The Spot Test:
an easy method to determine auxotorophy, sensitivity or resistance to chemicals
and physical conditions in many strains
HIROZAKU
INOUE
In
many cases we need to test phenotype of large numbers of strains. For example, to
make
the gene mapping we test several phenotypes of many progeny (usually
200
to 400) derived from a cross. The spot
test is very useful in such
experiment.
Typical
example; cross: cyh-1,
his-3, A x un-3, ad-3A, a
Objective:
to know distance (recombination rate) between each marker
(1)
isolate 200 ascospores randomly and transfer each of them to
mini-culture
tube (8x75mm) containing panthotenate, histidine and adenine
at
appropriate concentrations. Culture at
25 C for one week.
(2)
score non-growing tube and calculate germination rate (%).
(3)
prepare 1 ml water per middle-size tube (12x100mm) for number of
growing
tubes and autoclave them. Also autoclave
long toothpicks and
pasteur
pipettes.
(4)
make conidial suspension of each tube using toothpick. Conidial
suspension
should be thin and homogeneous.
(5)
make 4 different 90 mm plates, each including 20 ml medium (containing
1
% sorbose, 0.2 % sucrose, 1.5 % agar, commonly);
#1
plate contains histidine and adenine.
#2
plate is same to #1 plate except for addition of cycloheximide,
#3
plate contains only histidine,
#4
plate contains adenine.
(6)
make 50 sections per one plate using a sign pen and number each
section.
(7)
suck a little amount of conidial suspension using a pasteur pipette and
spot
it onto center of a section of same number in each different plates.
Incubate
plates at 25 C, except plates for un-3 are at 36 C.
(8)
score growth of each spot after 2 days.
You
can determine the genotype of each progeny and calculate recombination
rate
between each marker.
In
this system 50 progeny's phenotype will be determined by using one
plate. We also use this for tests of mutagen
sensitivity.