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SOP on Maintenance and usage of Reference cultures

SOP on Maintenance and usage of Reference cultures

Objective:

  • SOP on Maintenance and usage of Reference cultures.

Responsibility:

  • Officer or above of Microbiology Laboratory:

Accountability:

  • Head – Microbiology section.
  • Head – Quality control.

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.

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cultures

Opening of Lyophilized ampoule:

  • Disinfect the outside of the 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.

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