Preparation of Agar Plates
Growth and Check of Bacterial Strains
Bacteria can be propagated on liquid or solid
media. The use of liquid allows large quantities
of bacteria to be harvested but does not permit
easy selection or determination of phenotype of
single cells. The technique of "streaking" cells onto a solid media provides simple isolation
of colonies arising from single cells. Colonies selected for the desired
phenotype are then used to inoculate liquid broth. A single colony inoculum
is preferred because bacteria can undergo many types of mutations naturally.
The instability of some of the mutations, especially transposons and
phages, can allow some cells to lose characteristics important to the
selection scheme and may complicate the analysis. It is always wise to
check the parent strain for proper phenotype that reflects the genotype
and then use "picks" from single colonies
to start liquid cultures.
*adjust pH to 7.0 with NaOH and bring to 1 L; sterilize
by autoclaving for 20 minutes at 121°C at 15 psi
Media can be contaminated by contact with non-sterile
surfaces or by air borne organisms. Remove lids and coverings
carefully avoiding contact with any part of the cover
that may contact the media. Lids and coverings should
be held with media side down at all times. Air borne
contaminants are usually falling downward. Replace the
coverings carefully so that the rim of the container
makes contact only with sterile surface of the inside
of the cap. The use of a flame helps maintain aseptic
materials. Working near a flame can decrease air borne
contamination. The flame is also used to singe surfaces
to maintain sterility. The mouth of the tube or flask
is passed through the flame before and afer pouring.
The cap or cover is also passed through the flame prior
to replacing on the container.
PROTOCOL (work as a team)Label the bottoms of 4 Petri dishes with the type of agar and antibiotic type/concentration; also put the date and team number. The bottoms are labeled because the lids can easily get separated; also, the plates are usually handled inverted.
Before starting, make sure you remove gloves and pull back hair.
We would like to thank New England Biolabs for their generous support of our laboratory program
Copyright and Intended Use
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Created by David R. Caprette (email@example.com), Rice University 14 Jul 08
Author: Beth Beason Abmayr, Ph.D., Rice University
Updated 2 July 2014