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|  MIL-F-U252 ( `R )
4. 3.6.1 Test organisms. - The following test organisms shall be
used: -
American Type
Culture
Quartermaster Culture
Collection No.
Collection No.
QM.459
6205
a Chaetomlum globomun
QM-432
11730
b Aspergillus verslcalor
QM-380
9643
c Aspergillus flavus
9644
W-391
d Penicillium luteum
10404
w-387
e Rhizopus nigricans
These organisms may be obtained from the American Type
Culture Collection, 2029 M Street, N. W. Washington 6A D. C.
or for Service use, From Quartermaster General Laboratories,
2800 South 20th Street, Philadelphia 45, Pa.
4.3.6.2 Maintaining stock cultures.- Cultures of these fungi
shall be maintained separately on an appropriate medium such
as potato dextrose agar. The stock cultures may be kept for
not more than 4 0
months in a refrigerator at approximately 3
0
o
to 10 C 37 to 50 F) . Subcultures Incubated at 28 to 30 C
(82 to 86 oF) for 7 to 20 days, shall be used in preparing the
spore suspension.
4.3.6.3 Spore suspension. - A spore suspension shall be pre-
pared of each of t he five fungi to be used (see 4.3.6.1) as
fOllows: A sterile 10 milliter portion of distilled water
shall be poured into a subculture of the fungus. A sterile
platinum needle shall then be used to scrape the surface growth
from the culture of the test organisms. The spore charge shall
then be poured into a sterile 125 ml glass stoppered Erlen-
meyer flask containing 50 ml of sterile distilled water and
10 to 15 solid glass bevels, 5 mm in diameter. The flask
shall be shaken vigorously to liberate the spores from the
fruiting bodies and to break the spore clumps. The shaken
suspension shall then be filtered through a thin layer of
sterile glass wool in a glass funnel to remove mycellal frag-
ments. The filtered spore suspension shall then be centri-
fuged at 2500 r.p.m. and the supernatant discarded. The re-
sidue shall be resuspended in 50 ml of sterile distilled water
and centrifuged. The spores shall be washed in this manner
three times. The final washed residue shall be diluted with a
sterile mineral salts solution, conforming to Table XIX In such
a manner that the resultant spore suspension shall contain
1,000,000
200,000 spores per milliliter as determined with a
counting c #amber. This operation shall be repeated for each
organism used in the test and equal volumes of the resultant
spore suspensions shall be blended to obtain the final mixed
spore suspension. The spore suspension may be prepared fresh
54
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