Aseptic Technique

Marlene P. Tubieros

Introduction

Bacteria are everywhere, and some are good for us while others are harmful. Thats why
minimizing our exposure to harmful bacteria when undergoing medical procedures is so
important. Healthcare providers regularly use aseptic techniques to achieve this.

Aseptic technique is a method designed to prevent contamination from microorganisms.

It involves applying the strictest rules and utilizing what is known about infection prevention to
minimize the risks that youll experience an infection.

Objectives

To acquire the skill of aseptic technique in the field of Microbiology.

To prevent contamination of cultures and media from microbes in the environment.
To transfer cultures from one medium by inoculating another medium.

Materials and Methods

1. Using a marking pen, label the test tubes correctly.

2. Sterilize your inoculating loop by passing it at an angle through the flame of the alcohol
lamp until the entire length of the wire becomes glowing red/orange from the heat.
(NOTE: Never lay the loop down once it is sterilized or it may become re-contaminated.)
3. Using your other hand, pick up the tube containing the culture and gently shake it to
disperse the culture.
4. Loosen the cotton plug of the test tube which contains your bacteria culture with the little
finger curled towards the palm of your right hand.
5. Do not put down the cotton plug.
6. Flame the neck of the test tube of bacteria culture by passing the neck forwards and back
through a hot alcohol lamp flame.
7. Using the inoculating loop, get an ample amount of culture in the test tube. Carry out the
transfer of cultures as quickly as possible, with tubes open to the air for the minimum
length of time. (NOTE: Work close to the alcohol lamp flame to reduce the chances of
contamination.)
8. Again, flame the neck of the test tube that contain your cultured bacteria and return the
cotton plug using your little finger.
9. Get the test tube of sterile nutrient broth. Carefully remove the cotton plug) with your
little finger of the hand holding the inoculating loop.
10. Flame the neck of the test tube of sterile nutrient broth.
11. Insert the inoculating loop into the sterile broth and inoculate it by gently moving
the loop back and forth in order to disperse the cells.
 12. Remove the inoculating loop from the test tube and flame the mouth of the tube
again then replace the cotton plug.
13. Sterilize the loop by flaming it. Now you may lay it down on the lab bench or
return it to its container.

Result and Discussion

Aseptic technique aims to prevent pathogenic organisms, in sufficient quantity to cause

infection, from being introduced to susceptible body sites by the hands of staff, surfaces or
equipment. It protects patients during invasive clinical procedures by utilising infection
prevention measures that minimise the presence of micro-organisms. Aseptic transfer is
transferring living microbes from one place to another without contamination of the culture, the
sterile medium, or surroundings.

In addition, aseptic technique is of utmost importance to maintain pure stock cultures while
transferring cultures to new media. Aseptic technique is also essential for isolation of a single
species of microorganism from a mixed culture to obtain a pure culture. Furthermore, proper
aseptic technique prevents microbes used in the laboratory from accidentally being released into
the environment and/ or infecting people working in the laboratory. This is especially relevant
when pathogens are being handled

1. Explain why the following steps are essential during subculturing.

A. Flaming the inoculating prior to and after each inoculation.

Holding the loop in the flame of the Bunsen burner kills all contaminating organisms,
thus sterilizing the loop
Inoculation loops need to be sterilised before and after every use to reduce the
chances of contamination as much as possible to achieve the most accurate results
possible within tests.

B. Cooling the inoculating instrument prior to obtaining the sample

If the inoculating instrument is too hot it may kill the bacteria you are trying to obtain on
contact

Conclusion

Aseptic technique is a fundamental and important laboratory skill in the field of

microbiology. Microbiologists use aseptic technique for a variety of procedures such as
transferring cultures, inoculating media, isolation of pure cultures, and for performing
microbiological tests. Proper aseptic technique prevents contamination of cultures from foreign
bacteria inherent in the environment. For example, airborne microorganisms (including fungi),
microbes picked up from the researchers body, the lab bench-top or other surfaces, microbes
found in dust, as well as microbes found on unsterilized glassware and equipment, etc. may
potentially contaminate cultures, thus interfering with the lab results. Using proper aseptic
technique can greatly minimize or even eliminate the risk of contamination.

References

http://www.biotopics.co.uk/microbes/tech1.html
http://www.austincc.edu/microbugz/aseptic_technique.php