TRYPTICASEIN SOY AGAR: RATIONALE, PREPARATION, AND USES - BIOLOGY - 2025

The soy agar tryptone or Trypticase Soy Agar is a solid culture medium, and nutritional nonselective. It is designated by the letters TSA for its acronym in English Trypticase Soy Agar. It is composed of triptein, soy peptone, sodium chloride, and agar-agar.

Due to its high nutritional power, it is ideal for the cultivation of moderately demanding and non-demanding microorganisms. Medium without additional supplements is not recommended for primary cultures, but it is very useful for subculturing pure strains and keeping them viable, among other uses.

Trypticasein soy agar wedges in tubes with bakelite caps. Source: Photograph taken by the author MSc. Marielsa Gil.

Also, this agar serves as a base for the preparation of enriched media such as blood agar, especially when it is required to observe the hemolysis patterns and to mount the optoquine and bacitracin taxa, required in the diagnosis of Streptococcus pneumoniae and Streptococcus pyogenes respectively.

On the other hand, when combined with antibiotics it is useful to isolate clinically important facultative and strict anaerobic microorganisms from samples with mixed flora.

Finally, the composition of trypticasein soy agar and its performance meet the requirements established by the different pharmacopoeias (European, Japanese and North American).

Basis

For the proper development of bacteria, the presence of energy supplies is necessary, such as amino acids, vitamins, puric and pyrimidic bases.

In this sense, triptein and soy peptone provide these nutrients to microorganisms, thus allowing their full development. However, for fastidious bacteria it is necessary to supplement this agar with defibrinated blood or warmed blood to increase its enrichment.

On the other hand, if antibiotics are added to the medium, it becomes a selective medium. 0.6% yeast extract can also be added to favor the isolation of species of the genus Listeria, while the addition of cystine tellurite and lamb's blood is ideal for Corynebacterium diphteriae.

Finally, the sodium chloride provides the osmotic balance to the medium and the agar provides the solid consistency.

Preparation

Trypticasein Soy Agar Plates

To prepare trypticasein soy agar, 40 g of the dehydrated commercial medium should be weighed on a digital scale. It is dissolved in one liter of distilled water contained in a flask.

The mixture is left to rest for 5 minutes and then it is taken to a heat source to help dissolve the medium. It should be stirred frequently and boiled for 1 to 2 minutes. Subsequently, the medium is sterilized in the autoclave at 121 ° C for 15 minutes.

Allow to cool to 50 ° C and distribute into sterile Petri dishes. Allow to solidify, invert, order in plaqueros and store in the refrigerator.

The final pH of the medium should be 7.3 ± 0.2.

It should be noted that the color of the dehydrated culture medium is light beige and it should be stored between 10 to 35 ° C, in a dry place.

For its part, the prepared agar is light amber in color. Prepared plates should be stored in a refrigerator (2-8 ° C) until use.

Plates must reach room temperature before use.

Trypticasein soy agar plates supplemented with blood

Blood agar is prepared by adding 5% defibrinated blood at the time of cooling the trypticasein soy agar to 50 ° C. The mixture is homogenized by rotating with gentle movements.

Serve in sterile Petri dishes. The middle color should be cherry red.

Trypticasein soy agar plates supplemented with warmed blood

To prepare the TSA-based blood agar, proceed the same as the procedures already described, but when leaving the autoclave it is left to rest until the temperature of the medium is approximately between 56 to 70 ° C. At that time the blood is placed and mixed until the medium turns brown.

Serve in sterile Petri dishes. The color of the medium is chocolate brown.

Trypticasein Soy Agar Wedges

The agar preparation procedure is the same as that described for plates, with the difference that instead of serving the medium on Petri dishes, it is distributed between 10 to 12 ml in tubes with Bakelite covers before sterilizing.

Subsequently, the tubes are autoclaved at 121 ° C for 15 minutes. When they leave, they are inclined with the help of a support and allowed to solidify.

The prepared wedges are sown by surface area and serve to keep certain non-demanding microorganisms viable for a specified time.

Applications

Trypticasein Soy Agar is used in the following cases:

-As a base to prepare the classic blood agar that is used routinely in most laboratories.

-The isolation of demanding bacteria.

-Observation of the hemolysis pattern.

-Execution of diagnostic tests.

-As a base to prepare special blood agar for Corynebacterium diphteriae, with cystine tellurite and lamb's blood.

-As a base to prepare lamb blood agar, plus kanamycin-vancomycin for the growth of anaerobes, especially Bacteroides sp.

- For the maintenance of non-demanding strains (Bacterioteca).

- Aerobic microbial count in the study of the microbial limit of water, environment, food and cosmetic samples.

Sown

Samples can be sown directly onto the surface of Trypticasein Soy Agar supplemented with blood or other additives. It is sown by exhaustion.

Whereas, trypticasein soy agar plates without additives are generally used to subculture microbial strains (bacteria or yeast).

QA

Sterility control

To check the sterility of the different media prepared with Trypticasein Soy Agar, it is recommended to do the following: from each prepared batch, 1 or 2 uninoculated plates or tubes should be incubated at 37 ° C for 24 hours to demonstrate their sterility. In all cases it must remain without growth.

If contamination is found, the entire batch must be discarded.

Growth control

The following bacterial strains can be used to study the proper functioning of trypticasein soy agar: Escherichia coli ATCC 8739, Staphylococcus aureus ATCC 6538, Pseudomonas aerugiosa ATCC 9027 and Enterococcus faecalis ATCC 29212.

The strains are seeded and incubated aerobically at 37 ° C for 24 hours.

In all cases growth must be satisfactory.

Fungi such as Candida albicans Complex ATCC 10231 and Aspergillus niger ATCC 16404 can also be used. Good growth is expected for both strains.

Growth control and hemolysis pattern of trypticasein soy agar supplemented with blood

To verify the proper functioning of the blood agar prepared with this base, the following strains can be used: Streptococcus pyogenes ATCC 19615, Streptococcus pneumoniae ATCC 6305 and Streptococcus pneumoniae ATCC 49619.

They are seeded and incubated at 37 ° C in microaerophilicity for 24 hours.

In all cases, growth should be satisfactory, taking into account that beta-hemolysis (light halo around the colony) should be observed in S. pyogenes and alpha hemolysis (greenish halo around the colony) should be observed in both strains of S. pneumoniae. colonies).

References

1. Wikipedia contributors. Trypticase I am agar. Wikipedia, The Free Encyclopedia. December 17, 2018, 15:47 UTC. Available at:
2. Britannia Laboratories. Triptein soy agar. 2015.Available at: britanialab.com
3. Neogen Laboratories. Tryptic soy agar. Available at: foodsafety.neogen.com
4. Forbes B, Sahm D, Weissfeld A. (2009). Bailey & Scott Microbiological Diagnosis. 12 ed. Argentina. Editorial Panamericana SA
5. BD Laboratories. Trypticase I am Agar. 2014.Available at:.bd.com