ASEPTIC TECHNIQUES INOCULATION TECHNIQUES

Tubed Media
Culture media  Slant/Slope
- nutrient materials prepared for the growth of - inoculating loop
microorganisms in the laboratory - streak the surface from the bottom going
Inoculum upward in a zigzag manner
- microbes introduced into a culture medium to
initiate growth  Butt/Deep
Inoculation - inoculating needle
- process of transferring or isolating - stab from the center and withdraw thru the
microorganisms from clinical specimen or same route
from pure culture to a culture medium
Culture  Butt-Slant
- microbes that grow and multiply in or on a - inoculating needle
culture medium - stab the center of the butt
- streak the slant in a zigzag manner
TYPES OF CULTURE
 Pure culture - has only 1 species/strain of A. Simple Streak Method
microorganism - pure culture
 Mixed culture - has ≥2 species/strains of - introduce inoculum on one
microorganisms side of the plate
 Contaminated - unwanted microorganisms - Spread across the surface of
are accidentally grown ex. fungi in bacterial the agar
medium - Streaks should be parallel to each other
- Streaks should not overlap the other lines
Aseptic techniques
- procedures/ techniques used to prevent B. Radial Streak Method
contamination - used in hospitals
- pure culture
Colony - Introduce the inoculum on
- visible growth on surface of solid medium one side of the plate
- visible mass of microbial cells arising from one cell or - streak on the opposite
from a group of the same microbes side in a concentric
fashion
Fishing out - Sterile cotton swab
- process of picking out a single colony so as to
prepare a smear or subculture C. Multiple Streak Method
- Pure stock culture
INOCULATION METHODS - 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.
Inoculator is made up of platinum/nickel/nichrome
wire D. Overlap Method
- sensitivity or antimicrobial test
1. Streaking - pure culture is swabbed 3x in MHA
- using inoculating loop (plated media), > Mueller-Hinton (antibiotic
inoculating loop (slant) susceptibility testing)
2. Stabbing - use sterile cotton swab
- using inoculating needle
3. Swabbing E. Clock Method (mixed culture)
- using sterile swab - 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
- Streaked back and forth into each quadrant
(turning plate at 90 degrees angle)
- Incubate at 37oC for 18-24 hours
- Fish out isolated colony from the 3rd set of
streaks
ASEPTIC/STERILE TECHNIQUES
PREPARATION
 Disinfect working area
 Clean and sterilize glassware
 Keep dirty things off table
 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
- Duration: 40 to 60 seconds
1. Before touching a patient
2. Before clean/aseptic procedure
3. After body fluid exposure risk
4. After touching a patient
5. After touching patient surroundings
 Wear gloves , cap, mask and laboratory gown
 Use aseptic techniques in inoculating plated
medium, butt-slant, butt/deep, slant/slope
 Flame sterilize inoculating loop
 Incubate 18-24 hours at 37oC
From Broth to Butt-Slant
 Flame sterilize inoculating needle
 Remove cap/cotton using dominant hand’s
4th + 5th finger
 Flame mouth of the tube
 Insert needle into culture (should NOT be hot)
 Reflame tube and replace cap/cotton
 Flame sterilize inoculating needle
 Incubate 18-24 hours at 37oC
From Broth to Slant
 Flame sterilize inoculating loop
 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
 Flame sterilize inoculating loop
 Incubate 18-24 hours at 37oC
PLATE-TUBE INOCULATION:
From Plate to Broth
 *same crap*
 Incubate for 24 hours at 370C
To isolate microorganisms from a Pure Culture:
TUBE-PLATE INOCULATION:
From Broth to Plate
 *same crap*
 Incubate INVERTED for 18-24 h at 37oC
To isolate microorganisms from a Mixed Culture:
TUBE-PLATE INOCULATION:
From Broth to Plate
 *same crap*
*overlap streaks only once or twice*
 Incubate INVERTED for 18-24 h at 37oC

1. Flame sterilization is an easy method to Fishing out colonies from solid medium:
ensure sterile transfer of a culture from a  Incubate for 24 hours at 370C
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
 Flame sterilize inoculating loop
 Remove cap/cotton using dominant hand’s
4th + 5th finger
 Flame mouth of the tube
 Insert loop into culture (should NOT be hot)
 Reflame tube and replace cap/cotton
Steps in Preparing PLATED Media 5. STERILIZE by autoclaving at 121C for 15mins.
1. WEIGH dehydrated medium at 15psi.
> Aluminum boxes 6. FORM the tubed media. (allow the culture
> spatula media to solidify).
2. DISSOLVE culture medium in ERLENMEYER
FLASK by constant stirring and with the aid of After the culture medium solidifies, place gum
heat until the culture medium becomes clear label on each of the test tube
and straw colored ▪ Indicate the name of the culture medium, group
3. PLUG the mouth of Erlenmeyer flask with number, year and section
GAUZE and cover with aluminum foil. Secure ▪ Place in a beaker
the foil using ▪ Wrap in aluminum foil. Secure with masking
masking tape. (Grp. Number, Yr.& Sec. and tape. Write your group number and section.
name of culture medium) ▪ Place inside the biological ref.
> Plug should not be too tight or too
loose
4. STERILIZE by autoclaving at 121C for 15mins.
at 15psi. (place a strip of moist heat indicator
before autoclaving)
5. DISPENSE in PETRI DISHES (20 ml/petri dish)
6. FORMATION
- Allow the plated culture medium to solidify
on a flat surface
- Petri dish can be partially opened to prevent
too much moisture.
After the culture medium solidifies, place gum label
on the cover of the petri dish
▪ Indicate the name of the culture medium, group
number, year and section
▪ Wrap in aluminum foil. Secure with masking
tape. Write your group number and section .
▪ Place inside the biological ref in INVERTED
position.

Steps in Preparing TUBED Media

1. WEIGH the dehydrated medium.
2. DISSOLVE culture medium in a BEAKER by
constant stirring and with the aid of heat until
the culture medium becomes clear and straw
colored.
3. DISPENSE in test tubes.
- for solid or liquid tubed media DISPENSE first
before STERILIZATION
(Screw capped test tube – butt slant -
dispense 7mL
Loffler's test tube – slant - dispense 5mL
Wasserman test tube – butt – dispense 3 mL
Kahn test tube - Nutrient broth – 2 ml
DO NOT USE PIPETTE TO DISPENSE TUBED
MEDIA
4. PLUG
- plug the mouth of test tubes using cotton
- place the test tubes in a beaker cover with
aluminum foil
- secure with masking tape. Write the Grp.
Number year and section and the name of the
culture medium