Pure Culture Preparation and

Plating Technique
 Introduction

Culture mixed sample Isolated colony Pure culture

Spreading technique Subculture
Streaking technique
Pouring technique
 Introduction
• Pure culture: represents the growth of a
single species of microorganism.
• Subculture: transferring of microorganism
from one culture medium to another by
subculturing, using specific procedures and
aseptic technique.
• Asepsis: means free from a toxic condition
resulting from the presence of
microorganisms.
 Culture Transfer Instruments

• A pipette is an instrument often used to

transfer aliquots of culture, to prepare serial
dilutions of microorganisms, and to dispense
chemical reagents.
• Inoculating needles and loops are used to
aseptically transfer microorganisms from
broth, slant, or agar cultures to other media.
 Maintenance of pure culture
• Short-term maintenance:
- generally between one to three months.
- can often be achieved by storing slant cultures
in the refrigerator at 4° to 10°C.
• Long-term maintenance:
- The best way to preserve many stock cultures
for long periods is through lyophilization
(freeze-drying).
 Plating Techniques
Spread-Plate Technique
1. A small volume of dilute bacterial mixture
containing 100 to 200 cells or less is
transferred to the center of an agar plate.
2. spread evenly over the surface with a sterile,
L-shaped glass rod.
• The glass rod is normally sterilized by dipping
in alcohol and flamed to burn off the alcohol.
 Spread-Plate Technique

1 2
Spread-Plate Technique
 Plating Techniques
Streak- Plate Technique
1. In this technique, the bacterial mixture is
transferred to the edge of an agar plate with
an inoculating loop and then streaked out
over the surface in one of several patterns.
2. The key principle of this method is that by
streaking, a dilution gradient is established
on the surface of the plate as cells are
deposited on the agar surface.
Streak- Plate Technique
 Plating Techniques
Pour-Plate Technique
1. The original sample is diluted several times to
reduce the microbial population sufficiently
to obtain separate colonies upon plating.
2. The small volumes of several diluted samples
are added to sterile petri plates and mixed
with liquid media agar that has been cooled
to about 48° to 50°C.
3. used with bacteria and fungi.
Pour-Plate Technique
Pour-Plate Technique
An inoculated plate is always incubated in an
inverted position, Why????
Procedure for Isolation of Pure
Cultures