The Spot Test: an easy method to determine auxotorophy, sensitivity or resistance to chemicals and physical conditions in many strains




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



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



(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.


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