SOP Title: Maintenance of Various Cultures used in Microbiological Analysis

SOP Title: Maintenance of Various Cultures used in Microbiological Analysis

1. Objective:

To lay down the procedure for Maintenance of Various Cultures used in Microbiological Analysis in Microbiology section of Quality Control Department.

2. Scope:

This procedure is applicable for Maintenance of Various Cultures used in Microbiological Analysis section of Quality Control.

3. Responsibility:

3.1 Quality Control Department: To prepare and review the SOP. To follow the procedures for Maintenance of Various Cultures used in Microbiological Analysis as per this SOP.

• Quality Assurance Department: To review and approve the SOP and Annexure.

 

4. Accountability:

Head Quality Control Department, Head Quality Assurance Department.

 

5. Procedure:

 

5.1	Safety Precautions:
5.1.1	Restrict the access to handle all live Microbial Cultures only to qualified and authorized persons.
5.1.2	All prepared Culture media and their components should be stored away from light and exposure to direct sunlight should be avoided at all times.
5.1.3	If any culture and surrounding debris is spilled (e.g. glass, cotton wool plugs) must not be touched with unprotected hands.
5.2	Procedure for Maintenance of culture:
5.2.1	Microorganisms used for Microbiological analysis should be procured from National or International Institute.
5.2.2	These Cultures are generally supplied in vacuum sealed glass ampoules or vials in freeze-dried condition or Sub Cultured on slants.
5.2.3	On receipt of the Culture, check the name of the organism and strain number as per the requirement. Check for physical damage if any.
5.2.4	Record the receipt details of Mother Culture in Annexure.
5.2.5	Use hand gloves, Nose mask and Overgown while handling live Cultures.
5.2.6	Report any incidence of spillage of live Microbial Culture or Microbial contamination to the Quality Control Head who will review the situation and take necessary measures.
5.2.7	Transfer the Cultures to Microbiological area after following Entry and Exit Procedure of Microbiological Section.
5.2.8	Carry out tests for identification and purity check of Culture slant or vials  as per SOP.
5.2.9	If the purity check fails, inform the Department Head and file an incident. Repeat the purity check.
5.2.10	If purity check fails again, inform the Department Head and discard the Culture as per Procedure for Destruction of Cultures and Microbial waste-SOP. Procure new Mother cultures.
5.2.11	If the purity test passes then proceed for the Maintenance of Cultures such that each Culture should never be used for more than five passages from its Original Mother Culture. The mother culture which is received from the supplying agency should be considered as Second passage.
5.2.12	Prepare specific growth medium as per Procedure for Media Preparation and Sterilisation- SOP.
5.2.13	Disinfect hands by using 70% v/v Iso propyl alcohol.
5.2.14	Disinfect the outer parts of Culture slant, ampoules, tubes or vials with 70% v/v isopropyl alcohol.
5.2.15	Break the Seal and remove the cotton plug from the neck of tube, vial or ampoule. Flame the mouth of tube by using a burner to sterilize the mouth of the tube to prevent contamination.
5.2.16	Transfer with a Nichrome wire a loopful of each culture in separate 10 ml tube containing 0.9% v/v sterile saline solution or Buffered Sodium Chloride Peptone pH 7.0. Refer SOP for preparation of reagents.
5.2.17	Mix the inoculated tube well to disperse the microorganisms and incubate at required temperature for 18-24 hours. Bacterial cultures at 30-35°C and fungal cultures at 20-25°C.Consider this as Culture suspension.
5.2.18	Before Sub Culturing ensure that fresh slants should attain room temperature if stored in refrigerator as microorganisms may not grow properly on cold temperatures. (For slant preparation refer SOP). Streak a loopful of Culture from 24 hours old Culture suspension on the respective media slants and incubate at 20-250 C for fungi and 30-350C. Streak 2 Slants each.
5.2.19	Incubate Bacterial cultures at 30-35°C for 48 hours and fungal cultures at 20-25°C for 72 hours Aspergillus brasilliensis can be incubated upto 120 hours for luxuriant growth. Use SCDA slants for bacteria and SDA slants for Fungal cultures.
5.2.20	First slant will be Mother Culture slant, second will be Working Culture slant, incubate at specified temperature as above.These slants along with initial inoculated Mother Culture suspension (point no.5.2.16) should be used only for culture identification and Purity check.
5.2.21	Proceed for the Identification and Purity Check of cultures as per SOP.
5.2.22	Label the slants and preserve the culture suspension in the refrigerator at 2° C to 8° C.
5.2.23	Streak each of the cultures on separate Nutrient Agar Plates from the culture suspension (Point No. 5.2.16) or the above slants (refer point No. 5.2.20) for obtaining pure isolated colonies which should be used for Identification tests. Incubate Bacterial cultures at 30-35°C for 48 hours and candida albicans at 20-25°C for 48-72 hours. Aspergillus brasilliensis should be incubated at 20-25°C for upto 120 hours for luxuriant growth.
5.2.24	Perform the Identification and Purity Check as per SOP.
5.2.25	If all the cultures are complying with Identification and Purity tests then proceed further to prepare  Master Culture Slants from culture suspension (Refer point No. 5.2.16)
	Preparation Of Master Slants
5.2.26	Prepare Six Master Culture Slants from the 10 ml of Culture suspension(Refer point No. 5.2.16). Master slants should be labelled as M1, M2, M3, M4, M5 and M6. Store at 2 – 80C.
5.2.27	Streak a loopful of the mother Culture suspension on the surface of 6 agar slants of Soyabean Casein Digest Agar for each Bacterial Culture and Sabourauds Dextrose Agar with chloramphenicol (SCA) or without antibiotic (SDA) for each fungal culture(Third passage). Incubate as per point No. 5.2.23 and observe the growth. If growth is not proper then streak again. These six Master slants shall be used for preparation of working and reference slants for next 12 months. Each Master slant is valid for 2 months.
5.2.28	Label the Agar Slants as Master Slants as follows. Eg. E. coli. For example MS/EC/01/03. MS -Master Slant EC – code number of organism 01 – serial No. of slant and 03 – passage No.
5.2.29	Incubate at 30 to 35°C for 48 hrs (Bacterial Cultures) and 20 to 25°C for 72-120 hours (Fungal Cultures).
5.2.30	Check for growth, if satisfactory growth is not observed then inform the Department Head and Subculture again on fresh agar slants.
	Preparation Of Working and Reference Slants
5.2.31	Prepare 2 SCDA slants for bacteria and SDA agar slants for fungi for each Culture from Master Slant. Prepare Media as per SOP.
5.2.32	Incubate the slants at 30 to 35°C for 48 hrs (Bacterial Cultures) and 20 to 25°C for 72-120 hours (Fungal Cultures). Consider this as Passage 4.
5.2.33	Keep one slant as Working Slant and another as Reference Slant, Working Slant should be use for routine analysis for 1 month from date of preparation.
5.2.34	Before due period, from Reference Slant further Sub Culture on two SCDA and SDA slants, use one for routine analysis as Working Slant  and another one as Reference Slant (Passage 5).
5.2.35	Working Slants should be used for a period 1 Month. If there is any accidental contamination with Working Slant, then prepare new working slant by using reference slant and use as a working slant for routine analysis.
5.2.36	Allocate the Reference Number to each Working Slant and Reference Slant for example WS or RS /EC/01/04 or 05. WS – Working Slant RS – Reference Slant EC- code no. of Organism. 01 – serial No. of Master Slant and 04 or 05 – passage No.
5.3.37	Record the sub Culturing details in Annexure and label the slants with as per annexure. Store the Working and Reference Slants at 2° C to 8° C.
5.2.38	Discard the expired Working Slant and Reference Slant as per procedure for Destruction of Cultures and Microbial Waste-SOP. The Master slant shall be discarded only when the incubation period of working and reference slant is completed and proper growth is observed.
5.2.39	If there is delay in new Culture, use 5th passage Reference Slant as a Working slant for another one month. Refer Point No. 5.2.34.
5.2.40	Procure a new Mother Culture before the 5th passage validity gets over.

 

 

6. Definitions / Abbreviations:

• Definitions :
• Abbreviations :

Abbreviation	Expansion
v/v	Volume by volume
No.	Number
°C	Degree Centigrade
SCA	Sabouraud Chloramphenicol Agar
SDA	Sabouraud dextrose agar
SCDA	Soyabean casein digest agar
MS	Master Slant
RS	Reference Slant
Ec	code no. of organism

*Note – Ready to use SOP available in “DOWNLOAD” Section.