Tryptic Soy Agar (TSA):Principle, Composition, Preparation And Uses

Introduction

Trypticase soy agar or tryptone soya agar (TSA) and Trypticase soy broth or tryptone soya broth (TSB) with agar are growth media for the culturing of bacteria. They are general-purpose, nonselective media providing enough nutrients to allow for a wide variety of microorganisms to grow. They are used for a wide range of applications, including culture storage, enumeration of cells (counting), isolation of pure cultures, or simply general culture.

Principle

The Tryptone Soy Agar (TSA) contains the magical ingredients that supports almost all types of Fastidious i.e. those bacteria that have some specific requirement for the growth as well as the Non-Fastidious bacteria i.e. those bacteria which can grow easily on any medium and have no special requirements, are the Soybean and Casein Digest supported by various components.

TSA contains enzymatic digests of casein and soybean meal, which provide amino acids and other nitrogenous substances, making it a nutritious medium for a variety of organisms. Glucose is the energy source. Sodium chloride maintains the osmotic equilibrium, while dipotassium phosphate acts as buffer to maintain pH. Agar extracted from any number of organisms is used as a gelling agent.

The medium may be supplemented with blood to facilitate the growth of more fastidious bacteria or antimicrobial agents to permit the selection of various microbial groups from pure microbiota. As with any media, minor changes may be made to suit specific circumstances. TSA is frequently the base medium of other agar plate types. For example, blood agar plates (BAP) are made by enriching TSA plates with defibrinated sheep blood, and chocolate agar is made through additional cooking of BAP. Nutrient agar is also similar to TSA.

One liter of the agar contains:

• 15 g tryptone
• 5 g “soytone” – enzymatic digest of soybean meal
• 5 g sodium chloride
• 15 g agar

Preparation Of Typtic Soy Agar

Composition Of Tryptic Soy Agar

Material Required

• Sterile Conical Flask / Erlenmeyer Flask
• Spatula
• Pancreatic digest of casein
• Papaic digest of soybean meal
• Dextrose (Glucose)
• Dipotassium hydrogen phosphate
• Sodium Chloride
• Agar-Agar
• Measuring Cylinder
• 1N HCl
• 1N NaOH
• pH Strip
• Weighing Scale
• Distilled Water
• Butter Paper

Preparation Procedure

1. Weigh the quantity of Pancreatic digest of casein, Papaic digest of soybean meal, dextrose, Dipotassium Hydrogen Phosphate, sodium chloride, and Agar-Agar using the weighing scale for 1000 ml of Trypticase Soy Agar medium.
2. Take a clean and dry Conical Flask/ Erlenmeyer flask.
3. Pour 500 ml of distilled water to the flask and add the weighed quantity of Pancreatic digest of casein, Papaic digest of soybean meal, dextrose, Dipotassium Hydrogen Phosphate, and sodium chloride.
4. Now add the weighed quantity of Agar-Agar.
5. Mix well the content and Heat it with continuous agitation to dissolve the constituents.
6. Now add more distilled water to the medium and make the volume 1000 ml.
7. Check the pH of the solution using pH strip, it should be 7.3 ± 0.2. If required, adjust the pH by adding either 1N HCl (acid) or 1N NaOH (base) as per the case.
8. Mix well the content and apply the Non-absorbent cotton plug to the flask.
9. Autoclave the content at 121 °C and 15 psi pressure for 15 minutes.
10. Allow the content to cool down to 40-45 °C and pour in the empty media plates under the strict aseptic atmosphere (preferably in Laminar Air Flow) and allowed it to cool at room temperature.
11. Use the prepared media plates to inoculate the specimen to be cultured and then place in the incubator at optimum temperature.

Storage

Tryptic Soy Agar formulations should be stored away from direct light at 4°C to 8°C. For plated media, the medium side should be uppermost to prevent excessive accumulation of moisture on the agar surface.

Result Interpretation on Tryptic Soy Agar

After incubation, most plates will show an area of confluent growth. Because the streaking procedure is, in effect, a “dilution” technique,diminishing numbers of microorganisms are deposited on the streaked areas. Consequently, one or more of these areas should exhibit isolated colonies of the organisms contained in the specimen. In addition, growth of each organism may be semiquantitatively scored on the basis of growth in each of the streaked areas. Hemolytic reactions should be noted for organisms inoculated on the medium containing blood.The tubed slant containing pure cultures can be used for additional studies or as stock cultures as desired

Uses Of Tryptic Soy Agar

• The medium is used for a wide range of applications, including culture storage, enumeration of cells (counting), isolation of pure cultures, or simply general culture.
• Tryptic Soy Agar supports the growth of a wide variety of organisms including fastidious and non- fastidious such as Neisseria, Listeria, and Brucella, etc.
• Tryptone Soya Broth with added dextrose, sodium chloride, and agar is recommended for the cultivation of Salmonella Typhi.
• TSA is frequently the base medium of other agar plate types. For example, blood agar plates (BAP) are made by enriching TSA plates with defibrinated sheep blood, and chocolate agar is made through additional cooking of BAP.
• It can be used to determine, X, V, and XV factor in Hemophilus species using strips that have the factors X, V, and XV in the inoculated plates.
• Tryptic soy agar with the addition of salt can be helpful in determining the halotolerance level of microorganisms. 
• It is commonly used for environmental testing in the pharmaceutical field.
• It is helpful in testing water, cosmetics, industrial, food, and beverages.
• Tryptic Soy Agar may also be used as a medium for maintaining or subculturing reference strains, e.g., Enterobacteriaceae and staphylococci.

Further References

1. FDA. Bacteriological analytical manual, 2002. Retrieved January 27, 2003, from FDA website: http://www.cfsan.fda.gov.
2. NCCLS. Quality assurance for commercially prepared microbiological culture media. 2nd ed. NCCLS document M22-A2. Wayne, PA: NCCLS, 1996.
3. APHA Technical Committee on Microbiological Methods for Foods. Compendium of Methods for the Microbiological Examination of Foods. APHA, Washington, D.C.
4. American Public Health Association. Standard Methods for the Examination of Water and Wastewater, APHA, Washington, D.C.
5. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.
6. MacFaddin, J.F. 1985. Media for Isolation, Cultivation, Identification, Maintenance of Bacteria, Vol. I. Williams & Wilkins, Baltimore, MD.
7. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.
8. MacFaddin, J.F. 1985. Media for isolation-cultivation-identification-maintenance of medical bacteria, vol. 1, Williams & Wilkins, Baltimore.
9. Fries, S.M. 1995. Diagnosis of group A streptococcal pharyngitis in a private clinic: comparative evaluation of an optical immunoassay method and culture. J. Ped. vol. 126, number 6.
10. Campos, J.M. 1995. Haemophilus. In: P.R. Murray, E.J. Baron, M.A. Pfaller, F.C. Tenover, and R.H. Yolken (ed.), Manual of clinical microbiology, 6th ed. American Society for Microbiology, Washington, D.C.