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

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 the 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 subculturing 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 labeled 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 the first time; “B” for the 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, the 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 a sterile pipette. Avoid frothing or creating aerosols.
5.4.7 Mix well and inoculate the contents into 100 mL of sterilized liquid medium in a 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 a 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 a 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 have 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 the 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 lactophenol cotton blue staining preparation for fungal identification.
5.5.4 Growth & Colony morphology in selective media.
5.5.5 Gram staining reaction / simple staining/ lactophenol 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/ lactophenol 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 the 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 decolorizer on the smeared slide and allow to stand for 30-60 seconds.
5.6.7 Wash the Grams decolorizer with running tap water and air dry.
5.6.8 Add 3-4 drops of counterstain i.e. safranin solution 2.5% (w/v) on the slide and allow to stand for one minute.
5.6.9 Wash the safranin solution with running tap water and air dry.
5.6.10 Observe the slide under the microscope with 10X, 40X and with Oil immersion objective and record the observations.
5.6.11 Observe the other two tubes for the 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 labeled as a Stock culture (SC1) and the other two as Subculture (A1) and Subculture (B1) where (A1) is used only for the subculturing purpose and (B1) for routine use.
5.7.6 After one month, two subcultures shall be prepared from Subculture (A1). One is labeled as Subculture (A2) and Subculture (B2)
5.7.7 Subculture (A2) shall be used only for the subculturing 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 a Stock culture (SC2) and the other two as Subculture (A1) and Subculture (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 subculturing 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 Soybean 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 labeled 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 - Millilitre
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

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