STOCK CULTURE

03 Jun 2010

SS Lab 1
 STOCK CULTURE

AIM :
• Introduction
• Preparation of Stock Culture
• Maintenance of Stock Culture

SS Lab 2
 INTRODUCTION
• Pure culture
– traceable to ATCC*
– consist of only one type of microorganism
• Primary culture
– subculture from pure culture
• Stock culture
– propagation of primary culture
• Working culture
*American Type Culture Collection SS Lab 3
 PREPARATION
KWIK STICK
(PURE CULTURE) INCUBATE

INCUBATE STOCK CULTURE

CRYOGENIC BEADS REFRIGERATE INCUBATE

FREEZE AGAR SLANT

(PRIMARY CULTURE)

WORKING
REVIVAL CULTURE

SS Lab 4
CULTURE MAINTENANCE
KWIK STICK
(PURE CULTURE)

PRIMARY CULTURE
(CRYOGENIC BEADS)

STOCK STOCK STOCK

CULTURE CULTURE CULTURE

WORKING WORKING WORKING

CULTURE CULTURE CULTURE

POSITIVE NEGATIVE
SS Lab 5
CONTROL CONTROL
KWIK-STIK

SS Lab 6
 KWIK-STIK
• Each pack contain lyophilized pellet of a known
organism
• Tear off pull-tab portion of the label and attach to
Stock Culture Record Book
• Pinch the bottom of ampule in the cap to release
the hydrating fluid
• Hold vertically and tap to facilitate flow of hydrating
fluid through shaft into bottom containing the pellet

SS Lab 7
 KWIK-STIK
• Crush the pellet and mix in hydrating fluid by
pinching
• Immediately saturate swab in suspension
• Inoculate on agar slant
• Immediately incubate the agar slant
• Decontaminate used Kwik-Stik before disposal

SS Lab 8
 KWIK-STIK
Bacteria Yeast & Mold
• TSA • SDA or PDA
• 35oC • 22 - 25oC
• 24 – 48 hours • 5 – 7 days
• TSA may be used with
additional 1 day
incubation

SS Lab 9
MICROBANK

SS Lab 10
 MICROBANK
• Each Vials contain cryogenic fluid and 25 beads
• Unused vials may be stored at 4oC or room
temperature
• Cryogenic fluid should be clear
• Different vial colour is for easy identification
only

SS Lab 11
 MICROBANK
• Use aseptic technique
• Inoculate pure culture on agar slants
• Incubate at 35oC for 18 – 24 hours (depending
on type of organism)
• Transfer several loopfuls to a cryogenic vial
• Mix by inverting a few times (do not vortex) and
allow to stand at room temp for 15 – 20 minutes
• Remove excess cryogenic fluid with pasteur
pipette
• Immediately store vials in freezer -70 to -20 oC

SS Lab 12
 MICROBANK
• Remove one bead using sterile needle or
straight wire or forceps into revival broth
• Return vial to freezer immediately
• Incubate to revive culture (according to type of
organism)
• Streak broth on agar slants
• Incubate to create primary or stock culture

SS Lab 13
 STREAKING METHOD
• Uses more media • Uses less media
• Use up more • Easy to manage
incubation space

Agar Plate Agar Slant

SS Lab 14
 STREAKING METHOD
• Various styles and function

To propagate To confirm different colonies

SS Lab 15
 STREAKING METHOD
• Various styles and function
• To isolate or create pure culture

SS Lab 16
STREAKING METHOD

SS Lab 17
 1. PURE CULTURE
Media Temp Time
Bacteria TSA 35 + 1oC 18 – 24 hours
Y&M PDA 22 - 25oC 5 – 7 days

• Inoculate media with Kwik-Stik

• Incubate
• Transfer several loopful to cryo solution
• Allow to stand for 30 seconds
• Remove excess fluid with sterile pipette
• Store vials in freezer (no expiry date)  primary
SS Lab 18
 2. REVIVAL
• Remove one bead with sterile forceps or needle
• Place bead in TSB
• Incubate
• Verify the organism (refer positive control
procedure for the respective organism)
• Check viability before used to make stock culture
• Maintain a Stock Culture record

SS Lab 19
 3. STOCK CULTURE
• Streak on TSA slants
• Incubate
• Store by refrigerating TSA slants at 2-8 oC
• Expiry is 6 months from date of refrigeration
• Maintain a Stock Culture record

SS Lab 20
 4. STOCK CULTURE
• Wash off colonies on TSA slants with Buffer
• Inoculate a quantity of the suspension to
determine the microbial concentration
• Dilute with buffer if necessary
• Store by refrigerating suspension at 2-8 oC
• Expiry is 1 month from date of refrigeration
• Maintain a Stock Culture record

SS Lab 21
 5. WORKING CULTURE
• Remove stock culture from refrigeration
• Incubate (to revive organism)
• Check viability
• Expiry is 1 week from date of revival
• Maintain a Stock Culture record

SS Lab 22
 6. QC RECORDS

Culture : E. coli Batch No. :

Pure culture Primary culture Stock culture Working culture
Date
Quantity Expiry Quantity Expiry Quantity Expiry Quantity Expiry

1 Jan 1 -

2 Jan 10 vials -

3 Jan 6 tubes 6 mth

5 Jan 1 tube 1 wk

12 Jan 1 tube 1 wk

19 Jan 1 tube 1 wk

26 Jan 1 tube 1 wk

SS Lab 23
 Positive Control

• To verify storage condition does not have any

deleterious effects on efficacy of petrifilm
• To verify petrifilm is capable to promote growth of
specific microorganism
• To verify petrifilm is capable to produce the correct
reaction with colour indicator
• To verify diluent used does not have any deleterious
effects on microorganism
• To verify incubation temperature does not have any
deleterious effects on growth

SS Lab 24
 Negative Control

• To verify petrifilm is sterile (not contaminated) eg due

to plating technique
• To verify petrifilm does not promote growth of other
microorganism (which was inhibited) eg fungal
• To verify petrifilm is specific for the respective
microorganism eg S.aureus
• To verify petrifilm does not produce colour reaction
with microorganism other than the one specified eg
E.coli

SS Lab 25
 The End

Question & Answer

SS Lab 26