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SOP for Procurement, Maintenance and Sub-culturing of Standard Microbial Cultures


Standard operating procedure to procure, subculture and maintain the microbial culture.

1.0 PURPOSE:

The purpose of this SOP is to lay down a procedure for revival, maintenance and disposal of microbial cultures.

2.0 SCOPE:

This SOP describes the procedure for revival, maintenance and disposal of microbial cultures in microbiology laboratory.

3.0 RESPONSIBILITY:

Officer/Executive - Quality Control.

4.0 ACCOUNTABILITY:

Head –QC department.

5.0 PROCEDURE:

5.1 The microbial strains shall be handled in the microbiology laboratory by taking all the precautions as listed below.
  • The sub culturing activities shall be carried out in the laminar air flow station.
  • Microbiologists handling cultures shall wear nose mask and hand gloves.
  • Spillages shall be disinfected with 10% solution of Virosil Pharma [Hydrogen peroxide].
  • The inoculation loop shall be incinerated using the flame in between every loop transfer.

5.2 Procurement of Culture:

5.2.1 The microbial cultures required for testing in Microbiology laboratory shall be procured either from the following or any other authentic source (Microbiologics-USA, IMTECH- Institute of Microbial Technology-Chandigarh).
NCIM (National Collection for Industrial Microorganisms)
Division of Biochemical Sciences
National Chemical Laboratory,
Pune-411 008.
5.2.2 Cultures shall be received either in the form of lyophilized ampoules/culture slants/ culture sticks.
5.2.3 On receipt of the cultures, the organism name, and the ATCC / NCIM shall be cross checked with the Culture certificate and the physical verification of any damage shall also be checked.
5.2.4 After the check, the cultures shall be labelled as mentioned in 3.0, and shall be stored in the refrigerator at temperature 2-8°C until further processing.
5.2.5 On receipt of reference culture, assign a lot number to the culture. The lot number will be “A” if the culture is received first time; “B” for second time; “C” for the third time and so on……
5.3 Label for Mother culture Slant

Name of the organism & strain no.:
Date of received:
Use Before :
Signature of Microbiologist:

5.4 Revival of Cultures

5.4.1 The lyophilized ampoules/culture slants/ culture sticks shall be removed from the refrigerator and shall be allowed to acclimatize to the ambient temperature.
5.4.2 In case of mother cultures in lyophilized ampoules, the ampoule shall be surface disinfected with 70% IPA and allow it to dry under LAF.
5.4.3 Mark at the middle of the ampoule with a sterilized ampoule cutter.
5.4.4 Wrap thick sterilized cotton around the ampoule and break it carefully at the marked area.
5.4.5 Gently remove the pointed top of the ampoules. Snap opening will draw the cotton plug to one end, hasty opening will release fine particles of dried organisms into the air of the laboratory.
5.4.6 Carefully remove the cotton plug and add about 0.3 to 0.4 ml of specified liquid medium with sterile pipette. Avoid frothing or creating aerosols.
5.4.7 Mix well and inoculate the contents into 100 mL of sterilized liquid medium in test tube.
5.4.8 In case of mother cultures in slants, the transfer shall be made by the inoculation loop from the culture and shall be inoculated in 100 mL of sterilized liquid medium in test tube.
5.4.9 In case of cultures in sticks, the stick shall be removed and inoculation shall be done by immersing the stick in 100 mL of sterilized liquid medium in test tube. The stick shall be manually rotated in the medium.
5.4.10 The used lyophilized ampoules/culture slants/ culture sticks shall be immersed in 10% solution of Virosil Pharma [Hydrogen peroxide].
5.4.11 The disinfected lyophilized ampoules/culture slants/ culture sticks shall be decontaminated in the autoclave at 121-124°C for 30 minutes.
5.4.12 The inoculated liquid medium with cultures in test tube shall be incubated as per the conditions described in the Table-I.
5.4.13 During the incubation period, the enriched tubes shall be checked for any growth in terms of turbidity for bacterial and yeast culture and growth of mycelium in case of fungal cultures at an interval of every 24 hours.
5.4.14 During every 24 hours observation of enriched tubes, if growth is observed, the cultures shall be then proceeded to the next stage of purity check and culture transfer.
5.4.15 If growth is not observed within the stipulated time period as mentioned in the Table-I, the clause 4.1 to 4.8 shall be followed for the next set of cultures.

5.5 Purity check

5.5.1 After the growth observed in the enriched medium, purity check shall be done for all the cultures and simultaneously shall be streaked in the maintenance medium as described in the Table-II. In case of culture of Clostridium sporogenes ATCC-11437 stabbing shall be done in the Cooked Meat Medium / Thio Glycollate Agar (TGA) butt.
5.5.2 Purity check shall be done for all the cultures for the following parameters as listed below.
5.5.3 Gram staining reactions for bacterial cultures/ simple staining for yeast cultures and lacto phenol cotton blue staining preparation for fungal identification.
5.5.4 Growth & Colony morphology in selective media.
5.5.5 Gram staining reaction / simple staining/ lacto phenol cotton blue staining shall be performed for the organisms and noted.
5.5.6 The parameters of purity check as mentioned in Table-III shall be followed for the cultures.
5.5.7 The growth observed in the selective media, the colony morphology, Gram staining shall be observed and recorded.
5.5.8 Based on the gram staining reactions/simple staining/lacto phenol cotton blue staining, growth in selective media and the colony morphology, the culture shall be confirmed for its purity as described in Table-III.
5.5.9 If the culture fails in the purity check, the supplier shall be intimated and a fresh culture shall be asked for and clauses mentioned under 4.0 & 5.0 shall be performed again to confirm the culture purity.
5.5.10 The mother culture in the enriched medium shall be discarded after the growth observed in the culture slants.
5.5.11 The master slant/butt shall be discarded only at the end of the 12 months period.
5.5.12 At the end of the twelve months period, fresh lyophilized ampoule/slant/culture stick shall be purchased from the supplier and the procedure shall be performed again for the revival and the culture transfer.
5.5.13 The mother cultures received in the slants shall be used for the next 12 months from the date of receipt if preserved at 2- 8°C. After expiry date the cultures shall be discarded after 12 months.
5.5.14 At the end of the 12 months period, fresh lyophilized ampoule/slant/culture stick shall be procured as mentioned in clause 2.1 and the procedure shall be performed again for the revival and the culture transfer.

5.6 Gram’s Staining Technique:

5.6.1 Prepare a thin smear of the culture to be examined on a clean and dried glass & heat fix it by gently passing on the flame. Allow the slide to cool.
5.6.2 Add 3-4 drops of Primary stain, i.e. Gram's crystal violet solution on the smear with and allow to stand for one minute.
5.6.3 Wash the crystal violet solution with running tap water and air dry.
5.6.4 Add 3-4 drops of Gram's iodine solution and allow to stand for 1-2 minutes.
5.6.5 Wash the Gram's iodine solution with running tap water and air dry.
5.6.6 Add 3-4 drops of Grams decolouriser on the smeared slide and allow to stand for 30-60 seconds.
5.6.7 Wash the Grams decolouriser with running tap water and air dry.
5.6.8 Add 3-4 drops of counter stain i.e. saffranin solution 2.5% (w/v) on the slide and allow to stand for one minute.
5.6.9 Wash the saffranin solution with running tap water and air dry.
5.6.10 Observe the slide under microscope with 10X, 40X and with Oil immersion objective and record the observations.
5.6.11 Observe the other two tubes for growth of the organisms and check for purity of culture (only one type of colony should be present).

5.7 Culture Transfer (Frequency: Once in a month).

5.7.1 The cultures in the master SCDA/SDA slants/ TGA butt / Cooked meat medium shall be treated as the first passage/generation.
5.7.2 From the Master slant/butt, cultures shall be streaked/ stabbed onto the maintenance media slants/butts and incubated as per the conditions mentioned in the Table-II.
5.7.3 Follow the schedule of culture transfer regularly.
5.7.4 After checking the purity of mother cultures 3 subcultures slant of each microorganism shall be prepared.
5.7.5 One culture tube shall be labelled as Stock culture (SC1) and the other two as Sub culture (A1) and Sub culture (B1) where, (A1) is used only for subculturing purpose and (B1) for routine use.
5.7.6 After one month, two sub cultures shall be prepared from Subculture (A1). One is labelled as Sub culture (A2) and Sub culture (B2)
5.7.7 Subculture (A2) shall be used only for sub culturing purpose and (B2) for routine use.
5.7.8 This procedure shall be repeated for 4 months to finally to get set subculture (A4) and (B4).
5.7.9 After 4 months, prepare 3 subcultures from stock culture (SC1).
5.7.10 Label one culture tube as Stock culture (SC2) and the other two as Sub culture (A1) and Sub culture (B1).
5.7.11 Repeat the above steps from clause 7.3 to 7.6 for further subculturing up to (SC3).
5.7.12 Record the all the sub culturing details.
5.7.13 Discard the cultures as per their expiry.
5.7.14 Incubate the following organisms for time and temperature on respective media as mentioned in below Table II:
5.7.15 After luxuriant growth label the subculture slants as follows: 
Name of the organism & strain no.:
Subculture No. :
Date of sub culturing:
Subculturing due date :
Signature of Microbiologist:

5.8 Environmental Isolates Transfer

5.8.1 The environmental isolates cultures shall be cultured on Agar slants.
5.8.2 Bacterial isolates shall be cultured on Soyabean Casein Digest Agar slants and for fungi Sabouraud Dextrose Agar Slants.
5.8.3 After subculturing the SCDA slants shall be inoculated at 30-35°C for 24-48 hrs and the SDA slants shall be inoculated at 20-25°C for 3-5 days.
5.8.4 After completion of the incubation the slants shall be checked for growth. Well developed colonies on the slants shall be labelled as mentioned in Clause 8.6 details shall be recorded.
5.8.5 The environmental isolates maintained in the culture shall be subcultured three months once.
5.8.6 Label for environmental isolate
Environmental Isolate no.:
Date of sub culturing:
Subculturing due date :
Signature of Microbiologist:

5.9 Storage of cultures

5.9.1 The master slant/butt, working culture slant/butt, working culture plates shall be labeled, the cotton of tubes and plates sealed with parafilm and store in refrigerator at 2- 8°C.
5.9.2 After the usage, the cultures shall be restored to the refrigerator at 2- 8°C.

5.10 Disposal of cultures

5.10.1 The cultures to be discarded after the usage shall be loaded in the container and shall be autoclaved at 121-124 0C for 30 minutes.
5.10.2 After the decontamination activity is over, the media shall be incinerated.

6.0  ABBREVIATIONS

6.1  SOP - Standard Operating Procedure
6.2  LAF - Laminar Air Flow
6.3  % - Percent
6.4  °C - Degree centigrade
6.5  gm - gram
6.6  ml - Milli litre
6.7  CFU - Colony Forming Unit


Table-I



Sr. No.
Name of culture
Enrichment Medium
Incubation time &
temperature
1
Staphylococcus aureus
ATCC-6538
SCDM
35-37°C for 24-48 hrs
2
Escherichia coli
ATCC-8739
SCDM
35-37°C for 24-48 hrs
3
Escherichia coli
ATCC-11105
SCDM
35-37°C for 24-48 hrs
4
Salmonella sps; preferably salmonella abony  NCTC-6017
SCDM
35-37°C for 24-48 hrs
5
Pseudomonas aeruginosa
ATCC-9027
SCDM
35-37°C for 24-48 hrs
6
Bacillus subtilis
ATCC-6633
SCDM
35-37°C for 24-48 hrs
7
Clostridium sporogenes
ATCC-11437
FTGM*/ Cooked meat medium
35-37°C for 24-48 hrs
8
Candida albicans
 ATCC-10231
SCDM
20-25°C for 48-72 hrs
9
Saccharomyces cerevisiae
ATCC-10231
SCDM
20–25ºC for 48 -72 hrs
10
Aspergillus niger
ATCC 2601
SCDM
20-25°C for 48-72 hrs
*FTGM-Fluid Thio Glycollate Medium
Table-II


Sr. No.
Name of culture
Maintenance Medium
Incubation time &
temperature
1
Staphylococcus aureus
ATCC-6538
SCDA Slant
35-37°C for 24-48 hrs
2
Escherichia coli
ATCC-8739
SCDA Slant
35-37°C for 24-48 hrs


Sr. No.
Name of culture
Maintenance Medium
Incubation time &
temperature
3
Escherichia coli
ATCC-11105
Vitamin B12 Culture agar Slant
35-37°C for 24-48 hrs
4
Salmonella sps; preferably salmonella abony  NCTC-6017
SCDA Slant
35-37°C for 24-48 hrs
5
Pseudomonas aeruginosa
ATCC-9027
SCDA Slant
35-37°C for 24-48 hrs
6
Bacillus subtilis
ATCC-6633
SCDA Slant
35-37°C for 24-48 hrs
7
Clostridium sporogenes
ATCC-11437
TGA Butt/ Cooked meat medium
35-37°C for 24-48 hrs
8
Candida albicans
 ATCC-10231
SDA/SCA Slant
20-25°C for 48-72 hrs
9
Saccharomyces cerevisiae
ATCC-10231
SDA/SCA Slant
20–25ºC for 48 -72 hrs
10
Aspergillus niger
ATCC 2601
SDA/SCA Slant
20-25°C 5 days
  
Table-III


Sr. No.
Name of culture
Confirmatory purity check parameters
1
Staphylococcus aureus
ATCC-6538
Gram positive cocci, yellow pigmented colony growth in Mannitol Salt Agar
2
Escherichia coli
ATCC-8739
Gram negative rods, metallic sheen colony growth in  Eosin Methylene blue agar
3
Escherichia coli
ATCC-11105
Gram negative rods and growth in Vitamin B12 Culture agar.
4
Salmonella sps; preferably salmonella abony  NCTC-6017
Gram negative rods, black colony growth with surrounding metallic sheen  in Bismuth Sulphite agar



Sr. No.
Name of culture
Confirmatory purity check parameters
5
Pseudomonas aeruginosa ATCC-9027
Gram negative rods, growth in Cetrimide agar
6
Bacillus subtilis
ATCC-6633
Gram positive bacilli and irregular edges in the colony growth in soyabean casein digest agar.
7
Clostridium sporogenes
ATCC-11437
Gram positive bacilli and growth in TGA butt
8
Candida albicans
ATCC-10231
Elliptical shaped cells in simple staining
9
Saccharomyces cerevisiae
ATCC-10231
Oval shaped cells and growth in SDA
10
Aspergillus niger
ATCC 16404
As described in the Picture-I by Lacto phenol cotton blue staining


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


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