Maintenance of Microbial Cultures

Learn how to procure, maintain the Microbial Cultures by sub culturing and destruction of
microbial cultures.
Ankur Choudhary | Microbiology Be the first to comment!

1.0 Equipments required

LAF
Wire loop

2.0 Material Required

Microbial Culture
Sterile Saline solution
Sterile slants

3.0 Utilities Required

NA

4.0 Procedure:

4.1 Procurement of cultures:

4.1.1 Prepare an indent for Microbiological cultures with their strain numbers
and get it verified from your HOD and finally Approved from Manager - QA
4.1.1 Send the indent copy to Purchase department for further action.
Purchase department must arrange the cultures as per the specification
given in indent copy, from approved source with their authentic certificate.
4.1.2 Generally Microbial cultures must be procured from any of the source
listed below-
American Type Culture Collection
12301, Park Lawn Drive,
Rockville, MD 20852
or
Institute of Microbial Technology
Sector 39A, Chandigarh - 160 036
or
National collection for Industrial Micro Organism
Division of Biochemical Sciences
National Chemical Laboratory
Pune 411 008

4.1.3 Following is the list of standard culture with ATCC Nos. and their
equivalent number of other agencies.
Sr Name of ATCC NCIB NCIM MTCC
. Culture
N
o
1 E. coli 8739 8545 2065 4313
2 Bacillus subtilis 6633 8054 2063 441
3 Candida 10231 - 3471 227
albicans
4 Cl. sporogenes 19404 5326 5125 2684
5 Ps. aeruginosa 9027 - 2200 1688
6 A. niger 16404 - 1196 1344
7 Salmonella NCTC - 2257 733
6017
8 S. epidermidis 12228 8853 2493 3615
9 Staph. aureus 6538 8625 2079 737
4.1.4 After receipt, record all the details and labeled as mother culture
suitably batch numbered and store at 2 80C.

4.2 Method of Sub culturing

4.2.1 Operate LAF as per SOP for operating instruction for LAF.
4.2.2 Wipe out the outside surface of the culture slant tubes / lyophilized
ampoules with cotton soaked with 70% IPA.
4.2.3 Get fresh slant/stab tube and wipe out outside surface of the tube with
cotton wool soaked in 70% IPA.
4.2.4 Hold on the extreme end and heat the nichrome wire loop on the
reducing flame till red-hot.
4.2.5 Allow to cool the wire loop and slowly open the cotton plug of old
culture tube and gentle warm the mouth of the tube.
4.2.6 Pick up the smear/growth with the help of loop after removing cotton
plug of the slant tube.
4.2.7 Hold the nichrome wire loop with smear/culture growth, plug the
culture slant tube, and put aside the old slant culture.
4.2.8 Open the fresh slant tube, warm the mouth of the tube holding in
other hand and carefully transfer the culture that is held on the loop.
4.2.9 Spread the culture spirally on the surface of the slant, warm the mouth
of slant and plug it.
4.2.10 For anaerobic bacteria, insert the loop into a deep stab of slant, warm
the mouth of slant and plug it
4.2.11 Label the culture tube, incubate at specified temperature and period,
and then store it in refrigerator at 2 80C

4.3 Maintenance of Microbial culture

4.3.1 If the Mother culture is lyophilized, score the middle of the ampoule
with an ampoule cutter.
4.3.2 Disinfect the ampoule with sterile 70% v/v IPA solution which is
moistened with lint free cotton.
4.3.3 Wrap the sterile cotton cloth around the ampoule and break it
carefully.
4.3.4 Aseptically add about 0.2 ml of sterile saline solution with the help of
micropipette and mix well.
4.3.5 Transfer a loop full suspension in a duplicate sterile media slant and
incubate at a temperature as stated in table No. 1, and observe the growth
after incubation
4.3.6 If the culture is in vegetative form (slant culture), check the purity of
the cultures by staining method and record the observation in Format
4.3.7 If the cultures are not showing their physical characteristic, discard the
culture as per SOP for destruction of Microbial wastes.
4.3.8 After ensuring the purity of Mother culture, prepare three Master
Culture of each culture by transferring a loop full suspension from mother
culture to fresh slant, and treat as a MC1, MC2 and MC3.
4.3.9 Incubate both the tubes at specified temperature and period, as
indicated in Table 1 and observe the growth after incubation.
4.3.10 Check the purity of culture by staining and record their physical
characteristic as per format
4.3.11 If the cultures are not showing their physical characteristic, discard
the culture as per SOP for destruction of Microbial Waste
4.3.12 If the culture showing their physical characteristics as stated in Table
No: 1, transfer a loop full growth from MC1 to a fresh seven set of slant and
treat as M1, M2, M3, M4, M5, M6 and M7 respectively.
4.3.13 Incubate the all the slant at specified temperature and period, and
assign batch No. I.e. MC1/M1, MC1/M2, MC1/M3 up to MC1/M7.
4.3.14 After incubation observe the growth and label the culture as per
sample given in Annex-1, store the culture at 2 8 0C for their specific period.
4.3.15 Use the prepared culture as per Schematic diagram specified in
annexure-1.
4.3.16 For the 1st month, 2nd month, 3rd month, 4th month, 5th month and
6th month use M1, M2, M3, M4, M5 and M6 respectively for preparation of
weekly cultures. If one of the above culture get contaminated use M7 culture.
4.3.17 Transfer a loopful growth from Monthly Cultures i.e. M1, M2, M3, M4,
M5, & M6 after ensuring the purity of cultures to a five fresh slant and treat
as a W1, W2, W3 W4 and W5. Assign the batch number i.e. MC1/M1/W1,
MC1/M1/W2, MC1/M1/W3, MC1/M1/W4 and MC1/M1/W5.
4.3.18 After incubation observe the growth and label the culture as per
sample given in Annex-1, store the culture at 2 8 0C for their specific period.
4.3.19 For 1st, 2nd, 3rd and 4th Week of every month use W1, W2, W3 and W4
cultures respectively. If one of the above culture get contaminated use W5
culture. Prepare daily working cultures after ensuring the purity of Weekly
culture by transferring a loopful growth to a fresh four slants and treat as a
D1, D2, D3 and D4. Assign batch number i.e. MC1/M1/W1/D1 to
MC1/M1/W1/D4.
4.3.20 Incubate all the slant at specified temperature and period (Table 1),
observe the growth and labeled properly. Store the culture at 2 8 0C for their
specific period.

4.4 Usages and Destruction of Microbial Cultures

4.4.1 For Daily Microbiological Analysis use only Daily Working Culture. Use
one D1 culture for two days, D2 for next two days like this, D3 and D4
respectively.
4.4.2 After completion of work discard the culture slant by the addition of
disinfectant solution for 30 minutes and then autoclave at 121 0C / 15 psi
Pressure for 30 minutes and record the destruction details.
Specimen Label
Culture Name :
Strain No. :
Batch No. :
Gen. No. :
Sub. Date :
Use before :
Storage Cond. :

Table 1
Sr. Name of Media Incubat Physical Incubat
No. the culture slant to ion characteri ion
be used Temp. stics Period
1 E.coli Nutrient 30 Gram ve, 24 - 48
NCIM 2065 agar 350C short rods hrs
2 Staph.aureu Nutrient 30 Gram +ve, 24 - 48
s agar 350C cocci in hrs
MTCC 737 clusters
3 Salmonella Nutrient 30 Gram ve 24 - 48
NCIM 2257 agar 350C rod shaped hrs
4 Ps.aeruginos Nutrient 30 Gram ve 24 - 48
a agar 350C rod shaped hrs
NCIM 2200
5 C.albicans Sabouraud 20 Oval 48 72
NCIM 3471 dextrose 250C shaped hrs
agar
 6 S.epidermidi Nutrient 30 Gram +ve 24 - 48
s agar 350C cocci shape hrs
NCIM 2493
7 B. subtilis Nutrient 30 Gram +ve, 24 - 48
NCIM 2063 agar 350C rods hrs
8 Cl. Cooked 30 Gram +ve, 24 - 48
sporogenes Meat 350C rods hrs
NCIM 5125 Medium
9 A. niger Sabouraud 20 Mycelial 48 120
1196 dextrose 250C spores hrs
agar
10 Environment Nutrient 30 Gram +ve, 24 - 48
al flora EF- 1 agar 350C cocci shape hrs
(Kocuria
marinus)
MTCC 8772
11 Environment Nutrient 30 Gram +ve, 24 - 48
al flora EF- 2 agar 350C cocci shape hrs
(Staphyloco
ccus
arlettae)
MTCC 8773
12 Environment Nutrient 30 Gram -ve, 24 - 48
al flora EF- 3 agar 350C rod shape hrs
(Ralstonia
mannitolilyti
ca)
MTCC 8774

5.0 Precaution

5.1 After transferring the culture, always return the cotton plug or caps to
the correct tubes.
5.2 Never forget to sterilize the inoculating loop before returning it to the
holder.
5.3 Always flame the tips of the tube before you insert the loop and after
withdraw the tube.
5.4 The plug after removal must be kept in hand. Never place the plug on
the work surface or touch it to anything except the lamed lip of culture tube.
5.5 Do not dip the needle into the agar while inoculating slant.
5.6 Never leave the culture tube open for longer time than the time needed
to transfer the culture.

6.0 Frequency
6.1 First Generation Once in Year
6.2 Second Generation Once in Six month
6.3 Third Generation Monthly

7.0 Abbreviation

SOP : Standard Operating Procedure

QA : Quality Assurance
QC : Quality Control
NA : Not Applicable
LAF : Laminar Air Flow
IPA : Isopropyl Alcohol