1.0 Objective
To maintain and transfer the stock culture.2.0 Scope
To control over bacterial cultures use for the microbiological testing purpose.3.0 Responsibility
3.1 Doing: Tech.Assistant ( Microbiologist) / Executive3.2 Checking: Executive/Manager
4.0 Accountability
Head of the Department5.0 Procedure
5.1 Media requirement:
1. Soybean casein digest agar2. Soybean 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 inoculum broth
10.Calcium pantothenate culture agar
11.GYE agar
12. L.leichhmani inoculum-broth
13. L. Leichhmani culture agar
5.2 Material requirement
a. Sterile spectulab. Scissors
c. Burner
d. Cryovial of 1 ml and 2 ml
e. 20% Sterile glycerol
f. Sterile water
g. Sterile micro tips
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.
5.6 Reconstitution of lyophilized 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 the respective fluid medium in duplicate.
Note.: For A. niger spread in potato dextrose agar and pack the plate in a sterile bag.
c. Incubate at respective time and temperature as mention above.
5.7.1 Sporulating organisms are as follows
a. B.subtilis
b. Cl.sporogenes
5.7.2 For B.subtilis
1. Cultivate the lyophilized spore in the 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 deg C 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 lyophilized culture in fluid thioglycollate medium and incubate anaerobically.
2. Prepare two roux bottle of 2 flasks.
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 flasks into two 9.0 ml fluid SCD tubes for viability and count test.
2. Add 0.2 ml into two cryovials containing 0.2 ml 20% sterile glycerol and keep it at -20 degC
Label master subculture code M2 & M3 on cryovial as a master subculture.
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 takes 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 the 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 a slant and record the date of withdrawal and activation on the slant.
5.9 Viable count
a. Take a viable count of each culture, when the lyophilized 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 a Deep freezer at –20°c to check the viability and effect on culture.
d. Make several serial dilutions 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 was 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 °C
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 102 cfu/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 °C 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 to autoclaving.
5.16 Handling of ATCC/MTCC/NCTC culture
1. Proceed within 2 hours of removal from Deep freezer and dispose of 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 °C = Degree centigrade6.2 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
MMICROBIOLOGICALDEPARTMENT
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
H. Morphology character
Date : Done by Checked by
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