Aseptic Technique

MéthodeAseptic Technique 

Aseptic Technique :

To deal with the problem of contamination, microbiologists follow a basic set of procedures known as sterile or aseptic technique Sterile technique is used for

culturing and transferring cultures, and for streaking plates to isolate and purify strains.

Aseptic technique

While learning sterile technique, another important microbiology skill is plate streaking. The purpose of streaking agar plates is to generate individual colonies for examination or study.

The technique is also used to isolate individual microbial species from a mixture of microbes in a sample. In order to generate single colonies on a plate by streaking, you must have good streaking technique.

Consider that an inoculating loop dipped into a pure culture of Halobacterium may pick up 10,000,000 cells. If a plate is streaked correctly, some of these cells will

be spread out enough to grow individual colonies.

Bacteria growing on agar

DéfinitionHalobacterium sp. NRC 1

Halobacterium sp. NRC 1 is a member of the Archaea and grows in extremely salty environments including salt marshes, hypersaline lakes (e.g., the Great Salt Lake).

Halobacterium is grown on medium so salty that few other organisms would survive it. In fact, Halobacterium will lyse at total salinities below 1.0 2.0 M. (For

comparison, sea water has a total salinity of 0.6 M, while human blood contains only 0.14 M NaCl).

For these reasons and the fact that Halobacterium is not known to cause any human disease, these microbes are ideal for beginning practice of sterile technique. Using these microbes allows students to practice the hand motions of sterile technique without the concern that they will culture a pathogen or contaminate their workspace with the microbe they are using.

It is essential to learn good sterile technique and use the same precautions as if working with a known pathogen.

Halobacterium sp. NRC 1 is a member of the

MéthodeMaterials

Included in the kit

40 petri dishes

8 bottles Halobacterium Agar, 135 mL each

Halobacterium liquid culture

128 sterile inoculating loops

autoclavable disposal bag

hand lenses, or dissecting microscope(s)

wax markers or lab pens

Bunsen burners

resealable plastic bags or plastic food storage boxes

Incubator, 42 or 37

Disinfectant for benchtop (e.g., alcohol or bleach solution) If an incubator is not available, the plates may

be stored on the lab bench. If the plates are incubated at 42 C, results may be obtained in 7 days, and if at 37 C, 7 14 days. If the plates are incubated at room

temperature on the bench, it may take 2 weeks or longer to see the results, depending on the temperature of your lab.

Preparing Media Plates

Preparing Media Plates

1. To melt the medium, slightly loosen the cap(s) and set the bottle(s) of medium in a pot of water and bring it to a boil. Make sure the water level is even with the level of the medium in the bottle(s). Leave the bottle in the boiling water until the medium has completely melted. This will take approximately 30 minutes.

2. Allow the medium to cool to 55 C either by allowing the pot of water to cool to that temperature or by letting the bottle(s) sit for several minutes at room

temperature. The bottle(s) should feel comfortably hot to the touch when around 55 C.

3. Disinfect the work surface. Wash your hands thoroughly. Unpack the petri dishes, being careful not to disturb their sterility. Align the sterile plates along the edge of a clean, level tabletop away from any draft or breeze.

4. Remove the cap and flame the mouth of a bottle of medium. Lift the lid of a petri plate just enough to pour in the molten medium. Carefully, pour to a

depth of about 5 mm per plate. Replace the lid immediately to prevent contamination.

5. Repeat Step 4 with the other bottle(s) of medium.

6. Let the petri plates stand undisturbed until they solidify (about 1 hour). Let the plates sit out until any condensation on the lid evaporates.

7. Dispose of the empty bottles in an autoclave disposal bag.

Microbiology [pdf]