Get the latest updates from us for free
Android App

SOP for Maintenance and Transfer of Stock Cultures


Standard operating procedure of control over bacterial cultures use for microbiological testing purpose.

1.0  Objective

To maintain and transfer the stock culture.

2.0  SCOPE

To control over bacterial cultures use for microbiological testing purpose.

3.0  Responsibility

3.1  Doing      : Tech.Assistant ( Microbiologist) / Executive
3.2  Checking : Executive/Manager    

4.0  Accountability

Head of the Department

5.0  Procedure

5.1  Media  requirement:

1. Soyabean casein digest agar
2. Soyabean casein digest broth
3. Sabouraud dextrose agar
4. Potato dextrose agar
5. Potato dextrose broth
6. Fluid thioglycollate medium
7. Anaerobic agar
8. E.coli mutant agar (maintenance medium)
9. Calcium pantothenate innoculam broth
10.Calcium pantothenate culture agar
11.GYE agar
12. L.leichhmani inoculam broth
13. L. Leichhmani culture agar

5.2  Material requirement

a. Sterile spectula
b. Scissors
c. Burner
d. Cryovial of 1 ml and 2 ml
e. 20% Sterile glycerol
f. Sterile water
g. Sterile microtips
h. H1 Anaerobic system mark
i. Anaerobic gas pack
j. Sterile normal saline
k. 0.9%saline + 0.5% Tween 80
l. Deep freezer
m. Roux bottles

5.3  On a receipt of  ATCC/MTCC/NCTC cultures label it and record it.
5.4 To minimise the risk of development of mutants, the passage should not be more than 5. Every work should be carried out under Biological Safety Cabinet to minimize the risk of contamination.
5.5  The detail of ATCC/MTCC/NCTC  strain fluid and solid medium  time and temperature requirement is as follows.

Media List

5.6  Reconstitution of lyophilised powder
a.  Reconstitute the powder with 1ml of sterile distilled water and vortex for 1 min and mix well.
b.  Add  0.25 ml in respective fluid medium in duplicate.
Note.: For A. niger spread in potato dextrose agar  and pack the plate in sterile bag.
c.  Incubate at respective time and temperature as mention above.

5.7  Procedure for sporulating organisms

5.7.1  Sporulating organisms are as follows
a. B.subtilis
b. Cl.sporogenes

5.7.2  For B.subtilis

1. Cultivate the liophilized spore in fluid medium .
2. Prepare two roux bottle for 2 flask of GYE agar.
3. Inoculate the culture of each flask on roux bottle and spread evenly with the help of sterile glass beads.
4. Incubate for 5 -7 days at 37 degC .
5. Collect the growth with the help of sterile normal saline and give the heat shock for 2 to 3 times at 70 degC for half an hour to kill the vegetative cell.
6. Mix with equal volume of 20% glycerol and fill the cryo vials
7. Store the vials in Deep freezer at – 20°C

5.7.3  For Cl. sporogenous

1. Cultivate the liophilized culture in fluid thioglycollate medium and incubate anaerobically .
2. Prepare two roux bottle for 2 flask .
3. Inoculate the culture of each flask on roux bottle and spread evenly with the help of sterile glass beads.
4. Incubate for 5 -7 days at 37 degC anaerobically.
5. Collect the growth with the help of sterile normal saline and give the heat shock for 2 to 3 times at 70 degC for half an hour to kill the vegetative cell.
6. Mix with equal volume of 20% glycerol and fill the cryo vials
7. Store the vials in Deep freezer at – 20°C.

5.7.4  After reconstitution of lyophilized powder proceed as follows.

1. Add 0.1 ml from two flask into two 9.0 ml fluid SCD tubes for viability and count test.
2. Add 0.2 ml in to two cryovials containing 0.2 ml 20% sterile glycerol and keep it at -20 degC
Label master sub culture code M2 & M3 on cryovial as a master sub culture.
3. Add 0.25 ml in 25 ml Fluid SCD Medium in duplicate and incubate at respective time & temperature as mentioned above for distribution in cryovials.

5.8  Distribution in cryo vials of other organisms

1. After incubation take two 25 ml flasks under Biological Safety Cabinet & take out 1.0 ml from each flask and add in 9 ml Sterile N Saline for viable count.
2. For A.niger harvest the spore with 0.9% Nacl and 0.5% polysorbate 80.
3. Add equal volume of sterile 20% glycerol slowly and mix smoothly in each flask having growth .
4. Distribute 1.5ml in 50 cryovial and label each vial as follow giving subculture code no.SC-1 to SC-50
Name of culture :
ATCC/MTCC/NCTC No. :
Date :
Passage No. :
Subculture no. :
5.8.1  Arrange in a cryobox and keep in Deep freezer at –20° C and label cryobox properly.
5.8.2  Record the reconstitution of cryotubes preparation.
5.8.3  Take out one cryovial of each culture every fifteen days give 1st activation  in liquid broth and second activation on slant and record the date of withdrawal and activation on slant.    

5.9  Viable count

a. Take viable count of each culture, when the lyophilised culture is reconstituted i.e Initial ATCC culture count.
b. After 24 hrs. incubation in fluid medium i.e preservation culture count.
c. After 24 hrs. freezing in Deep freezer at –20°c to check the viability and effect on culture.
d. Make several serial dilution by adding 1 ml to 9 ml sterile saline.
Note: For A.niger use 0.9% sterile saline and 0.5% Tween 80 mixture.
e. Perform plate count of all dilution in duplicate by taking 1 ml aliquot per plate.
f. Incubate at proper time and temp. according to culture, after achieving visible colony on plate find out the no. of organisms /ml.
g. Record the results.

5.10  Identification

An identification test of control stock carried out and recorded in annexure-2 .

5.11  Cultivation for growth promotion

a. The cryo tube taken from Deep freezer and recorded, 0.1ml of suspension inoculated in each 2X10ml relevant fluid medium using micropipette and cryo vial is discarded. Incubate tubes at proper time and temperature.
b. For A.niger : Inoculate 1 ml into 10cm petri dish with potato dextrose agar which already contains 1ml sterile water. Incubate in a bag for 7 days at 22.5 + 2.5 degC

5.12  Inoculation in fresh medium and slant  preparation:

a. Again inoculate in 2X10 ml relevant fluid medium using micropipette (0.1ml). Incubate at appropriate temperature for appropriate time and record.
b. Simultaneously streak each culture on relevant maintenance medium for preservation and record.

5.13  Preparation of inoculum

a.  Prepare dilution of suspension in sterile saline by serial dilution to get 102 cfu/ml.
5.13.1  For A.niger
The growth on potato dextrose agar plate. Harvested with 10 ml 0.9%Nacl + 0.05% Polysorbate 80 from 0.1ml suspension diluted to 20ml 0.9% NaCl + 0.05% polysorbate 80 and Further  it is diluted to get 10cfu/ml and  record.
5.13.2  Inoculum of 10-100 cfu/ml is used for inoculation for growth promotion testing of media.

5.14  Preservation of inoculum

1.  Keep inoculum at 2-8 oC temperature for preservation up to fifteen days. Before discarding check the viable count again.
2.  Prepare fresh inoculum every fifteen days from preserved cryo vial.
3.  Whenever the inoculum used take viable count of that organism on same day and record.

5.15  Precaution

1.  Latex gloves are worn when culture take out from Deep freezer.
2.  After harvesting gown is to be changed.
3.  All glassware and material contaminated with micro-organisms are send for autoclaving.

5.16  Handling of ATCC/MTCC/NCTC culture

1. Proceed within 2 hours of removal from Deep freezer and dispose the used cryovial as well as other contaminated material after treating it in sterilization cycle as per S.O.P. at 121° for 30 minutes.
2. Do every transfer and growth promotion work under safety cabinet.
3. Lock the Deep freezer after taking the cryovial.

6.0  Abbreviations

6.1  oC  = Degree centigrade
6.2  hrs.= hours
6.3  Nacl= Sodium chloride
6.4  cfu =Colony forming unit
6.5  ml = millilitre

ANNEXURE-1
MICROBIOLOGY DEPARTMENT
RECORD OF ATCC/MTCC/NCTC CULTURE

Date of Receipt
Name of
micro-organsims
ATCC/MTCC/NCTC no.
Exp.Date
Registered by
controlled by
used/discard by
Checked by
Remarks
Sign
























































ANNEXURE-II

MICRO BIOLOGICAL DEPARTMENT
RECORD OF CULTIVATION ,FREEZING AND CONTROL OF ATCC/MTCC/NCTC TEST STAIN AND ITS IDENTIFICATION

Date :

Name of  organisms  :

ATCC/MTCC/NCTC No.:

Medium used :

Incubation time and Temp:

A. Reconstitution of lyophilized Amp. With sterile  distilled water

B. Preparation of  2 (20% glycerol ) master stock from it code no. M2 and M3

C. Cultivation in 2 X 25ml  fluid medium passage 1  

D. Distribution in 50 cryo vials

E. Contamination control

F. Identification Test

G. Colony characteristic

H. Morphology character


Date :                                         Done by                                               Checked by      
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


Be the first to comment!

Post a Comment