Maintain and preserve of standard culture and its SOP

Maintain and preserve of standard culture; Purpose

To maintain & preserve the standard culture in order to use in specific microbiology test.

Maintain and preserve of standard culture;Scope

This SOP applies to preserve & maintain stock standard culture in Microbiology Laboratory of at XX Pharmaceuticals Ltd.

Definitions

Standard Culture

Standard culture is a specific microorganism of a specific strain that is recognized by BP/USP/Eur. Ph.  and certified by  ATCC/NCTC or any other standard culture bank. That culture is used in the different microbiological test such as Growth Promotion Test, Biological Assay of Antibiotics, Sterilizer Validation, Antimicrobial Preservative Effectiveness Test, Microbial Count suitability Test & Sterility Test Validation.

• ATCC: American Type Culture Collection
• NCTC: National Type Culture Collection

Responsibilities

Executive/ Senior Executive, Microbiology

• Preparation of Culture Media, sub-culture, preservation and record keeping.
• Follow the instructions of this procedure correctly.

Manager, Microbiology

• Ensure that this procedure is kept up to date.
• Ensure culture maintenance activities & proper documentation
• Ensure appropriate personnel from the section are trained on this procedure.
• Confirm that SOP is technically sound & reflects the required working practices.

Head of Quality Assurance

Take initiative to approval of this SOP

Procedure:

Instructions

• Standard microorganisms are usually non-pathogenic but those are unscrupulous pathogen. So before handling it, wear protective items such as sterile latex free gloves, laboratory coat and eye protection (if necessary).
• Move always gently. Don’t move vigorously into the test area.
• Disinfect the whole surface with the help of 70% IPA or any others suitable disinfectants. After completion of sub-culture or transfer of pellets.
• Take away all used items those are directly contacted with standard micro-organisms and Leave it at designated containers safely.
• Confirm the waste container is tightly capped until autoclaving.
• Never touch the apparatus directly in open hands those are used.
• Make sure that all personal ornaments, cell phone are left before entrance into the test room, to prevent unwanted contamination.

Sterilization of Apparatus & Glassware:

• Sterilize all glassware plus Latin Square Plate and others heat stable apparatus at 200⁰C for 60 minutes in hot air oven using a validated process.
• Use the glassware when the temperature reduce to 40⁰

Preparation of Culture Media:

• Select media & diluents as per instruction of BP or USP.
• Prepare required amount of Culture media of CSDA [Casein Soybean Digest Agar] & CSDB [Casein Soybean Digest Broth] and SDA [Sabouraud Dextrose Agar] as per indication by manufacturer instructions.
• Bring to boil to dissolve it completely.
• Distribute 10 ml of the media into each screw capped test tube.
• Sterilize at 121⁰C for 15 minutes.
• Cool media approximately to (45 to 50)⁰
• Allow to solidify agar media to prepare slant.
• Transfer broth media flasks into the test room.
• Dry surface of agar slant keeping into Laminar Air flow or Bio-safety Cabinet.
• Incubate sterilized CSDA [Casein Soyabean Digest Agar]/broth at (30 to 35)⁰C for 48 hours and SDA [Sabauroud Dextrose Agar] for 5 days at (22 to 25)⁰C for checking sterility of the media.
• After incubation observe each test tube for growth. If no growth found, the media is suitable for use.
• Store media at (2 to 8)⁰C into refrigerator until it use.

Preparation of Standard Microorganism:

Test Conditions

[] Disinfectant the surface of all equipments including LAF or BSC.
[] Rub hands with the help of 70% IPA.
[] Always maintain aseptic condition during handling of standard microorganisms.

Inoculation Technique

• Opening of Standard Culture vial/ampoule
• Read carefully label’s instructions of standard culture vial/ampoule.
• Aseptically break the ampoule/vial under Laminar Air Flow.
• Remove pellets from vial/ampoule aseptically as per instruction of the label.
• Tight the container for the next time use after removing pellets.

Inoculation of Standard Microorganisms:

• Label on the each tube with the name of standard culture.
• Reconstitute standard culture pellets as per instructions of manufacturer.
• Aseptically Transfer 1 or 2 pellets of dehydrated culture of bacteria and fungus in the test tubes containing CSB.
• Incubate test tubes containing bacterial culture at (30 to 35)⁰C for 3 days and the tubes containing fungi culture at (20 to 25)⁰C for 5 days. This will serve as mother culture.
• After incubation, observe the growth of standard culture that should be turbid or settle growth.
• Transfer & streak from the broth culture on surface of agar slant.
• Incubate test tubes containing CSA at (30 to 35)⁰C for 3 days & the SDA tubes containing fungi culture at 20 to 25⁰C for 5 days.
• Observe good growth, then preserve it at (2 to 8)⁰C in refrigerator with proper labeling.
• Discard previous culture by autoclaving at 121⁰C for 30 minutes.
• Subculture microorganism to freshly prepared culture media once in a month.

Preparation of spore suspension:

• Transfer sterilized media under Laminar Air Flow or Bio-safety Cabinet.
• Keep plate for incubation at 35⁰C for 7 days.
• After incubation period take out culture with aid of sterilized glass beads & pre-sterilized diluents (0.001 g/L containing Manganese sulfate) by rotating plate.
• Pour culture suspension along with few of those glass beads in a 100ml flask containing 50ml of sterilized diluents.
• Heat culture suspension at 70⁰C for 30 minutes or 80⁰C for 10 minutes in a water bath.
• Cool suspension & then keep inside a refrigerator not exceeding at 4⁰C
• Don’t use spore suspension more than 60 days.

Preparation of standard culture suspension (Vegetative form):

• Prepare culture suspension by regular subculture on Nutrient Agar slant or Sabouraud Dextrose Agar.
• Before using a culture maintained in a slant, subculture the organism in another slant containing a specified medium.
• Keep the slant for incubation at (30 to 35)⁰C for bacteria for (24 to 30) hours and at (20 to 25)⁰C for fungi for (3 to 5) days.
• Store slant in the refrigerator not exceeding at 4⁰C if not used immediately.
• Don’t use this suspension at more than 7 days.

Stock Maintenance of Standard Microorganisms:

• Check expiry date before use of standard microorganisms.
• Don’t use if the expiry date is exceeded & discard it after autoclaving.
• Raise requisition for standard culture before exceed of expiry date.

 Report Preparation

Maintain Register of standard culture maintenance Record, Annexure-I.

Download Annexure:

Download the annexure here: Standard Culture Maintenance Record