Lifting the lid: What's inside the TSA plate?

Culture media contains nutrients, growth promoting factors, energy sources, buffer salts, minerals, metals and gelling agents (for solid media). The ISO 11133 definition is:

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“Formulations of substances in liquid, semi-solid or in sold form, which contain natural and/or synthetic constituents intended to support the multiplication, or to preserve the viability, of microorganisms.”

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Culture media contains the nutrients needed to sustain a culturable microorganisms; beyond this, culture media vary in the many ingredients added, allowing the media to select for or against microbes.

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One of the most common media used in pharmaceutical microbiology is soyabean casein digest medium (SCDM) – alternatively referred to as TSA (depending on the precise formulation). Under appropriate temperature and atmospheric conditions, for a suitable time period, the medium is particularly effective at recovering organisms residential to human skin making the medium suitable for cleanroom monitoring.

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What is this complex medium?

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Lifting the lid

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Tryptone soya agar or tryptic soya agar (equivalent to soybean casein digest medium are common media medium for the isolation and cultivation of non-fastidious and fastidious micro-organisms – both bacteria and fungi. This medium is used for environmental monitoring and in-process bioburden testing and it contains the products of two peptones. The medium was first described by Leavitt and colleagues in 1955 (1). In terms of recovering organisms from an environment, papers describing the use of the medium begin to appear during the 1970s and by the 1980s the use of the medium becomes well-established (in 1970 the eighteenth edition of the USP introduced, in replacement to Sabouraud broth, soybean casein digest broth (2) selected because it possesses a higher pH and it was considered to be, comparatively, a better medium for the recovery of fungal contaminants).

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Readers may notice the use of terms ‘soy’ and ‘soya’ – these are the same things, the former being the preferred U.S. spelling and the latter the preferred European spelling.

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The medium may contain a neutralizer when used for environmental monitoring so that disinfectant residues can be neutralized. Often the medium will also be irradiated where it is used in aseptic processing environments. Alternatively, the medium may be supplemented with blood (5% defibrinated sheep’s blood or horse blood) to facilitate the growth of more fastidious bacteria (3).

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The medium, unmodified, is weak at recovering fastidious organisms and it is not suitable for recovering pathogens in the medical microbiology context (4).

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Formulation

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The general formula is (5):

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• Pancreatic digest of casein (a peptone): 15.0 – 17.0 gm/liter
• Enzymatic digest of soya bean (sometimes called soytone): 3.0 - 5.0 gm/liter
• Sodium chloride: 5.0 gm/liter
• Glucose (often in the form of dextrose): 2.5 gm/liter
• Dipotassium phosphate: 2.5 gm/liter
• Agar: 15.0 gm/liter

Adjusted to pH 7.3 ± 0.2 @ 25°C.

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The medium 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 provides the energy source. Sodium chloride maintains the osmotic equilibrium, while dipotassium phosphate acts as buffer to maintain pH. Agar is added to form a gel (agar being a phycocolloid extracted from a group of red-purple marine algae including Gelidium, Pterocladia and Gracilaria).

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But what precisely do these ingredients contribute and what exactly are they?

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Peptones

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Peptones are the products of peptic digestion, using enzymes and the word is a genetic term for the partial hydrolysis of proteins. Pancreatic digest of casein is a general purpose growth peptone used as a nitrogen source for cultivating microorganisms. Caseins, which account for approximately 80% of the total proteins in mammalian milk, exist in a micelle form aggregated by four phosphorylated proteins: αS1-, αS2-, β- and κ-caseins (αS1-, αS2-, β- and κ-CN). The common source of casein used for culture media production is bovine (cows). The digest is liberated by the activity of proteolytic enzymes like trypsin (6). Trypsin is an enzyme in the first section of the small intestine in animals. It starts the digestion of protein molecules by cutting long chains of amino acids into smaller pieces.

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There are several different types of protein hydrolysates, depending on the origin of the protein, (beef, casein - from bovine or caprine milk, fish, bovine collagen, etc.) or vegetable (mainly soy). Different forms of protein hydrolysis will generate different peptones. For example, pancreatic enzymes hydrolysis of bovine milk casein from the origin to tryptone or trypticase - peptone rich in tryptophan).

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Tryptone and trypticase are very similar, and in many cases the selection does not make any difference in terms of the performance of the medium. However, some microbiologists maintain that trypticase is better for the cultivation of more fastidious organisms.

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The second source is an enzymatic digest of soybean meal (derived from a species of legume native to East Asia; soybean meal is produced as a co-product of soybean oil extraction used in human and animal food production). This is sometimes referred to as ‘soytone’,often produced using a porcine-based enzyme for the digestion.

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The resultant product has a high tryptophan content and it is biologically low in carbohydrates. The product also provides nitrogen, vitamins, minerals, and amino acids: aspartic acid; methionine; threonine; isoleucine; serine; leucine; glutamic acid; tyrosine; proline; phenylalanine; glycine; histidine; alanine; lysine; cysteine; arginine; and valine. The medium is complex ?but also undefined, in that not all of the components or the specific proportions are known.

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Characteristics

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In a dehydrated form, the powder is homogeneous, free-flowing, and off-white to beige in color. Typically characteristics are (7):

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• Total nitrogen: ≥11.5%
• Amino nitrogen (AN): 4.4 – 5.0%
• Ash: ≤6.5%
• Moisture: ≤6.0%
• pH (2% Solution) 6.6 – 7.5

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Growth promotion

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Testing culture media to ensure that it is suitable is an important part of quality control. In addition to agar gel strength and pH, growth promotion demonstrates the ability of a medium to support the growth of a range of organisms often from low starting inoculum. With growth promotion testing, the common test organisms are:

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o?? Bacillus subtilis (ATCC 6633) – spore-forming Gram-positive rod.

o?? Candida albicans (ATCC 10231) – dimorphic fungus.

o?? Aspergillus brasilensis (ATCC 16404) – filamentous fungus.

o?? Staphylococcus aureus (ATCC 6538) – Gram-positive coccus.

o?? Pseudomonas aeruginosa (ATCC 9027) – Gram-negative rod.

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The use of these organisms ensures that testing meets the different pharmacopeia chapters which describe the use of the medium.

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Tim Sandle is the author of Pharmaceutical Microbiology: Essentials for Quality Assurance and Quality Control.

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References

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1.????? Leavitt JM, Naidorf IJ, Shugaevsky P.? The undetected anaerobe in endodontics; a sensitive medium for detection of both aerobes and anaerobes.? NY J Dentist 1955; 25: 377-82

2.????? United States Pharmacopeia, 18th ed.; United States Pharmacopeial Convention: Rockville, MD, 1970; pp 851-857

3.????? Kurzynski TA, Meise CK.? Evaluation of sulfamethoxazole-trimethoprim blood agar plates for recovery of group A streptococci from throat cultures.? J Clin Micro 1979; 9:189

4.????? MacFaddin, J.F.1985. Media for isolation-cultivation-identification-maintenance of medical bacteria, vol.1, Williams & Wilkins, Baltimore, Maryland USA

5.????? Downes,F.P.,and K. Ito (ed.). 2001. Compendium of methods for the microbiological examination of foods, 4th ed. American Public Health Association, Washington, D.C. USA.

6.????? Fraser, Dean; Powell, Richard (1950). The Kinetics of Trypsin Digestion, The Journal of Biological Chemistry. 187 (2): 803–820

7.????? Vanderzant, C., and D. F. Splittstoesser (eds.). 1992. Compendium of methods for the microbiological examination of food, 3rd ed. American Public Health Association, Washington, D.C.