ACTIVITY #2

ASEPSIS and ASEPTIC TECHNIQUE

INTRODUCTION

Aseptic technique is a precautionary procedure done to prevent

contamination by microorganisms. Two general procedures can be under taken
in performing aseptic technique procedures and theses are Sterilization (removal
of all microbial growth and eradication of spores) and Disinfection (process of
removing pathogenic microorganism/s and contaminants). These procedures
are employed separately or in combination to ensure purity of the
microorganism/s being studied. Asepsis on the other hand, is aseptic procedure
usually practiced in hospitals and clinics to eliminate growth of pathogenic
organisms.

Disinfection can be accomplished through pasteurization, blanching, use

of detergent/soap and use of chemical disinfectants such as hypochlorite (4%)
and isopropyl alcohol (70%).

On the other hand, Sterilization can be achieved by: direct flaming, use of
dry heat (oven dry), use of steam under pressure (autoclaving), irradiation,
filtration and tyndallization.

Objectives:

At the end of the experiment, the students are expected to:

1. Practice and demonstrate the different basic aseptic techniques

procedures done in the laboratory;

2. Fill in the table by studying the situation and determine the procedure
needed to have asepsis; and

3. Be familiarized with the parts and operation of the two most commonly
used laboratory equipment being used in sterilization.
 METHODOLOGY

Materials

Aluminum foil *Alcohol lamp

Cotton *Test tube rack
Chlorox & hand soap Test tubes
95% 7 70% ethanol Petri plates
Inoculating needle * Pipette
Inoculating loop
Lighter Isolation chamber
Masking Tape Autoclave
Medical Gauze Laboratory oven
Methods

Pay attention as your laboratory instructor demonstrate the different basic

aseptic technique procedures being employed in the laboratory: Practice and
master these procedures. Ask the laboratory aide/technician to enumerate the
parts and demonstrate to the class the operation and maintenance of the
equipment being used in sterilization (autoclave and oven).

1. Hand and Surface disinfection

Remove all jewelries on hand and using your personal ethyl alcohol, wet
your hands from finger to your elbow. Air dry your hands for one minute before
performing any microbiological procedure.

(picture courtesy of DOH campaign for disease free country)

 To perform surface disinfection, Wet a piece of cotton with 5% chlorox and
wipe the surface of your working area (open air table toop isolation)or the same
inside the isolation chamber.

NOTE: if there is spillage of microorganisms, cover the area with cotton

soaked in chlorox/disinfectant then wipe the contaminated area with fresh
cotton soaked in disinfectant solution.

2. Direct Flaming (inoculating needle and inoculating loop)

Pass the needle/loop slowly over the hottest portion of the flame (bluish)
three times back and forth from tip to base/edge of handle until the inoculating
needle or inoculating loop becomes red-hot.

After flaming, avoid the inoculating needle and inoculating loop to come
into contact with any unsterilized surface. Allow inoculating needle and
inoculating loop to cool for a while before using it.

NOTE: Flaming of the inoculating needle and inoculating loop must be done
before and after using it.
3. Inoculating a test tube ( tube to tube).

Hold the two test tubes in one hand. Using the middle and ring fingers of
your free hand, remove the cotton plug of the source test tube. Avoid the cotton
plug to touch any surface. Hold the source test tube in inclined position and flame
the mouth.

Hold the mouth of the test tube about three (3) inches above the flame
and insert a pre-cooled inoculating loop/ inoculating needle to obtain a small
amount of inoculum. Withdraw the inoculating needle/ inoculating loop carefully,
avoid touching the wall and mouth of the test tube. Gently flame the mouth of
the test tube and recap/ reinsert the cotton plug.

Remove the cotton plug of the other test tube to be inoculated, flame the
mouth and hold it three (3) inches above the flame. Insert the inoculating needle/
inoculating loop (without touching the mouth and wall) and streak in zigzag
pattern. Flame the mouth of the tube and insert the plug. Flame the inoculating
needle/ inoculating loop to kill the microorganism left on it.
4. Inoculating a petri plate (tube to plate)

Hold the petri plate with one hand. Place thumb on the center cover of the
petri plate and supporting the bottom with middle and ring finger. Using the index
and little finger, gently rotate the plate to allow the sides of the plates to pass over
the flame. Hold the petri plate two (2) inches near the flame, using your thumb lift
the cover; your index finger will serve as the pivot on the other end support the
bottom with your middle and ring finger. Introduce the inoculum into the petri
plate and inoculate the plate (point or streak). Replace the cover and flame the
sides of the plate by gently rotating it.

5. Using the Laboratory Oven

Dried glass wares like test tube, beaker, pipette, petri plate and beakers are
sterilized using hot air.

To prepare glass wares for oven drying, wash glass wares in detergent, rinse
them thoroughly and air dry. Wrap glassware (test tube, pipette and petri plate)
with aluminum foil or paper (used coupon bonds- use the clean side of the
paper). For beakers and flasks (graduated cylinder and Erlenmeyer) cover the
mouth of the flasks with aluminum foil.

Note: The use of this equipment will be demonstrated by the laboratory

technician. Make sure to identify the parts and their functions as well as operation
before the end of the laboratory time.
 Open the lid/cover of the laboratory oven and place glassware inside the
oven with minimal space in between them. Close the lid/cover and lock it. Set
the temperature setting to 210oC. Plug the AC wire of the oven to the wall socket
and switch it on. Monitor the temperature inside the oven. When the temperature
reached 180oC, reset the temperature setting to 180oC for 2 hours.

After the desired time has been reached, turn off the oven and unplug it
from the wall socket (other brands laboratory oven can be operated with timer).
Monitor the temperature until reaches 40 degrees or lower. DO NOT OPEN the
door of the lab oven while it is HOT.

6. Using the Autoclave

Glassware with liquid like culture media and distilled water are sterilized
using steam under pressure. Since autoclave requires steam, water is added
inside the chamber. Check for the maximum allowed water capacity of the unit
before operating autoclave.

To prepare the glassware, ensure that the liquid inside the glassware (test
tube, beaker or Erlenmeyer flask) does exceed two thirds full of the glassware size
to avoid spillage during sterilization. Use cotton plug for culture media and wrap
it with aluminum foil to prevent being moistened while being sterilized. Close the
lid and lock properly.

To start operating the unit, locate the exhaust valve and keep it open. Plug
the autoclave to the AC wall socket. Check the sterilization setting (The usual
autoclave setting for sterilization is 121oC or 15 psi (pound per square inch)for 15
minutes). Switch the unit on. Close the exhaust valve if vapor/steam starts to come
out of the exhaust valve noozle. Monitor the gauge until the pressure reaches
15psi (121 degrees) then start timer for 15 minutes. Set the switch off after 15
minutes, do not open yet, let pressure inside reached zero before removing
autoclaved materials.
 Name Rating
Course/Year
Lab Sched
Date

ACTIVITY #2

ASEPSIS and ASEPTIC TECHNIQUE

1. Laboratory Sterilizer

Characteristics Autoclave Hot Air oven

Temperature
requirement

Duration of
sterilization

Materials to
be sterilized

2. Define:

a. Inoculum

b. Packeting

c. Irradiation
d. Blanching

3. Identify the activity/industry or sterilization/disinfection being applied in:

Activity/Industry Method being applied

a. Canning
b. Injection of drug
c. Sterilization of surgical
instruments
d. Taking a bath
e. Surgery
f. Washing (clothes)
g. Cooking

4. Can we consider a microorganism as both beneficial and pathogenic? Why?

5. Name foods that are subjected to sterilization procedure prior to its processing
and packing.