Aseptic technique (sometimes called "sterile technique") is the basic skill in isolation
and culture of microorganisms. The purpose of this technique is to avoid contamination
of the culture with any other organism other than the plant pathogen that causes the
disease under study.
A major source of contamination is from the air. Airborne particles contain fungal and
bacterial spores. The person who does the isolation or transfers a culture is also a source
of contamination.
To avoid contamination the following precautions should be taken:
1. Avoid drafts of air over working surfaces as much as possible
2. Clean bench tops before working
3. Open cultures, as briefly as possible, only when absolutely necessary
4. Pour culture media when cool to avoid convection of airborne particles
5. Avoid sneezing, coughing and speaking while working over a culture media
All tools that touch the culture must be sterilized, meaning that all microorganisms on
them are killed. Usually tools are flame-sterilized. Transfer needles and loops held in
the flame until red-hot, and then allow it to cool before use. Scalpels and forceps are
dipped in alcohol, then quickly moved through a flame to allow the alcohol to burn
off. The temper of the steel of forceps and scalpels will be destroyed if they are
heated until red-hot. All heated tools should be cooled in air or by plunging in sterile
media before touching a fungal or bacterial colony that you are going to transfer.
Media used for isolation must be autoclaved before use.
During some lab sessions you will have occasion to isolate pathogens from diseased
tissues and grow them in pure cultures.
Materials needed:
A clean work area wiped with bleach or ethanol
Infected plants or parts of plants
A solution of 10% household bleach (0.5% NaOCl). Don't get it on your clothes!
Sharp scalpel, forceps, transfer needles, Bunsen burner, alcohol.
Sterile nutrient media
1. Examine diseased plant material carefully and note the size and shape of lesions.
The lesions may be small (spots) or large. Cut off and discard parts of the plant
that will not be used for isolation. Extremely dirty roots should be rinsed with
running water.
2. Clean the bench area well. Place the tissue pieces on a clean paper towel. Use
flame-sterilized tools at all times for cutting and handling the tissue.
3. Small spots: cut out the entire spot, leaving a margin of healthy tissue around it.
Larger lesions: cut pieces from the edge of the lesion, so that you have a piece of
healthy tissue and a piece of diseased tissue (see figure).
4. The first objective is to free the tissue from surface contaminants that are
ubiquitous. Immersing all or part of the infected stem or leaf in the bleach
solution does this. Make sure that no air bubbles are trapped on the plant tissue;
these bubbles can protect microorganisms from exposure to the chemical. The
length of time to immerse the tissue depends on the rate of penetration of the
bleach: woody stems sections can be immersed for 5 minutes or more, delicate
roots or leaves should be immersed only for 30 seconds to 1 minute. For fleshy
tissues such as fruits, cleanse the outer surface by wiping with 10% bleach, then
peel back the top layer of tissue and cut out pieces of lesion from within.
5. Tissues are now surface-sterilized and should be handled only with sterile
instruments. The objective is to keep them free from contaminants. Since bleach
is rapidly dissipated, it is not necessary to rinse thick tissues after the bleach
treatment. Delicate tissues should be rinsed in sterile distilled water.
6. Remove the tissue pieces and blot them on a paper towel. Then put several pieces
on a plate with media.
Based on Laboratory Exercises in Plant Pathology.
American Phytopathological Press