the significance of each measurement of toxicity. 
Many of the techniques now used have been devel- 
oped with the express purpose of reducing biological 
variability. 
Testing With Spore Germination.—Early tests of 
Reddick & Wallace (1910) and McCallan and co- 
workers (McCallan 1930; McCallan, et al. 1941b; 
McCallan & Wilcoxon 1938, 1939; Wilcoxon & Mc- 
Callan 1939) and Horsfall, et al. (1940) were spore 
germination tests involving the deposition of chemicals 
on glass slides, evaporation to dryness, and the addi- 
tion of a drop of water containing spores of the test 
fungus. The American Phytopathological Society 
(1943) published instructions for this technique. 
One problem encountered with this technique was 
the variation in size of the water drops. Montgomery 
x Moore (1938) attempted to solve this by drawing 
a circle on the glass slide with a diamond pencil, 
Young (1944) etched a circle with hydrofluoric acid, 
and Miller (1949) drew a circle with a wax pencil. 
Others have used depression slides (Barratt & Horsfall 
1947, Kirby & Frick 1953, and Prusova 1962), small 
petri dishes (Tamura 1954), microbeakers (Shafer 
1952 and Spencer 1962), or raised cover slips on slides 
‘Peterson 1941). 
Another problem was that of obtaining an accurate 
letermination of the amount of toxic material on the 
lide. Elaborate laboratory sprayers have been built 
o uniformly deposit the chemical. An alternate solu- 
ion by McCallan & Wilcoxon (1938) and Peterson 
(1941) involved mixing the toxicant with the spore 
uspension directly and not previously forming a dry 
leposit on the slide. Procedures for this test tube 
lilution technique were described by the American 
hytopathological Society (1947). 
Although germinating spores on glass slides are 
asily examined microscopically, glass is not the ideal 
ubstrate for spore germination. Marsh (1936) ob- 
erved that fungus spores germinated better on glass 
lides that had been coated with cellulose. Trans- 
arent materials other than glass have been success- 
ully used. McIntosh ( 1961) and Spencer (1962) used 
ramsparent plastic polymers. Neely & Himelick 
1966) germinated spores on nonwaterproof cello- 
hane discs placed on filter paper pads previously 
aturated with the toxic solution or suspension. The 
ellophane discs were transferred to slides for micro- 
sopic determination of fungistatic activity or trans- 
red to agar for determination of fungicidal activity. 
Many fungus spores do not germinate well in 
‘ater drops. Davies, et al. (1948) found that shaking 
1e culture greatly improved the uniformity of spore 
ermination. Darby (1960) described ‘a shaker 
ethod with a 4-hour germination period. Gattani 
1954) observed that spores germinate better on agar 
lan in water drops. He mixed the fungicide with 
arm agar, allowed the agar to solidify, seeded the 
agar with spores, and counted the germinating spores 
by placing the petri dish under a microscope. Heyns, 
et al. (1965) placed seeded, toxicant-agar preparations 
on microscope slides. 
Rapid tests involving swelling of spores prior to 
spore germination also have been used to measure 
fungitoxicity. Mandels & Darby (1953) described a 
3-hour test with the failure of spores to swell as the 
measurable character. Koopmans (1959) based his 
results on the loss of turgidity of powdery mildew 
spores in toxic solutions. 
Macroscopic Tests.—Counting germinating spores 
has always been a tedious, fatiguing task. In vitro 
tests utilizing macroscopic observations of fungus 
colony growth have often been used. Lee & Martin 
(1927) and Wellman & Heald (1940) placed fungus 
spores in known aqueous concentrations of the toxi- 
cant for varying lengths of time before transferring 
them to bouillon or agar. Their tests are based on 
reduction in number of fungus colonies formed. 
The most commonly used macroscopic tests involve 
germination of spores on toxicant-agar preparations 
in petri plates with colony counts being made or the 
absence of colonies noted (Palmiter & Keitt 137s 
With the roll culture technique of Manten, et al. 
(1950), the toxicant-agar layer is formed along the 
sides of round bottles rather than in petri dishes. A 
closely related method involves placing known quanti- 
ties of toxicant at specific spots on seeded agar plates. 
The fungicide diffuses through the agar. The dia- 
meter of the clear area where spore germination was 
inhibited is measured. The fungicide may be placed 
in holes in the agar (Mildner, et al. 1963) or added 
with saturated filter paper pads (Thornberry 1950 
and Leben & Keitt 1950) or string (Kuhfuss 1957). 
In vitro techniques measuring toxicity of chemicals 
to fungus hyphae are often useful, especially with 
fungi that fail to sporulate or sporulate poorly in the 
laboratory. A common procedure is to incorporate 
toxic matcrials into agar and then seed with uniform 
mycelial colonies in agar blocks (Carpenter 1942), on 
filter paper pads (Sharvelle & Pelletier 1956), or on 
agar-coated cover slips (Bomar 1962). The reduction 
in fungus growth is then measured. Fungus cultures 
on agar plates also have been dusted or sprayed with 
toxic materials (Henry & Wagner 1940), submerged 
beneath toxic materials (Grosser & Friedrich 1947), 
or placed near toxic materials (Moreau & Moreau 
1959), after which the resulting inhibition of growth 
was measured. 
The fungus mycelium growing in toxic solutions 
has been linearly measured (Mason & Powell 1947) 
and weighed (Le Tourneau & Buer 1961). Forsberg 
(1949) placed a fungus-infested string in toxic dilu- 
tions for varying periods of time, then transferred the 
string to agar to determine fungicidal activity. 
Mandels & Siu (1950) describe fungitoxicity tests 
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