Tryptic soy broth rationale, preparation and uses

3221
Basil Manning

The tryptic soy broth It is a liquid culture medium, highly nutritious and non-selective. Due to its great versatility, it is one of the most widely used liquid culture media in the microbiology laboratory..

It is also known by the name of trypticase soy broth or casein-soy digested broth, whose abbreviation is TSB for its acronym in English. Tryptic am Broth or CST for its acronym in Spanish. Its uses are very varied due to its composition. It is composed of triptein, soy peptone, sodium chloride, dipotassium phosphate and glucose.

Trypticasein soy broth seeded with a strain of Pseudomonas aeruginosa ATCC 27853 where pigment production is observed. Source: Photo taken by the author MSc. Marielsa gil.

It is capable of reproducing clinically important pathogenic bacteria, including those that are nutritionally demanding and anaerobic bacteria. Some opportunistic and contaminating fungi can also develop in this environment.

Due to its high nutritional power, it has high sensitivity to detect microbial contamination, for this reason it was chosen by the USDA Animal and Plant Health Inspection Service for the microbiological analysis of vaccines..

Likewise, trypticasein soy broth meets the requirements of the different pharmacopoeias (European EP, Japanese JP and North American USP) for the microbiological study of products at an industrial level, such as cosmetics and food..

On the other hand, it is worth mentioning that despite its great utility this medium is relatively inexpensive, making it affordable for most microbiology laboratories. It is also very easy to prepare.

Article index

  • 1 Rationale
  • 2 Preparation
    • 2.1 -Tripticasein soy broth
    • 2.2 -Variants of trypticasein soy broth
  • 3 Use
  • 4 Sown
  • 5 Quality control
  • 6 Limitations
  • 7 References

Basis

Triptein, peptone and glucose provide essential nutritional properties to make it an ideal medium for rapid microbial growth.

In approximately 6 to 8 hours of incubation, growth can already be seen in most microorganisms. However, there are slow-growing strains that can take days to grow..

Sodium chloride and dipotassium phosphate act as osmotic balance and pH regulator respectively. The presence of growth is evidenced by the appearance of turbidity in the medium; if there is no growth the medium remains translucent.

Due to its light color it is possible to observe the production of pigments, such as the one shown in the image located at the beginning of the article, which corresponds to the pigment produced by Pseudomonas aeruginosa. 

Preparation

-Trypticasein soy broth

To prepare trypticase soy broth, 30 g of the dehydrated commercial medium must be weighed on a digital scale. Then it is dissolved in a liter of distilled water contained in a flask.

The mixture is left to rest for 5 minutes and later it is taken to a heat source to help dissolve the medium. It should be stirred frequently while boiling for 1 minute.

Once dissolved, it is distributed into tubes of the appropriate size as needed. Tubes with cotton plugs or with Bakelite caps can be used. Subsequently, the tubes are sterilized with the medium in the autoclave at 121 ° C for 15 minutes..

The pH of the medium must remain at 7.3 ± 0.2

It should be noted that the color of the dehydrated culture medium is light beige and should be stored between 10 to 35 ° C, in a dry place. While the prepared broth is light amber in color and should be stored in a refrigerator (2 to 8 ° C).

-Trypticasein Soy Broth Variants

Modified trypticasein soy broth can be prepared by adding bile salts and novobiocin in order to make it selective for the isolation of E. coli. Another option for the same purpose is to prepare trypticase soy broth supplemented with vancomycin, cefixime and tellurite (2.5 µg / ml)..

On the other hand, more glucose (0.25%) can be added to tryptic soy broth when the objective is to stimulate the formation of biofilms..

Use

It is nutritious enough to allow the growth of fastidious or fastidious bacteria such as Streptococcus pneumoniae, Streptococcus sp and Brucella sp, no need to supplement with blood or serum.

Likewise, some fungi can develop in this broth, such as Candida albicans complex, Aspergillus sp and Histoplasma capsulatum.

Furthermore, this medium under anaerobic conditions is ideal for recovering bacteria belonging to the genus Clostridium, as well as non-sporulated anaerobic bacteria of clinical importance..

If 6.5% sodium chloride is added, it can be used for the growth of Enterococcus and other Group D Streptococcus..

At the research level, it has been very useful in various protocols, especially in the study of biofilm or biofilm-forming bacteria. It is also used to prepare the bacterial suspension at 0.5% of Mac Farland necessary to carry out the antibiogram by the Kirby and Bauer method..

In this case, 3 to 5 colonies of similar appearance are taken and emulsified in 4-5 ml of trypticasein soy broth. It is then incubated for 2 to 6 hours at 35-37 ° C and subsequently adjusted to the desired concentration using sterile saline. Trypticasein soy broths should not be used from 18 to 24 hours of incubation.

Sown

The sample can be seeded directly or pure colonies taken from selective media can be subcultured. The inoculum should be small so as not to cloud the medium before incubation.

Normally it is incubated at 37 ° C in aerobiosis for 24 hours, but these conditions may vary depending on the microorganism being sought. It can also be incubated under anaerobic conditions at 37 ° C for several days if necessary. For example, in fastidious or slow-growing microorganisms it can be incubated for up to 7 days.

In the microbiological analysis of pharmaceutical substances - such as vaccines - the protocols are stricter. In these cases, the broth without growth is not discarded until it reaches 14 days of continuous incubation..

QA

From each batch prepared, 1 or 2 uninoculated tubes should be incubated to demonstrate their sterility. Must remain unchanged.

Known strains can also be planted to assess their behavior. Among the strains that can be used are:

Aspergillus brasiliensis ATCC 1604, Candida albicans ATCC 10231, Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 6538 or 25923, Escherichia coli ATCC 8739,  Streptococcus pyogenes ATCC 19615, Streptococcus pneumoniae ATCC 6305, Pseudomonas aeruginosa ATCC 9027, Salmonella typhimurium ATCC 14028.

In all cases, growth must be satisfactory under the conditions of the appropriate atmosphere and temperature for each microorganism..

Limitations

-The fermentation of glucose causes the pH of the medium to decrease due to the production of acids. This can be unfavorable for the survival of some microorganisms sensitive to acidity..

-It is not recommended for the maintenance of strains, because in addition to acidity, bacteria deplete nutrients after a few days with the consequent accumulation of toxic substances that make the environment inhospitable.

-You must work taking care of all the sterility protocols, since the broths are easily contaminated.

-After preparing trypticasein soy broths, do not try to transfer broth to another sterile tube, because this type of maneuver is very vulnerable to contamination.

References

  1. Cona E. Conditions for a good susceptibility study by agar diffusion test. Rev. chil. infectol. 2002; 19 (2): 77-81. Available at: scielo.org
  2. Britannia Laboratory. Triptein soy broth. 2015.Available at: britanialab.com
  3. MCD Laboratory. Trypticasein Soy Broth. Available at: electronic-systems.com
  4. Neogen Laboratory. Triptych Soy Broth. Available at: foodsafety.neogen.com
  5. Forbes B, Sahm D, Weissfeld A. (2009). Bailey & Scott Microbiological Diagnosis. 12 ed. Editorial Panamericana S.A. Argentina.
  6. Rojas T, Vásquez Y, Reyes D, Martínez C, Medina L. Evaluation of the magnetic immunoseparation technique for recovery of Escherichia coli O157: H7 in heavy creams. ALAN. 2006; 56 (3): 257-264. Available at: scielo.org.ve
  7. Gil M, Merchán K, Quevedo G, Sánchez A, Nicita G, Rojas T, Sánchez J, Finol M. Biofilm formation in isolates of Staphylococcus aureus according to antimicrobial susceptibility and clinical origin. Vitae. 2015; 62 (1): 1-8. Available at: saber.ucv.ve
  8. Narváez-Bravo C, Carruyo-Núñez G, Moreno M, Rodas-González A, Hoet A, Wittum T. Isolation of Escherichia coli O157: H7 in Double Purpose Cattle Faeces Samples from Miranda Municipality, Zulia State, Venezuela. Rev. Cient. (Maracaibo), 2007; 17 (3): 239-245. Available at: scielo.org

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