This Standard Operating Procedure for Preparation of Tryptic Soya Broth outlines the steps for the preparation of Tryptic Soya Broth, a commonly used medium in microbiology for the cultivation of a wide variety of microorganisms.

PURPOSE

This Standard Operating Procedure for Preparation of Tryptic Soya Broth has been established to provide the guidelines for preparation of Tryptic Soy Broth.

SCOPE

This procedure is applicable to microbiological section of Quality Control Lab.

RESPONSIBILITIES & AUTHORITIES

1. Microbiologist is responsible for proper preparation of Standard Operating Procedure for Preparation of Tryptic Soya Broth culture media.
2. Quality Control Manager is responsible for implementation of this Standard Operating Procedure for Preparation of Tryptic Soya Broth

REFERENCES & REQUIREMENTS

1. Manufacturer COA
2. USP general chapters

DEFINITION’S & ABBREVIATION’S

1. Dehydrated culture media: It is a master mix of different nutrients and minerals which are essential for the growth of micro-organisms in form of powder or granules.
2. Culture medium: It is a liquid or gel designed to support the growth of microorganisms.
3. Sterilization: It is a term referring to any process that eliminates (removes) or kills all forms of life; including transmissible agents (such as fungi, bacteria, viruses, spore forms, etc.)
4. Bacteria and fungi are grown on or in microbiological media of various types. The medium that is used to culture the microorganism depends on the microorganism that one is trying to isolate or identify. Different nutrients may be added to the medium, making it higher in protein or in sugar. Various pH indicators are often added for differentiation of microbes based on their biochemical reactions: the indicators may turn one color when slightly acidic, another color when slightly basic. Other added ingredients may be growth factors, NaCl, and pH buffers which keep the medium from straying too far from neutral as the microbes metabolize.

PROCEDURE

Materials & Equipment’s

1. Tryptic Soy Broth, Merck or equivalent.
2. Distilled water, from Gallop water distillation plant.
3. Media bottle, 250ml, 500ml, 1000ml, according to desired volume of preparation.
4. Spatula, stainless steel or equivalent.
5. Magnetic Stirrer, compatible to Stirring Hot Plate.
6. Stirring Hot Plate
7. Weighing Balance
8. Autoclave

Safety Measures

1. Dehydrated media are hygroscopic and are sensitive to moisture, heat and light.
2. They are adversely affected by drastic changes in temperature e.g. hot/cold cycling temperatures which may occur between day and night laboratory temperatures in winter.
3. Sealed, unopened Culture Media containers should be stored at room temperature below 25°C and near to 45% relative humidity.
4. Opened containers should have the cap or lid carefully and securely replaced. It is important that opened containers are stored in a dry atmosphere at room temperature.
5. Opened containers of dehydrated powders will be affected by high humidity, so an adjacent cold room or an adequate storage cupboard is preferable for storage of dehydrated culture media.
6. After use, make sure the container is tightly closed and return it to the designated storage area.
7. Discard the medium if the powder is not free flowing, if the color has changed or if it appears abnormal in any way.
8. Prepare the medium in a vessel about twice the final volume of the medium to allow adequate mixing.
9. Open the culture medium container away from draughts and moisture.
10. Avoid inhaling the powder and prolonged skin contact.
11. Weigh the dehydrated culture media quickly, accurately and without creating ‘clouds of dust’, reclose the container as soon as possible.
12. Always use freshly prepared distilled or deionized water for media preparation.
13. Media containing agar should be heated to dissolve the agar before autoclaving.
14. All prepared culture media and their components should be stored away from light and exposure to direct sunlight should be avoided at all times.

Procedure

1. Pick the container of dehydrated Tryptic Soy Broth from storage area and read the amount use for preparation of 1liter Tryptic Soy Broth which is mentioned on the label of the container and calculate the amount which will be used for your desired volume of preparation using unitary method.
2. Weigh 30.00g dehydrated Tryptic Soy Broth (Merck) for 1liter quickly and accurately according to your preparation without creating ‘clouds of dust’, reclose the container as soon as possible.
3. Gradually add the required volume of distilled water while stirring continuously to avoid lumps formation.
4. Check the pH of the solution using a pH meter. The optimal pH range for Tryptic Soya Broth is around 7.2 to 7.4.
5. If necessary, adjust the pH by adding small amounts of 1N sodium hydroxide (NaOH) or 1N hydrochloric acid (HCl) while stirring and checking the pH until it falls within the desired range.
6. Transfer the prepared Tryptic Soya Broth solution into autoclavable containers or tubes, leaving some space at the top to allow for expansion during autoclaving.
7. Autoclave the solution at 121°C (250°F) and 15 psi for 15 minutes to sterilize the medium and kill any existing microorganisms.
8. After autoclaving, allow the Tryptic Soya Broth to cool down to room temperature in a clean and controlled environment.
9. Once cooled, store the sterile Tryptic Soya Broth in a clean, dry, and sealed container.
10. Label the containers with the preparation date, expiration date (typically 1 month from the preparation date), and batch/lot number.
11. Transfer it in media bottle of about twice the volume of your final volume of the medium to allow adequate mixing.
12. Add distilled water about half of your final volume and heat along with constant stirring on Stirring Hot Plate.
13. Remove the stirrer and add remaining distilled water when medium is dissolved completely.
14. Dispense 100-100ml in 250ml media bottles.
15. Always wear appropriate personal protective equipment (PPE) such as lab coats, gloves, and safety goggles while handling chemicals and microbiological media.
16. Follow standard laboratory safety protocols and procedures.

DOCUMENTS/RECORDS

1. Weighing balance Log Book.
2. Autoclave Log Book
3. SOP for operation of Weighing Balance
4. Standard Operating Procedure for Preparation of Tryptic Soya Broth of Autoclave
5. Document the preparation process, including the weights, volumes, pH adjustments (if any), autoclaving details, and storage information.
6. Maintain a record of the expiration date and batch/lot number for traceability purposes.