1. Aseptic Technique & Streaking for Isolation

2. What is aseptic technique?
Bacteria are everywhere, and some are good for us while others are harmful.
Bacteria, viruses, and other microorganisms that cause disease are called pathogens. To protect patients from harmful bacteria and other pathogens during medical procedures, healthcare providers use aseptic technique.
Aseptic technique means using practices and procedures to prevent contamination from pathogens.
It involves applying the strictest rules to minimize the risk of infection. Healthcare workers use aseptic technique in surgery rooms, clinics, outpatient care centers, and other health care settings.

3. What is aseptic technique used for?
Following aseptic technique helps prevent the spread of pathogens that cause infection.
Healthcare professionals commonly use aseptic technique when they’re:
handling surgery equipment.
helping with a baby’s birth.
handling dialysis catheters and performing dialysis.
inserting a chest tube.
inserting central intravenous (IV) or arterial lines.
inserting other draining devices.
performing various surgical techniques.

4. Personal Protective devices
These are items or practices that protect you from contaminating your body with a pathogen. These include:
Wearing gloves.
Washing hands after removing gloves (last thing done as you leave lab).
Wiping down all surfaces with 10% bleach.
There are optional devices that we do not require in our lab:
Lab coats
Face masks
Hair nets

5. Aseptic technique is also a fundamental and important laboratory skill in the field of Biotechnology.
Microbiologists use aseptic technique for a variety of procedures such as:
transferring cultures
inoculating media
isolation of pure cultures
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 researcher’s 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.

6. Using proper aseptic technique can greatly minimize or even eliminate the risk of contamination.
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.

7. Significance of Flaming:
Flaming the loop : 
Holding the loop in the flame of the Bunsen burner kills all contaminating organisms, thus sterilizing the loop.  
The loop should glow red-hot for a few seconds. After flaming, make sure to slightly cool the loop before picking up organisms from the inoculum culture (the culture that is to be transferred.) 
When transferring a culture from a plate, cool the loop by touching on the very edge of agar. 
When transferring from a broth, the red-hot loop will make a sizzling noise as soon as you insert it into the culture.  The loop will automatically cool once it makes contact with the broth. wait a 1 or 2 seconds before removing the loopful of inoculum from the tube.

8. Flaming the Mouth of the Test Tube / Beaker / Flask: 
Passing the mouth of a tube through the flame of a Bunsen burner creates a convection current which forces air out of the tube. 
This prevents airborne contaminants from entering the tube.
The heat of the Bunsen burner also causes the air around your work area to rise, reducing the chance of airborne microorganisms contaminating your cultures.  

9. Streaking for isolation
Bacteria in natural circumstances are almost always found as mixtures of many species. For most purposes, it is necessary to isolate the various organisms in pure culture before they can be identified and studied. The most important technique for this purpose is "streaking" on the surface of a solid nutrient medium. It is extremely important that you master this technique.
There is an important principle to learn here: a single bacteria, physically separated from others on the surface of the medium, will multiply and give rise to a localized colony of descendants.

10. The purpose of streaking bacteria for isolation is to create a region in which the bacteria are so dilute that when each bacterium touches the surface of the agar, it is far enough away from other cells so that an isolated colony can develop. This allows for isolation of multiple organisms.

11. Step #1
Sterilize a wire loop by heating it until red hot in a flame; allow it to cool for several seconds. Test for coolness by touching the agar at the edge of the plate.

12. Divide you dish into 3 sections

13. Step #2
Pick up a loopful of liquid inoculum or bacterial colony from the surface of an agar plate. Starting in the largest section on the plate, streak lightly back and forth with the loop flat, making close, parallel streaks back to the edge of the plate.

14. Step #3 Sterilize the loop and cool again.
then with the edge of the loop, lightly make another set of nearly parallel streaks about 1/8 inch apart, in one direction only, from the inoculated area to one side of the uninoculated area, so that about 1/2 the plate is now covered.

15. Step #4 Flame and cool the loop again.
make another set of streaks in one direction, perpendicular to and crossing the second set of streaks, but avoiding the first set.

17. In the final set of streaks, you should be depositing single bacteria at different places on the agar.
After incubating for 24 hours, these should produce individual colonies.