Forest Pathology Lab 1: Aseptic or
Sterile Technique (link to handout)
In most microbiological procedures, it is necessary to protect instruments, containers,
media, and fungal or bacterial
cultures from contamination by microorganisms constantly present in the environment.
Sterile technique involves sterilization of tools, glassware, and media before
use as well as measures to prevent subsequent contamination by contact with nonsterile
objects.
Isolation of plant disease fungi using sterile technique:
Media preparation: Dry nutrient ingredients such as malt extract,
potato dextrose, or cornmeal are combined with agar (a solidifying agent
much like gelatin) and distilled water in a bottle or flask. The bottle
is then heated to 121 C and 15 psi in an autoclave (large steam pressure
cooker) to dissolve the solids and to sterilize the medium. The liquid
medium is then poured into sterile petri plates in a microbiological hood.
The plates are left in the hood to cool, solidify, and remain sterile.
Preparation of diseased plant material: With the use of clippers
or a scalpel, small (3mm x 3mm), thin pieces of symptomatic material are
cut from the plant or tree part that is diseased. Usually outer bark is
discarded because it is contaminated with other microorganisms. The pieces
are placed in a small petri dish and transferred to a microbiological hood.
Working in a microbiological hood: Before using the hood, roll up sleeves,
tie back long hair, and wash hands. Its advisable to wear a lab coat to protect
clothing and latex gloves to protect hands and prevent contamination. In the
hood, wipe the work surface with 70% alcohol. Allow to dry before lighting burner.
Submerge your plant pieces in a 4% bleach solution to sterilize the outside
surfaces. 
After
30 to 60 seconds (depending on size) remove each piece using forceps (tweezers)
and place in a sterile, covered dish of sterile distilled water to rinse bleach
from the surface. Once all pieces are sitting in rinse water, dip the forceps
in alcohol and then into the flame. Remove a piece from the water, slightly
lift the lid off an agar plate, and push the piece into the agar near the edge
of the plate. Close the lid and turn the plate one quarter turn. Flame forceps
and repeat, putting 4 or 5 pieces onto each agar plate. When finished and with
burner off, wipe down the work surface with 70% alcohol.
Always keep petri dishes closed as much as possible to minimize contamination
even in the hood. Always sterilize tools between each step. The purpose
of sterile technique in isolating plant disease fungi is to prevent growth
of other contaminating microorganisms that might outgrow the actual disease-causing
fungus and make it difficult to identify on the agar plates.