SOP of Reference cultures Maintenance and usage

SOP of Reference cultures Maintenance and usage

• Objective

SOP on Maintenance and usage of Reference cultures.

•  Scope

This SOP is applicable for Maintenance and usage of Reference cultures. Of (Pharmaceutical Company Name).

• Responsibility
  • Chemist or above of QC laboratory.
  • Head – Microbiology Section.

• Accountability
  • Head – Quality Control.

• Abbreviations and Definitions

SOP                             : Standard operating procedure

No.                              : Number

QC                              : Quality Control

QA                              : Quality Assurance

BI                                : Biological Indicator

• Procedure
  • Procurement of Lyophilized Cultures
    • Reference Cultures can be procured from following suppliers:
      • ATCC: American Type Culture Collection, 10801, University Bivd, Manassas, VA201110, USA.
      • NCIM: National Collection of Industrial Microorganism, National Chemical Laboratory, Pune 411008 INDIA.
      • MTCC: IMTECH (Institute of Microbial Technology) Sector 39-A, Chandigarh INDIA.
      • NCTC: National Collection of Type Culture, Central Public Health Laboratory, 61 Colindale Avenue, London NW9 5HT UK.
    • Opening of Lyophilized ampoule:
      • Disinfect the outside of ampoule with 70% I.P.A
      • Score the ampoule briskly with a sharp file about one inch from the top.
      • Disinfect the ampoule at the scored area with 70% I.P.A and flame.
      • Wrap a sterile gauge pad around the scored area and break the ampoule (using the thumb and forefinger) with a sharp snapping motion away from the body.
      • Flame the open end of the ampoule and proceed as below.
    • Reconstitution and maintenance procedure for Lyophilized Culture
      • Aerobic bacteria:
        • To the Lyophilized culture aseptically add 0.3-0.4ml of sterile Soya bean Casein Digest Medium (SCDM) with sterile pipette.
        • Transfer the reconstituted culture to the tube containing 5ml SCDM.
        • Transfer this culture to 7 sterile Soya bean Casein Digest Agar (SCDA) medium slants.This is transfer no. 1(T1) from the mother culture.
        • Incubate the slants at 32.5±2.5 ºC for 24-48 hours.
        • Simultaneously perform the purity check for this initial culture by mini API using the procedure given pack insert for specific type of organism.
        • Among the 7 slants 6 slants will serve as T1- Primary stock culture and 1 slant will serve as stand by for emergency.
        • Assign individual identification number to all 7, as T1SCA, T1SCB, T1SCC, T1SCD, T1SCE, T1SCF, and T1SCG. Give the expiry date as 1 year from the date of availability of these slants.
        • Transfer the culture from T1SCA to 2 sterile Soya bean Casein Digest Agar (SCDA) medium Slants.
        • Incubate the slants at 32.5±2.5 ºC for 24-48 hours.
        • One of the 2 Slants will serve as T2 (Transfer No.1) Stock Culture (T2SCA) and other will serve as T2WCA.
        • Give the expiry date as 15 days from the date of availability of these slants.
        • Use the working culture slant for culture suspension preparation for routine use.
        • Use the stock culture slant for next transfer after 15 days.
        • Label the culture slant with following details.

Culture Label

Name Culture Type: Stock/Working Transfer No.: Strain No. : Equivalent ATCC No.: I.D. No. Due/ Expiry Date:

• Preserve all Slants in refrigerator At 2 to 8 ºC
• Record the details as per annexure No. Format No..
• By the end of 15th day of working culture, subculture the T2SCA and prepare one working culture and one stock culture slant. This is transfer 3(T3).
• Assign identification to these culture tubes as T3WCA and T3SCA for working and stock culture tubes respectively. Given 15 days due for next subculture.
• Likewise continue this sub culturing series (from T1SCA) up to T5 (Transfer 5).
• Use T1SCB for next sub culturing and continue up to T5.
• Use T1SCG as a stand by (in emergency).
• Records the details of stock culture and working culture details as per Annexure No.Format NO. ) and Annexure No. Format NO.
• Refer to Sub-culturing schematic representation for further details.
• Perform the purity check test during routine sub-culturing of the culture as per the procedure given in purity checks.

• Anaerobic Bacteria
  • Proceed as for the procedure given for aerobic bacteria with the following changes.
  • Use reinforced clostridial Agar (RCA) medium species for slant preparation instead of SCDA medium.
  • Incubate the culture slants under anaerobic condition in anaerobic jar.
  • Preserve the cultures slants by adding sterile mineral oil on the top of the slant.
• Yeasts
  • Proceed as for the procedure given for aerobic bacteria with the following changes.
  • Incubate the slants at 22.5+/- 2.5˚C for 72 hours.
• Molds
  • Proceed as for the procedure given for aerobic bacteria with the following changes.
  • Use sterilized purified water for reconstitution of lyophilized cultures.
  • Incubate the slants at 22.5+/- 2.5˚C for 5-7 days or until sporulation.
  • Perform the purity check by Lactophenol cotton blue staining for initial as well as during routine sub culturing.
• Purity checks
  • Purity checks for E. coli.
  • Gram’s staining:
• Prepare a smear with culture, on a clean glass slide.
• Heat fix and perform the Gram’s staining.
• coli shows Gram –ve, rod shaped cells.
  • Cultural Tests:
• Streak the culture on the surface of Levine Eosin-Methylene Blue Agar Medium contained in a Petridis.
• Incubate the slants at 32.5+/- 2.5˚C for 24 to 48 hours.
• Observe the plate after incubation.
• Colonies exhibits both characteristics metallic sheen under reflected light and blue black appearances under transmitted light.
  • Biochemical Tests:
• Inoculate a loopful of culture in 10ml peptone water and incubate at 30-35˚C for 24 hours and add Kovacs Reagent.
• Red coloured ring formation on the top layer indicates Indole positive reaction.
• coli is Indole positive.
  • Purity checks for Salmonella
    • Gram’s staining
  • Prepare a smear with culture, on a clean glass slide.
  • Heat fix and perform the Gram’s staining.
  • coli shows Gram –ve, rod shaped cells.
    • Cultural Tests
  • Streak the culture on the surface of xylose lysine deoxycholate Agar Medium contained in a Petridis.
  • Incubate the slants at 32.5+/- 2.5˚C for 24 to 48 hours.
  • Observe the plate after incubation.
  • The pure culture of salmonella exhibits well-developed, red colonies, with or without black centers.
    • Biochemical Tests:
  • By means of an inoculating wire transfer the culture to a butt slants tube of Triple Sugar Iron Agar Medium by first streaking the surface of the slants and then stabbing the wire well beneath the surface.
  • Incubate the slants at 32.5+/- 2.5˚C for 24 hours.
  • Blackening occurs on the surface of the slants and vacuole formation. Occurs in the media due to formation of Hydrogen Sulphide gas.
    • Purity checks for Pseudomonas aeruginosa
      • Gram’s staining
    • Prepare a smear with culture, on a clean glass slide.
    • Heat fix and perform the Gram’s staining.
    • Pseudomonas aeruginosa shows Gram –ve, rod shaped cells.
      • Cultural Tests
    • Streak the culture on the surface of xylose lysine deoxycholate Agar Medium contained in a Petridis.
    • Incubate the slants at 32.5+/- 2.5˚C for 24 to 48 hours.
    • Observe the plate after incubation.
    • The pure culture of Pseudomonas aeruginosa exhibits greenish colonies.

• Biochemical Tests:

• By means of an inoculating wire transfer a colonies on the oxidase dise.
• Development of pink colour, changing to purple indicates positive test.
  • Purity checks for Staphylococcus aureus.
    • Gram’s staining
  • Prepare a smear with culture, on a clean glass slide.
  • Heat fix and perform the Gram’s staining.
  • Staphylococcus aureus shows Gram +e, cocci (In clusters).
    • Cultural Tests
  • Streak the culture on the surface of Mannitol salt agar in a Petridis.
  • Incubate the slants at 32.5+/- 2.5˚C for 24 to 48 hours.
  • Observe the plate after incubation.
  • The pure culture of Staphylococcus aureus exhibits Yellow colonies with Yellow zones.
    • Biochemical Tests
  • Take a loop full culture and inoculate it into a tube, containing 0.5ml of mammalian preferably rabbit or horse plasma with or without suitable additives.
  • Incubate in water bath at 37˚C, examine the tube at 3 hours and after 24 hours.
  • Staphylococcus aureus shows clot formation indicating coagulase positive.
    • Purity checks for Bacillus subtilis
      • Gram’s staining
    • Prepare a smear with culture,on a clean glass slide.
    • Heat fix and perform the Gram’s staining.
    • Bacillus subtilis shows Gram +e, rod.
      • Cultural Tests
    • Streak the culture on the surface of Starch agar medium in Petridis.
    • Incubate the slants at 32.5+/- 2.5˚C for 24 to 48 hours.
    • Flood the surface of agar with iodine solution.
    • Yellow or gold color colonies appear around growth.
      • Purity checks for Candida albicans
        • Staining
      • Place a loopful of culture 1 on to a clean glass slide.
      • Make a thin smear and heat fix the smear.
      • Flood the smear with methylene blue and allow it to act for 15 to 60 seconds. Rinse the slide gently in tap water.
      • Candida albicans shows oval shaped cells with or without budding under oil immersion objective.
        • Biochemical Tests
      • Add 0.5ml serum into a 12×75 mm test tube
      • Add a loopful of culture.
      • Incubate the tube at 37˚C for two to three hours.(not more than 3 hours)
      • Place one drop of suspension onto slide.
      • Candida albicans shows germ tube when observed under microscope.
        • Purity checks for Aspergillus Niger
          • Staining
        • Place a small tuft of culture onto a clean glass slide.
        • Place a drop of Lacto phenol cotton blue on the suspension.
        • Separate the tuft using two needles.
        • Cover it with cover slip. Examine the slide under 45x objective.
        • Aspergillus Niger
        • Shows brownish conodiospore near the vesicle,globose vesicle, primary and secondary stigmata globos conidia separate hyphae.
          • Cultural Tests
        • Streak the culture on the surface of SDA Medium or PDA Medium contained in a Petridis.
        • Incubate the slants at 225+/- 2.5˚C for 120 hours.
        • The pure culture of Aspergillus Niger exhibits fluffy arial mycelial growth covered with black spores. Reverse of the colony is buff coloured.
          • Purity checks for Clostridium Sporogenes
            • Gram’s staining
          • Prepare a smear with culture,on a clean glass slide.
          • Heat fix and perform the Gram’s staining.
          • Clostridium sporogenes shows Gram +e, rod with terminal spores appear like drum stick.
            • Cultural Tests
          • Streak the culture on the surface of Rein forced clostridial Agar Medium in petridishes.
          • Incubate the slants at 32.5+/- 2.5˚C I an anaerobic jar for 24-48 hours.
          • The pure culture of Clostridium Sporogenes shows the growth.
            • Purity checks for Lactic acid Bacillus.
              • Gram’s staining
            • Prepare a smear with culture, on a clean glass slide.
            • Heat fix and perform the Gram’s staining.
            • Lactic acid Bacillus shows Gram +ve rods.
              • Cultural Tests
            • Streak the culture on the surface of YPS agar in petridishes.
            • Incubate the slants at 32.5+/- 2.5˚C for 24-48 hours.
            • The pure culture of Lactic acid Bacillus shows the growth.
              • All the remains of the original ampoules, used working cultures used plates and tubes.
              • For purity checks etc. should be treated as infected and must be decontaminated and discarded.
              • Common microbiological and safety procedures should be followed while handling cultures.
              • All cultures derived from lyophilized mother cultures should be used with in a year.

• Forms and Records (Annexures)
  • Culture receipt and information record
  • Stock culture record
  • Working culture Record

• Distribution
  • Master copy       –       Quality Assurance
  • Controlled copies  –       Quality Assurance, Production, Quality Control, Stores, Engineering and Human Resource
• History:

Date	Revision Number	Reason for Revision
	00	NEW SOP

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