium.

-Direct light damages the culture medium, it must be stored protected from light.

References

1. Britannia Laboratories. Thioglycollate USP with indicator. 2015.Available at: labBritania.com.
2. Chios Sas Laboratories. 2019. Thioglycollate broth. Available at: quios.com.co
3. Laboratories BD Fluid Thioglycollate Medium (FTM). 2003.Available at: bd.com/Europe
4. BBL media prepared in tubes for the cultivation of anaerobic microorganisms. Thioglycollate media. 2013.Available at: bd.com
5. Forbes B, Sahm D, Weissfeld A. (2009). Bailey & Scott Microbiological Diagnosis. 12 ed. Editorial Panamericana SA Argentina.