METHODS OF

ISOLATING PURE
CULTURES
1) STREAK AND SPREAD PLATE
• BY MEANS OF A TRANSFER
LOOP, A PORTION OF A
BACTERIAL SPECIMEN IS
PLACED ON THE SURFACE
OF AN AGAR MEDIUM AND
STREAKED OR SPREAD
OVER THE SURFACE
• THIS THINS OUT THE
BACTERIA ON THE AGAR
SURFACE SO THAT
INDIVIDUAL BACTERIA
ARE SEPARATED FROM
EACH OTHER
2. POUR PLATE METHOD
• DILUTION ( THINNING ) OF
THE SPECIMEN IN TUBES OF
LIQUID (COOLED) AGAR
MEDIUM
• MEDIUM IS MAINTAINED IN
A LIQUID STATE AT 45OC TO
ALLOW THOROUGH
DISTRIBUTION OF
INOCULUM WITHIN THE
MEDIUM
• THE INOCULATED MEDIUM
IS POURED INTO PETRI
DISHES, ALLOWED TO
SOLIDIFY, THEN INCUBATED
3. ENRICHMENT CULTURE
TECHNIQUE
• PROVIDES A SPECIALLY
DESIGNED CULTURAL
ENVIRONMENT ( A MEDIUM OF
KNOWN COMPOSITION AND
SPECIFIC CONDITIONS OF
INCUBATION) WHICH WILL
FAVOUR THE GROWTH OF THE
PARTICULAR TYPE OF
BACTERIUM
• USED WHEN THE BACTERIUM
TO BE ISOLATED IS PRESENT IN
RELATIVELY SMALL NUMBERS
AND GROWS SLOWLY THAN
THE OTHER SPECIES
4. SERIAL DILUTION TECHNIQUE
• USED IF ORGANISM
BEING SOUGHT IN A
MIXED CULTURE IS
PRESENT IN GREATER
NUMBERS THAN
OTHER ORGANISMS
• DONE IN A SERIES OF
DILUTIONS IN TUBES
WITH APPROPRIATE
MEDIUM
5. SINGLE CELL ISOLATION
• USE SPECIAL EQUIPMENT,
MICROMANIPULATOR,
ALLOWING THE OPERATOR
TO CONTROL THE
MOVEMENT OF THE
MICROPIPETTE OR
MICROPROBE (FINE NEEDLE)
IN A HANGING DROP SO
THAT A SINGLE CELL CAN
BE TAKEN INTO THE TUBE
AND TRANSFERRED TO A
SUITABLE MEDIUM FOR
GROWTH
 PRESERVATION
TECHNIQUES FOR PURE
CULTURE
 1. PERIODIC TRANSFER TO
FRESH MEDIA

***THINGS TO CONSIDER:
• PROPER MEDIUM FOR EACH SPECIES
• PROPER TEMPERATURE FOR STORAGE
• TIME INTERVAL FOR THE TRANSFER
2.
2. PRESERVATION BY
OVERLAYING WITH
MINERAL OIL
LYOPHILIZATION
• RAPID DRYING UNDER
FROZEN STATE
• THE CELL SUSPENSION IS
PLACED IN SMALL VIALS, WHICH
ARE IMMERSED IN A MIXTURE OF
DRY ICE AND ALCOHOL (-78OC)
• VIALS ARE CONNECTED TO A
HIGH VACUUM LINE
• ADVANTAGES:
1. LONG TERM SURVIVAL
2. LESS CHANGE IN
CHARACTERISTICS OF CULTURE
3. SMALLNESS OF STORAGE
CONTAINERS
FUNDAMENTALS OF LYOPHILIZATION PROCESS
4. STORAGE AT LOW
TEMPERATURE
 STERILIZATION

• IS THE COMPLETE DESTRUCTION OF

PATHOGENIC ORGANISMS
TYPES OF STERILIZATION:

1. STERILIZATION WITH HEAT

2. STERILIZATION WITHOUT
HEAT
STERILIZATION
WITH HEAT
(DRY)
OVEN ( 160 – 170OC FOR 2 HOURS )
INCINERATION
STERILIZATION
WITH HEAT
(MOIST)
BOILING WATER( 80OC; KILLING EFFECT
INCREASED WITH ADD’N OF 2%
SODIUM CARBONATE)
STEAM
 AUTOCLAVE
( 15 PSI FOR 15 MIN)
STERILIZATION WITHOUT HEAT
A) CHEMICAL – ETHYLENE OXIDE
B) RADIATION
2 MAIN GROUPS:
B.1) IONIZING RADIATIONS
- X-RAYS, GAMMA, BETA
– SHORT WAVELENGTHS (0.06 – 2 AO)
B.2) UV RAYS
- LONG WAVELENGTHS (180 – 3900 AO)
C) FILTRATION
ETHYLENE OXIDE STERILIZATION
(CHEMICAL)
FILTRATION

 Membrane filters (0.025 – 8

µm)
 Berkefield filters
 Asbestos pads (Seitz filter)
 Sintered glass