In Microbiology laboratory, Bacteria need to be cultured in the for various purposes like identification of the causative agent associated with the pathology, for research purposes, preparations of various vaccines as well as for the productions of certain proteins and enzymes, etc.
The universal culture medium used in the microbiology laboratories that supports the cultivations of almost all types of bacteria is the Trypticase Soy Agar (TSA) and the Trypticase Soy Broth Medium (TSB), also known as the Tryptic Soy Broth or Tryptone Soya Broth. This media provides all the basic components that are required by the bacterial cell for the rapid growth in the laboratory.
In this article, I’m gonna explain the procedure of preparing Trypticase Soy Broth (TSB) medium in Laboratory
The Tryptone Soy Broth (TSB) 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.
However, there are various types of media available that are based on the requirements of particular bacteria but the Trypticase Soy Broth (TSB) is the best artificial medium that fulfills the basic requirements of almost all types of bacteria and gives a satisfactory and rapid growth of most organisms.
PRINCIPLE OF TRYPTICASE SOY BROTH MEDIUM (TSB)
The Trypticase Soy Broth medium (TSB), however not commonly employed in routinely in laboratories, is an excellent medium to obtain the discrete colonies of the bacteria present in the specimen or to get the information about cultural characteristics of bacteria in a liquid medium, colony morphology and patterns of growth etc. This Liquid medium, used in bacteriology laboratory, constitutes the 5 essential components which are described below in detail –
⇒ Pancreatic digest of casein – The Pancreatic Digest of Casein is a rich source of Nutrients, Vitamins, and Nitrogenous substances that support the rapid growth of the bacterial cell.
⇒ Papaic Digest of Soybean – Similar to Pancreatic Digest of Casein, the Papaic Digest of Soybean is the Rich source of protein or peptides and nutrients that support the rapid growth of the bacterial cell.
⇒ Dextrose – The Dextrose present in the Trypticase Soy Broth (TSB) Medium provides a rich source of carbohydrate for the rapid growth of the Bacterial Cell.
⇒ Dipotassium hydrogen phosphate – It acts as the buffer in the medium that helps to maintain the stable pH of the Trypticase Soy Broth (TSB) medium.
⇒ Sodium Chloride – It maintains the osmotic pressure in the Broth medium so that the movement of molecules takes place in and out of the bacterial cell. It must be present in right proportion otherwise it will lead to the lysis of the bacterial cell.
The final pH of the solutions is adjusted to 7.1 – 7.5 preferably the 7.3 at 25 °C. The above-mentioned components can be modified by adding the various substances in a variety of ways as per the results required so that the rapid and satisfactory growth of the bacterial cell takes place.
The most commonly used modification of TSB medium is the Trypticase Soy Agar (TSA) medium which contains all the above contents and besides that, it contains Agar-Agar as a solidifying agent. The TSA is commonly used for Blood Agar medium containing TSA as a base with added Sheep’s Blood.
The methodology explained in this article is based on HiMedia Labs Tryptic Soya Broth (TSB) Medium which you can check here
REQUIREMENTS FOR THE PREPARATION OF TRYPTICASE SOY BROTH MEDIUM (TSB)
- Sterile Conical Flask / Erlenmeyer Flask
- Pancreatic digest of casein
- Papaic digest of soybean meal
- Dextrose (Glucose)
- Dipotassium hydrogen phosphate
- Sodium Chloride
- Measuring Cylinder
- 1N HCl
- 1N NaOH
- pH Strip
- Weighing Scale
- Distilled Water
- Butter Paper
PROCEDURE FOR THE PREPARATION OF TRYPTICASE SOY BROTH MEDIUM (TSB)
⇒ Weigh the quantity of Peptone Pancreatic digest of casein, Papaic digest of soybean meal, Dextrose (Glucose), Dipotassium hydrogen phosphate and Sodium Chloride using the weighing scale for 1000 ml of Trypticase Soy Broth medium as follows:
COMPONENTS QUANTITY (in grams)
Pancreatic digest of casein 17.0
Papaic digest of soybean meal 3.0
Dextrose (Glucose) 2.5
Dipotassium hydrogen phosphate 2.5
Sodium Chloride 5.0
Put the butter paper on the weighing scale and transfer the required quantity of Pancreatic digest of casein on to the paper using the spatula. Repeat the step to obtain the required quantity of Papaic digest of soybean meal, dextrose, Dipotassium Hydrogen Phosphate, and sodium chloride powder.
Note: Use the separate piece of butter paper to avoid the errors in measurements.
Alternatively, You can use the commercially available Trypticase Soy Broth (TSB) media powders and simply weigh the mixture of content as prescribed on the carton or box of the manufacturer. As per HiMedia, its 30 grams for 1000 ml medium.
⇒ Take a clean and dry Conical Flask/ Erlenmeyer flask.
Note: The size of the flask should be at least 1.5 times larger than the quantity of media you are preparing, for e.g. use 1500 or 2000 ml flask to prepare 1000ml of the solid medium.
⇒ 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.
⇒ Mix well the content and Heat it with continuous agitation to dissolve the constituents.
⇒ Now add more distilled water to the medium and make the volume 1000 ml.
⇒ 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.
⇒ Now Pour about 5-10 ml of the broth medium in Test tubes and apply the Non-absorbent cotton plug to all the tubes.
⇒ Autoclave the content at 121 °C and 15 psi pressure for 15 minutes.
⇒ Allow the content to cool down and then use the prepared broth tubes to inoculate the specimen to be cultured under a strict aseptic atmosphere (preferably in Laminar Air Flow) and then place in the incubator at optimum temperature.
PRECAUTIONS TO BE TAKEN WHILE PREPARING TRYPTICASE SOY BROTH (TSB) MEDIUM IN LABORATORY
⇒ Carefully measure the quantity of Pancreatic digest of casein, Papaic digest of soybean meal, dextrose, Dipotassium Hydrogen Phosphate, and sodium chloride powder as per the quantity of medium to be prepared.
⇒ Maintain the pH of the medium carefully. A single extra drop can change the pH of the solution.
⇒ Store the Medium at low temperature in dust and contamination free environment for later use.
⇒ Acid and Alkali are corrosive to the skin, handle with care.
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