This Standard Operating Procedure (SOP) outlines the steps for the preparation of Peptone Water, a common microbiological medium used for the cultivation and maintenance of a wide range of microorganisms.

Standard Operating Procedure (SOP) for Preparation of Peptone Water

PURPOSE

This procedure has been established to provide the guidelines for preparation of Peptone Water.

SCOPE

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

RESPONSIBILITIES’ & AUTHORITIES

Microbiologist is responsible for proper preparation of this culture media.
Quality Control Manager is responsible for implementation of this SOP.

REFERENCES & REQUIREMENTS

Manufacturers COA
USP general chapters

DEFINITIONS & ABBREVIATIONS

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.
Culture medium: It is a liquid or gel designed to support the growth of microorganisms.
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.)

PROCEDURE

Materials & Equipment’s

• Peptone Water, Merck, Oxide, HI media or equivalent.
• Distilled water, from water distillation plant.
• Media bottle, 250ml, 500ml, 1000ml, according to desired volume of preparation.
• Spatula, stainless steel or equivalent.
• Magnetic Stirrer, compatible to Stirring Hot Plate.
• Stirring Hot Plates
• Weighing Balance
• Autoclave

Safety Measures

• Dehydrated media are hygroscopic and are sensitive to moisture, heat and light.
• 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.
• Sealed, unopened Culture Media containers should be stored at room temperature below 25°C and near to 45% relative humidity.
• 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.
• 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.
• After use, make sure the container is tightly closed and return it to the designated storage area.
• Discard the medium if the powder is not free flowing, if the color has changed or if it appears abnormal in any way.
• Prepare the medium in a vessel about twice the final volume of the medium to allow adequate mixing.
• Open the culture medium container away from draughts and moisture.
• Avoid inhaling the powder and prolonged skin contact.
• Weigh the dehydrated culture media quickly, accurately and without creating ‘clouds of dust’, reclose the container as soon as possible.
• Always use freshly prepared distilled or deionized water for media preparation.
• Media containing agar should be heated to dissolve the agar before autoclaving.
• 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 Peptone Water from storage area and read the amount use for preparation of 1liter Peptone Water 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 2.55g dehydrated Peptone Water (Merck) for 1liter quickly and accurately according to your preparation without creating ‘clouds of dust’, reclose the container as soon as possible.
3. Check the pH of the solution using a pH meter. The optimal pH for Peptone Water is typically around 7.0.
4. 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.
5. Transfer the prepared Peptone Water solution into autoclavable containers or tubes, leaving some space at the top to allow for expansion during autoclaving.
6. Autoclave the solution at 121°C (250°F) and 15 psi for 15 minutes to sterilize the medium and eliminate any existing microorganisms.
7. Transfer it in media bottle of about twice the volume of your final volume of the medium to allow adequate mixing.
8. Add distilled water about half of your final volume and heat along with constant stirring on Stirring Hot Plate.
9. Remove the stirrer and add remaining distilled water when medium is dissolved completely.
10. Dispense 100-100ml in 100ml media bottles.

DOCUMENTS & RECORDS

• Weighing balance Log Book.
• Autoclave Log Book
• SOP for operation of Weighing Balance
• SOP for Operation of Autoclave