Lab 3 - Sterile Technique

Microbiology and Parasitology
Laboratory
Lab Lesson 3 Aseptic Transfers

and Inoculation Procedures
Sterile technique is a common sense system of practices used when handling
microorganisms in culture to prevent contamination of the cultures and those working with them.
These practices succeed only if the tools, culture vessels, and media being used are sterile to
begin with, and if the worker understands how to prevent contamination of the cultures and
workspace. Below are the key aspects of sterile technique.
Due to the ubiquity of microbes in everything microbes comes with it. It is imperative to
remove all forms of life from the glassware, equipment and media to be used in culturing
through sterilization. Sterilization may be accomplished by physical or chemical means.
Common physical means include flaming, dry heat, boiling, steam under pressure, filtration and
radiation. Physical method of sterilization is more practical since it leave no residue that is
inhibitory to microorganisms.
Have a work plan

Always work from a plan or a protocol. Knowing what you need to do and how you will
accomplish it efficiently will minimize the time that cultures are exposed and vastly reduce the
chances of making mistakes.
A clean workspace/ protective clothing

BEFORE and AFTER doing any work with microorganisms, it is critical to disinfect the bench
top or hood with a disinfectant such as alcohol (minimum 70%; ethanol or isopropyl) or other
available disinfectants. Lab coats and gloves should be worn if available. Your hands should be
washed thoroughly and frequently with soapy water.
Sterilization of bench top tools by flaming

At the lab bench, tools may be
quickly sterilized in a couple ways.
Forceps and hockey stick spreaders
should be dipped into an ethanol bath
and then touched to flame. Allow the
alcohol to burn off and the tool to cool
before contacting bacteria. Inoculating
loops and needles can be heated to
red hot flame to sterilize (Fig. 3-1a&b).
ALWAYS allow flamed tools to cool
before touching cultures.
Figure 3-1 (A) Sterilization of inoculating loops
and needles. Flame the loop to red hot
starting at the base of the wire and then
(B) pulling it back through toward the tip.
Protect culture media against airborne contaminants

Culture tubes and Petri plates should never be exposed directly to the air column or your
breath. Any time you must add or remove something from a culture tube, the cap should be
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Lab Lesson 3: Aseptic Transfers and Inoculation Techniques
15
Laboratory
removed and held right-side up to prevent contamination. Never set a cap on the benchtop.
The rim of glass culture tubes should be flamed briefly before and after you go into the tube with
a tool. Holding the tube at an angle also reduces exposure possibilities (Fig. 3-2a&b).
Figure 3-2 (a) Technique for

holding the tubes
and removing the
caps to minimize
airborne
contamination. (b)
Flame the tube
mouth before and
after intervention.
When using Petri dishes, always keep the lid over the surface of the agar and raise it
only enough to do your work (Fig. 3-3).
Figure 3-3. When working with Petri dish cultures, keep the over the agar surface at all times.
Handling of tube cultures
Test tubes are handled in the following

manner (fig. 3-4):
 The test tube is held in the left hand (for
a right-handed person).
 The instrument (loop, pipet, or needle)
is held in the right hand. Sterilize the
instrument before opening the test tube.
 The test tube cap is grasped by the little
finger of the right hand, and removed.
NEVER PLACE THE CAP ON THE Figure 3-4. Correct method of holding tubes when
WORKING BENCH! transferring culture.
 While continuing to hold the cap with the
little finger, the tube rim is lightly flamed
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16
Laboratory
and the instrument is manipulated

appropriately, and withdrawn.
 The tube rim is reflamed before the cap
is replaced on the test tube and the test
tube is put back into the rack.
ACTIVITIES
Objectives
At the end of the exercise the student will be able to:
1) Practice by performing the correct way of flaming the inoculating loops/needles.
2) Demonstrate proficiently the proper way of transferring culture from tube to tube and
tube to a petri dish.
Materials
 4 inoculating loop/needle
 4 petri dishes
 2 alcohol lamps
 2 test tube racks with 4 test tubes containing unknown cultures
Procedure
1. Carry out the proper way of flaming the inoculating loop/needle as follows:
a) Hold the loop like a pen. Incline the loop at 45 O and position it on the middle of the
flame.
b) Allow the loop to be become red hot and pass the entire loop until you reach the
holder. Cool the loop for 10 seconds before using it.
2. Practice transferring of culture from a test tube to another test tube and test tube to a
Petri dish. Follow the steps below.
I. Tube to tube transfer

a) Sterilize the loop. Cool for 10 sec.
b) Loosen the caps of the test tubes but do not remove it.
c) Grab the cap of the source tube (tube nearest the palm) between the 4 th and 5th
fingers and the blank (sterile) tube between the 3rd and 4th fingers.
d) Heat the mouth of both tubes.
e) Take-out a sample and transfer it to the blank tube. Avoid touching the inner
sides of the tube with the loop.
f) Heat the mouth of the tube before returning the caps. The last cap removed
should be the last to be returned.
g) Reflame the loop.
II. Tube to plate transfer

a) Sterilize the loop. Cool for 10 sec.
b) Grab the source tube. Loosen and remove the cap of the tube.
c) Heat the mouth and fish out a sample.
d) Reheat the mouth before returning the cap.
e) Open the lid of the plate.
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17
Laboratory
f) Streak on the surface

g) Cover the plate and heat the rim.
h) Reheat the loop before placing it down.
3. Demonstrate the sterile techniques you have practiced to your lab instructor.
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18
Laboratory
Name: _____________________________ Score: ____________________

Lab Schedule: _______________________ Term/Date: ___________________
Aseptic Transfers
and Inoculation Procedures Lab Report 3
Results and Observation Attendance
prelab: _______________
postlab: ______________
Demonstration
a) Flaming the loop/needle Score: _________

b) Aseptic transfer
1. Tube to tube Score: _________
2. Tube to petri dish Score: _________
Questions
Explain briefly the following questions.
1) The necessity of flaming the loop before and after using.
2) The importance of tilting the test tube at 450 or so when taking out an inoculum.
3) The value of sterile technique in nursing practice.
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19

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Microbiology and Parasitology

Lab Lesson 3 Aseptic Transfers

Have a work plan

A clean workspace/ protective clothing

Sterilization of bench top tools by flaming

Protect culture media against airborne contaminants

Figure 3-2 (a) Technique for

Handling of tube cultures

Test tubes are handled in the following

and the instrument is manipulated

I. Tube to tube transfer

II. Tube to plate transfer

f) Streak on the surface

Name: _________ Score:

a) Flaming the loop/needle Score: _________

2. Tube to petri dish Score: _________

1) The necessity of flaming the loop before and after using.

3) The value of sterile technique in nursing practice.

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