Get the latest updates from us for free
Android App

SOP for Preparation of Culture Inoculum


Standard operating procedure to prepare the culture inoculum of known concentration for analysis of Raw material and Finished products.

1.0  PURPOSE

       To lay down procedure for preparation of culture inoculum.

2.0  SCOPE

       This SOP is applicable for the preparation of culture inoculum of known density being used for various testing purpose.

3.0  RESPONSIBILITY

       Trained Microbiologist is responsible to prepare culture inoculum as per SOP.

4.0  ACCOUNTIBILITY

       Head of Department

5.0  PROCEDURE

5.1  General Instructions

5.1.1  Perform all activities under bio safety cabinet.

5.2  Procedure

5.2.1  Take working culture slants/tubes of required organisms and place in the workbench.
5.2.2  Take required number of media plates or tubes and place in the workbench.
5.2.3  Label the media plates / tubes with the name of organism and date.
5.2.4  Inoculate a loop full of inoculum from working cultures of into appropriately labeled soybean casein digest medium tubes. For anaerobic culture, use cooked meat medium instead of soybean casein digest medium. Incubate at the following conditions:
•  30 – 35°C for 24 hours for aerobic bacterial culture.
•  30 – 35°C for 24 to 48 hours for anaerobic bacteria culture.
•  20 – 25°C for 48 to 72 hours for yeast culture.
•  30 – 35°C for 120 hours for mold culture.
5.2.5  After completion of incubation, check the tubes for evidence of contamination.
5.2.6  Assume that the broth culture has 109 organisms per ml in case of aerobic bacteria and 108 organism/ml in case of anaerobic bacteria/yeast and mold.
5.2.7  Prepare set of 8 dilution tubes (or up to appropriate dilution to get 10-100 CFU/ml) with 9 ml 0.1% peptone saline for each culture and label them with culture name and dilution number in the following order – 10-1 to 10-8.
5.2.8  Take 1 ml of each culture and add to dilution tube labeled 10-1 and prepare serial dilutions to arrive at the dilution that may contain approximately 10-100 CFU/ml. Ensure that each dilution is thoroughly mixed during diluting process. Proceed for enumeration.
5.2.9  Enumeration by Pour Plate method.
5.2.9.1  Take eight sterile 90 mm plates per culture and label them with culture name, dilution number (last four dilutions in duplicate), date and initials.
5.2.9.2  Inoculate 1.0 ml of last four dilutions (in duplicate) on to appropriately labeled plates.
5.2.9.3  Add approximately 20 ml of sterile molten Soybean Casein Digest Agar cooled to approximately 45°C to each plate and swirl gently for even mixing of culture suspension.
5.2.10  Enumeration by Spread Plate method.
5.2.10.1  Take eight Soybean Casein Digest Agar plates per culture and label them with culture name, dilution number (last four dilutions in duplicate), date and initials.
5.2.10.2  Inoculate 0.1 ml of last four dilutions (in duplicate) on to appropriately labeled plates.
5.2.10.3  Perform spread plating with sterile spreaders.
5.2.11  Incubate the plates at following conditions
•  30 – 35°C for 24 to 48 hours for aerobic bacterial culture.
•  30 – 35°C for 24 to 48 hours for anaerobic bacteria culture.
•  20 – 25°C for 48 to 72 hours for yeast culture.
•  30 – 35°C for 72 to 120 hours for mold culture.
5.2.12  Store the dilutions at 2- 8°C.
5.2.13  After completion of incubation, count the colonies in plates and select the dilution with 10 –100 CFU/ml and its previous dilution (10-100 CFU/0.1ml), label and store at 2 – 8°C.
5.2.14  If the dilution stock of 10 – 100 CFU is exhausted then prepare fresh stock from previous dilution stored by adding 1 ml in 9 ml of sterile peptone saline. If required 0.1 ml of previous dilution can be directly used to inoculate 10 –100 CFU, for example in GPT of Agar Media by spread plate method.
5.2.15  Use this dilution for one week, prepare and standardize inoculum every week.
5.2.16  Record the details of inoculum preparation in format.

Related: Incubation Conditions for Common Media used for Fungus and Bacteria

6.0  ABBREVIATIONS

6.1  SOP – Standard operating procedure
6.2  CFU – Colony forming unit
Ankur Choudhary is India's first professional pharmaceutical blogger, author and founder of Pharmaceutical Guidelines, a widely-read pharmaceutical blog since 2008. Sign-up for the free email updates for your daily dose of pharmaceutical tips.
Email: .moc.enilediugamrahp@ofni Need Help: Ask Question


Be the first to comment!

Post a Comment