SOP for Culture dilution of Microbial Culture
This Document describes (Standard Operating procedure) SOP for Procurement, Maintenance and Culture dilution of Microbial Culture
SOP for Procurement, Maintenance and Culture dilution of Microbial Culture
I. Purpose & Scope:
- The purpose of this SOP is to lay down the procedure for Procurement, Maintenance and Culture dilution of Microbial Culture.
- This Standard operating procedure shall be applicable for Procurement, Maintenance and Culture dilution of Microbial Culture.
II. Responsibilities:
- PROCEDURE:
- PROCEDURE:
- PROCUREMENT :
- All new and existing cultures shall be procured from any of the sources listed in Annexure-I.
- Existing cultures shall be procured once in a year or new additional cultures when it is required.
- The Procured culture (Mother culture), when received, shall be immediately stored in refrigerator between 2 to 8°C.
- After receiving the cultures, perform the culture identification tests and prepare ‘Master Culture Banks (MCB) simultaneously.
- Record the details in ‘Culture receipt and identification test report.’ Refer Annexure-V.
- REVIAVAL OF LYOPHILIZED CULTURES :
- Prepare the suspension of culture as per instructions provided by the supplier with the culture.
- General procedure: Prepare the specified liquid growth medium. Refer Table-I for respective medium. Disinfect the ampoule with alcohol damped gauze. Wrap sterile gauze/ampoule cutter around the ampoule and break it carefully. Cut open the ampoule aseptically. Add about 0.2 ml of growth medium in the ampoule and mix well. Transfer this suspension to about 10 ml of growth medium for enrichment & incubate for the specified time & temperature. Discard the ampoule only after its decontamination, in
- Streak loopfull of the enriched suspension on pre-incubated agar slants/plates. Use the appropriate media for each microbial culture; refer ‘Table-I’ for ‘Maintenance media’. Store the original suspension at 2 -8°C in refrigerator.
- Incubate the bacterial cultures at 30-35°C and fungal cultures at 20-25°C. Anaerobic culture shall be incubated in anaerobic condition by using anaerobic culture jar.
- After incubation, observe the slants/plates and check the culture for purity with respect to colony morphology. Confirm the purity visually by checking the slants/ plates for any contamination.
- PROCUREMENT :
- PREPARATION OF SLANT:
- Weigh the required bulk quantity of appropriate agar media in clean & dry flask/bottle and dissolve it into specific required amount of purified water. Heat this whole content to boiling and digestion of agar media. Pour approximate 10-12 ml media in the each test tube; prepare required number of test tubes. Plug the test tubes with non absorbent cotton. Sterilize the media tubes in autoclave in media sterilization cycle.
- After sterilization, allow to cool the media at about 40 to 50°C and place the tubes in standing slanting position for solidifying.
- After solidification, pre-incubate the tubes at 30 to 35°C for 24 hours.
- Before using the slants for sub culturing, check the slant for sterility. If slants are not to be used immediately, store them in refrigerator at 2-8°C, till its use before date.
- INITIATION OF CULTURE :
- Prepare the ‘Master culture banks’ from the procured mother culture of micro-organisms.
- Aseptically open the container of the mother culture, under LAF and subculture it onto five tubes of the agar media slants by using a sterile loop.
- These five tubes shall be referred as ‘Master Culture Banks’. Label them as MCB-I, MCB-II, MCB-III, MCB-IV and MCB-V respectively.
- Affix the label on each tube with the name of the culture, strain number, passage number, sub-culturing done by and date of sub-culturing. Refer Annexure-VI.
- Incubate the tubes at the specified temperature and time as given in Annexure-II.
- Record the details of sub-culturing in the Annexure-III.
- If the procured culture slants/vials are of ‘First Passage’, then the prepared master culture banks (MCB slants) from these procured cultures are of the ‘Second Passage’.
- After completion of incubation period of Master culture banks, Store the Master culture banks slants at 2 to 8°C.
- Proceed for next sub-culturing as and when required by using Master culture banks. Refer the flow chart given below for sub-culturing.
Flow chart of Sub-culturing:
| Mother Culture (1st Passage) |
2nd Passage
MCB-I MCB-II MCB-III MCB-IV MCB-V
3rd Passage
Working Culture Reference culture
(WC-A1) (RC-A1)
4th Passage
Working culture Reference culture
(WC-A2) (RC-A2)
5th Passage
Working culture Reference culture
(WC-A3) (RC-A3)
After Completion of 5th Passage of MCB-I, Continued same pattern for
Next Master culture Bank (MCB-II, MCB-III, MCB-IV and MCB-V)
- PROCEDURE FOR SUBCULTURING :
- Remove the slants from refrigerator and bring them to ambient temperature.
- Clean the LAF work station with 70% IPA.
- Label the each tube of subculture as per Annexure-VI.
- The culture received from organization /institute is termed as original or mother Culture. At first prepare five master cultures (MCB) from original/ mother culture.
- From each ‘Master Culture Bank’, prepare two slants. One for ‘Reference culture (RC)’ and other for ‘Working culture (WC)’.
- Use media for subculturing of each organism as mentioned in ‘Table-1’.
Table-1
| Sr.
no. |
Name of strain | Growth Medium | Maintenance Media |
| 1. | Aspergillus brasiliensis | SCDM | SDA/ SCA |
| 2. | Bacillus subtilis | SCDM | SCDA |
| 3. | Candida albicans | SCDM | SDA/ SCA |
| 4. | Clostridium sporogenes | SCDM/RMC | SCDA/CLA |
| 5. | Escherichia coli | SCDM | SCDA |
| 6. | Staphylococcus epidermidis | SCDM | SCDA |
| 7. | Pseudomonas aeruginosa | SCDM | SCDA |
| 8. | Salmonella abony | SCDM | SCDA |
| 9. | Staphylococcus aureus | SCDM | SCDA |
- Incubate the slants of bacterial cultures at 30-35°C for 24 to 48 hours whereas that of yeast and mould cultures at 20-25°C for 72 to 120 hours. Observe for appearance of growth on each slant after incubation and record the results in Annexure-III.
- Gram’s staining of each strain shall be done after every passage.
- Other tests of biochemical characters using different specific media & gram staining shall be performed only after receipt of the new culture.
- The slants prepared from ‘MCB-I’ shall be used as, Reference culture (RC-A1) and Working culture (WC-A1).
- In same manner, prepared slants from ‘MCB-II’ will be Reference culture (RC-B1) and Working culture (WC-B1). From ‘MCB-III’, Reference culture (RC-C1) and Working culture (WC-C1). From ‘MCB-IV’, Reference culture (RC-D1) and Working culture (WC-D1). From ‘MCB-V’, Reference culture (RC-E1) and Working culture (WC-E1).
- Use ‘Reference culture slant’ for next sub-culturing purpose only and do not use it for routine analysis.
- Working culture shall be used for culture dilution purpose and in routine analysis.
- Each prepared ‘Reference’ and ‘Working’ culture should be used up to one month. If received ‘Mother Culture’ is of first Passage, then prepared ‘Master culture slants’ (MCB) is of 2nd Passage, Working and Reference slants prepared from Master culture bank (MCB) is of 3rd Subculture in the same manner from passage no.3 to passage no.4 and finally from passage no. 4 to passage no.5.
- After the culture has completed five passages, do not use it for further transfer. Use the next ‘Master Culture Bank’ (MCB) for subculturing. After usage period of Fifth passage, discard the slants and also the respective ‘Master Culture Bank’ (MCB) by decontamination cycle.
- If working cultures becomes contaminated or fail to grow, revert to the master culture for preparation of new working cultures.
- All the cultures must be stored in polythene bags or in plastic box at a temperature 2 to 8°C.
- The procedure for procurement of new mother cultures must be started, before starting the usage of last Master Culture Bank (MCB-V) or earlier if required.
- After receipt of new mother cultures, the previous mother cultures shall be discarded by decontamination cycle of autoclave.
- PRECAUTIONS:
- Maintain the aseptic conditions through the process to avoid contamination.
- All the culture related activities such as subculturing and culture dilution shall be carried out in Culture Handling Area.
- After completion of usage period of slant/Passage or if occurrence of any contamination in culture slant, the culture to be decontaminated and destructed as per respective SOP in media destruction autoclave.
- PREPARATION OF MICROBIAL CULTURE DILUTION:
- Allow the bacterial and fungal cultures slants to grow on their respective maintenance medium as per given in Table-1.
- By using sterile loop, aseptically scrub the growth from the slant (Monthly Working Culture Slant) and inoculate in 10 ml of appropriate sterile growth medium tubes, specified as ‘Growth Medium’ in Table-1.
- Aspergillus brasiliensis should not be inoculated, as its spores can be directly used for dilution purpose.
- Incubate the tube for enrichment at below mentioned temperature for 24 to 48 hours. Bacterial cultures shall be incubated at 30 to 35ºC and fungal cultures to be incubated at 20 to 25ºC.
- Aseptically pipette out 1 ml of enriched culture and inoculate in 9 ml saline in test tube. For Aspergillus brasiliensis directly take spores from working culture slant by using sterile loop and inoculate in 9 ml saline tube. Vortex the tubes to get homogenized suspension. These tubes shall be referred as 10-1
- Serially dilute each microbial suspension by transferring into another tube containing sterile saline (diluent) solution till the appropriate dilution.
- Perform the dilution process by following the below mentioned steps up to 10-8 Mix each dilution properly on vortex mixer for approximately 30 seconds before further dilution.
1 ml enriched culture + 9 ml saline → 10-1
1 ml of 10-1 + 9 ml saline → 10-2
1 ml of 10-2 + 9 ml saline → 10-3
1 ml of 10-3 + 9 ml saline → 10-4
2 ml of 10-4 + 18 ml saline → 10-5
2ml of 10-5 + 18 ml saline → 10-6
2ml of 10-6 + 18 ml saline → 10-7
2ml of 10-7 + 18 ml saline → 10-8
- Pipette out 1 ml of suspension from each dilution of 10-5 to 10-8 in two sterile petri plates.
- Add about 15 to 20 ml of sterile molten agar media (of temperature NMT 45°C) in each plate. Mix the medium and suspension by carefully swirling the plates. Allow the media to solidify.
- Use appropriate agar media for each microbial culture, as specified as ‘Maintenance Media’ in ‘Table-1’.
- Incubate the plates of bacterial cultures at 30° to 35°C for 24 to 48 hrs and plates of fungal and yeast cultures at 20° to 25°C for 48 to 72 hrs. Store the dilutions at 2 to 8°C.
- After completion of incubation, count the number of colonies in each plate and calculate the mean count of two plates. Select the dilution of count 10 to 100 cfu/ml and its previous dilution (10 to 100 cfu/0.1ml). Keep these dilutions at 2 to 8°C and use them for routine analysis such as growth promotion test.
- Record the details of culture dilution in Annexure-IV and label the dilution tubes as per Annexure-VII.
- After confirmation of count and selection of dilutions, discard the remaining dilutions by decontamination cycle of autoclave.
- In case, if required, 0.1 ml of previous dilution can be used to inoculate 10 to 100 cfu. (Example in ‘Growth promotion test’ of agar media by spread plate method.)
- FREQUENCY:
- Procurement of culture: Once in a year or if new / additional cultures required.
- Sub-culturing of ‘Reference Culture’: Once in month.
- Culture dilution: Once in month.
- Identification tests/ purity: At the time of culture receipt.
- ANNEXURE:
Annexure I: Source of Procurement of Culture
Annexure II : Incubation details for Sub culturing
Annexure III : Sub Culturing Record
Annexure IV : Culture dilution report of Microbial culture
Annexure V : Culture receipt and identification test report
Annexure VI : Sub Culturing label
Annexure VII : Culture dilution label
Annexure I
SOURCES OF PROCUREMENT OF CULTURES
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1) AMERICAN TYPE CULTURE COLLECTION, 12301 PARKLAWN DRIVE,
ROCKVILLE, MD 20852.
2) INSTITUTE OF MICROBIAL TECHNOLOGY
SECTOR 39-A CHANDIGARH-160 036
3) NATIONAL CHEMICAL LABORATORY. PUNE -411008
NATIONAL COLLECTION OF INDUSTRIAL
MICROORGANISMS (NCIM)
DIVISION OF BIOCHEMICAL SCIENCES
4) CENTRAL DRUG LABORATORY
KYD STREET CALCUTTA
5) NATIONAL COLLECTION OF TYPE CULTURES CENTRAL PUBLIC
HEALTH LABORATORY CORINDALE AVENUE LONDON NW 95HT
ENGLAND
6) BIOMARK LABORATORIES
APOLLO GREEN 441/3
SOMWAR PETH PUNE
7) MILLIPORE (I) PVT. LTD
P.B. NO. 5870 50 A SECOND PHASE
RING ROAD PEENYA
BANGALORE 560 058
Annexure II
INCUBATION DETAILS FOR SUB CULTURING
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| Name of culture | Strain No. | Nutrient media | Incubation temp. | Incubation time | Frequency of Sub culturing |
| Escherichia coli | MTCC 1687
(ATCC 8739) |
SCDA | 30 – 35°C | 24-48 hours | Monthly |
| Salmonella abony | MTCC 733
(NCTC 6017) |
SCDA | 30 – 35°C | 24-48 hours | Monthly |
| Pseudomonas aeruginosa | MTCC 1688
(ATCC 9027) |
SCDA | 30 – 35°C | 24-48 hours | Monthly |
| Staphylococcus aureus | MTCC 737
(ATCC 29737) |
SCDA | 30 – 35°C | 24-48 hours | Monthly |
| Staphylococcus epidermidis | MTCC 435
(ATCC 12228) |
SCDA | 30 – 35°C | 24-48 hours | Monthly |
| Bacillus subtilis | MTCC 619
(ATCC 6633) |
SCDA | 30 – 35°C | 24-48 hours | Monthly |
| Clostridium sporogenes | ATCC11437 | SCDA | 30 – 35°C | 24 -48hours | Monthly |
| Candida albicans | MTCC 227
(ATCC 10231) |
SDA/SCA | 20 – 25°C | 48 -72hours | Monthly |
| Aspergillus brasiliensis | MTCC 1344
(ATCC 16404) |
SDA/SCA | 20 – 25°C | 3-5 days | Monthly |
Annexure III
SUB CULTURING RECORD
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Name of Organism : _______________________ Passage No. : ___________
Source : _______________________ Procured on :__________
Strain No.:______________ Incubation Temp. and Time: _____________
| Date of
Sub Culturing |
Microbial
culture taken for subculture |
Passage No. | In house lot number of media. | Incubator ID | Subculture
for Reference culture strain `RC’ |
Subculture
for Working culture strain `WC’ |
Done by/on | Observed by and On | Checked by/on | Culture
Discarded on |
Annexure IV
Culture Dilution report of Microbial cultureS
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Date of culture dilution: _______________ Culture dilution valid up to: ____________
In house media lot no. : _________________ Working Culture Used:______________
Incubation Temp & Time : _____________ Incubator ID :__________________ Done By : ___________
| Name of organism | Culture dilution Result (cfu/ml) | Taken culture dilution | Observed
Count cfu/ml |
Observed
on |
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| 10-5 | 10-6 | 10-7 | 10-8 | ||||||||||||
| Plate1 | Plate2 | Avg. | Plate1 | Plate2 | Avg. | Plate1 | Plate2 | Avg. | Plate1 | Plate2 | Avg. | ||||
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Remark : Complies/Does not comply
Observed By: Checked By:
Date : Date :
Annexure V
CULTURE RECEIPT AND IDENTIFICATION TEST REPORT
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| Culture receipt report | |||||||||||||||||||||||||||||||||||||||||||||
| Name of strain: | |||||||||||||||||||||||||||||||||||||||||||||
| ATCC/ MTCC/ NCIM: | |||||||||||||||||||||||||||||||||||||||||||||
| Date of receipt: | |||||||||||||||||||||||||||||||||||||||||||||
| Culture procured from: | |||||||||||||||||||||||||||||||||||||||||||||
| Culture received by: | |||||||||||||||||||||||||||||||||||||||||||||
| Date of testing: | |||||||||||||||||||||||||||||||||||||||||||||
| Culture identification test report | |||||||||||||||||||||||||||||||||||||||||||||
| 1. Viability:
Procedure: Aseptically open the culture slant and transfer loopful of growth in sterile……….. tubes. Incubate the tubes at ………….. for ……………hrs. Observation: turbidity observed / not observed after ………. hrs of incubation. Conclusion: culture is viable/ non viable. |
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| 2. Subculturing:
Subculture the culture on sterile …………….slants/tube and incubate at ……….. for …………. hrs. Observation: growth observed / growth not observed. |
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| 3. Gram’s staining: | |||||||||||||||||||||||||||||||||||||||||||||
| 4. Motility: | |||||||||||||||||||||||||||||||||||||||||||||
5. Colony characteristics:
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Annexure V
CULTURE RECEIPT AND IDENTIFICATION TEST REPORT
Page No.: 2 of 2
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6. Biochemical tests:
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| 7. Remarks:
Done by: Checked by: Date: Date:
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Annexure VI
Sub Culturing Label
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Sub Culturing Label
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| Culture Name: ————————- | |
| ATCC: ——————————- | |
| Passage no.: —————————- | |
| Type of culture:—————————- | |
| Subculture on:—————————- | |
| Done by: ————————
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Annexure VII
Culture Dilution Label
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Culture Dilution Label |
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| Culture Name: ————————- | |
| ATCC: ——————————- | |
| Dilution.: —————————- | |
| Count :—————————- | |
| Dilution done on:—————————-
Dilution Valid Up to: ——————— |
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| Done by: ———————–
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