ASEPTIC TECHNIQUES

TRANSFER AND ISOLATION

TECHNIQUES OF MICROBES IN
THE ENVIRONMENT
Outline
• Terms
• Inoculation Methods and Techniques
• Aseptic Techniques
Terms
Culture media
- nutrient materials prepared for the growth of
microorganisms in the laboratory

-
Terms
Inoculum
 microbes introduced into a culture medium
to initiate growth

MICROORGANISMS
Terms
Inoculation
 process of transferring or isolating
microorganisms from clinical specimen
or from pure culture to a culture medium
Terms
Culture
- microbes that grow and multiply in or on
a culture medium
 Terms: Types of Culture
1.Pure culture
- has only 1 species/strain of
microorganism

2. Mixed culture
- has ≥2 species/strains of
microorganisms
Terms: Types of Culture
3.Contaminated
- unwanted microorganisms are accidentally grown

ex. fungi in bacterial medium

 Terms
Aseptic techniques
- procedures/ techniques used to
prevent contamination
Colony
- visible growth on surface of
solid medium
- visible mass of microbial cells
arising from one cell or from a
group of the same microbes
Fishing out
- process of picking out a single
colony so as to prepare a smear
or subculture
Inoculation Methods
 INOCULATOR IS MADE UP OF PLATINUM/NICKEL/ NICHROME WIRE
1. streaking- using inoculating loop (plated media)
inoculating loop (slant)

SLANT
2. stabbing – using inoculating needle

BUTT/DEEP
3. swabbing –using sterile swab
Aseptic/Sterile Techniques
Preparation
• Disinfect working area
• Clean and sterilize glassware
• Keep dirty things off table
 Aseptic Techniques: Handwashing
• BEFORE ANYTHING

• Single most important

measure to reduce
transmission of
microbes from person to
person, or from one site
to another on the same
patient
Challenge:
• Lax /
Katamad
• Wear gloves , cap, mask
and laboratory gown
• Use aseptic techniques in
inoculating plated
medium, butt-slant,
butt/deep, slant/slope
1. Flame sterilization is an easy method to ensure
sterile transfer of a culture from a source to
growth medium

Flaming the inoculator

- quick method of killing
microorganisms
- heat whole length of
wire
- red hot
 Tube-tube Inoculation: From Broth to Broth

• Remove cap/cotton using dominant hand’s 4th+5th finger

• Flame mouth of the tube
Incubate • Insert loop into culture (should NOT be hot)
18-24 h • Reflame tube and replace cap/cotton
At 37oC
Broth should NOT sizzle
Tube-tube Inoculation: From Broth to Broth

• Remove cap/cotton using • Insert loop into culture

dominant hand’s 4th+5th (should NOT be hot)
finger • Remove loop from
• Flame mouth of the tube culture & quickly transfer
to sterile broth/water)

Incubate 18-24 h
At 37oC
 Tube-tube Inoculation: From Broth to Slant

Incubate for
18-24 hours
at 370C

• Remove cap/cotton using dominant hand’s

4th+5th finger
• Flame mouth of the tube
• Inoculate in zigzag pattern. DO NOT STAB.
• Reflame tube and replace cap/cotton
 TUBED MEDIA
SLANT/SLOPE
- inoculating loop
- streak the surface from
the bottom going
upward in a zigzag
manner
 Tube-tube Inoculation: From Broth to Slant

• Remove cap/cotton using

Incubate for dominant hand’s 4th+5th finger
18-24 hours • Flame mouth of the tube
at 370C
Tube-tube Inoculation: From Broth to Butt-Slant
• Use NEEDLE
• Gloves still advised

Incubate for 18-24 h

at 370C
 TUBED MEDIA

BUTT-SLANT
- inoculating needle
- stab the center of
the butt

- streak the slant in a

zigzag manner
 TUBED MEDIA
BUTT/DEEP
- inoculating needle
- stab from the center and
withdraw thru the same
route
 Plate-tube Inoculation: From Plate to Broth

Incubate for
24 hours at
370C
 To isolate microorganisms from a Pure Culture:
Tube-Plate Inoculation: From Broth to Plate

Incubate
INVERTED for
18-24 h at 37oC
A. Simple Streak
Method
• pure culture
• introduce inoculum on
one side of the plate
• Spread across the
surface of the agar
• Streaks should be
parallel to each other
• Streaks should not
overlap the other lines
B. Radial Streak
Method
• used in hospitals
• pure culture
• Introduce the inoculum
on one side of the plate
• streak on the opposite
side in a concentric
fashion
• Sterile cotton swab
C. Multiple Streak Method

• Pure stock culture

• done when available culture medium is not
sufficient for the number of specimens
• divide plate into several sections and streak
each separately in a zigzag motion.
D. Overlap Method
- sensitivity or antimicrobial test
- pure culture is swabbed 3x in MHA
- use sterile cotton swab
 Pure culture

Incubate at 37oC for 18-24hrs

To isolate microorganisms from a Mixed Culture:
Tube-Plate Inoculation: From Broth to Plate

Incubate
INVERTED for
18-24 h at 37oC
E. Clock Method (mixed culture)
• To dilute the inoculum
sufficiently so that well
defined isolated colonies of
bacteria can be obtained from
mixed culture
• Introduce inoculum on one
side
• Streak half of the plate in a
zigzag motion
• Turn the plate clockwise at
90°
• Streak 1/4 of the plate
touching the last two lines of
the first streak
• Repeat the procedure
Clock Method
- mixed culture

• streaked
back and forth into each
quadrant (turning plate at 90
degrees angle)
 Fishing out colonies from solid medium:

Incubate for
24 hours at
370C
 Mixed Culture

Incubate at 37oC for 18-24 hours

Fish out isolated colony from the 3rd set of streaks