Downloaded from https://www.everyspec.com MIL-STD-81OC Maintain cultures of these fungi separately on an appropriate medium such as potato dextrose agar. However, the” culture of chaetomium globosum shall be cultured on strips of filter paper on the surface of mineral salts agar. (Mineral salts agar is identical to mineral salts solution described in 3.1.1, but contains in addition 15.Og of agar per “liter). The stock cultures may be kept for not more than 4 months at 6° *4° C (43° i7° F) at which time subcultures shal1 be made and new stocks shal1 be selected from the sub- cultures. If genetic or physiological changes occur, obtain new cultures as specified above.
Subcultures used for preparing new stock cultures or the spore suspension shal1 be incubated at 30”C (86° F) for 9 tO 12 days”Or .lOnger. Prepare a,spore suspension of each of the five fungi by pouring into one subculture of each fungus a 10-ml portion of a sterile soIution containing 0.05g per liter of ‘anontoxic wetting agent such as sodium dioctyl sulfOsuc- cinate or sodium Iauryl sulfate. Use a sterile platinum or nichrome inocula- ting wire to scrape gently the surface growth from the culture of the test ~. organism. Pour the spore charge into a sterile 125-ml glass-stoppered Erlenm6yer flask containing 45 ml of sterile water and 50 tO 75,solid glass beads, 5 mm in diameter.
Shake the flask vigorously to liberate the spores from the fruiting bodies and to break the spore clumps. Filter the dispersed fungal.spore suspension, through a 6 mm layer of glass wool contained in a glass funnel, into a sterile flask. This process should r,emovelarge mycelial fragments and clumps of agar which could interfere with the spraying process. Centrifuge the filtered spore suspension aseptically, and discard the super- natant liquid. Resuspend the residue in 50 ml of sterile water and centrifuge. Wash the spores obtained from each of the fungi in this manner three times.
Dilute the final washed residue with sterile mineral-salts solution in such a manner ‘that the resultant spore suspension shall contain 1,000,000 *200,000 spores per ml as determined with a counting chamber. Repeat this operation for each organism used in the test and blend equal volumes of the resultant spore suspension to obtain the final mixed spore suspension. The spore sus- pension may be prepared fresh each day or may be held at 6° *4° C (43° *7”F) for not more tlian7 days. 3.1.3 ,Viability of.inoculum control.
With each daily group of tests place each of three pieces of sterilized filter paper, l-inch square, on hardened mineral-salts ~gar in separate Petri dishe;.- Inoculate these with the spore suspension by spraying the suspension from a sterilized atomizer ~/ until initiation of droplet coalescence. Incubate these sit30”C (86”F) at ? relative humidity not less than 85.percent and examine them after 7 days of incubation. There shall be copious growth on all three of the filter paper control speci- mens. Absence of such growth requires repetition of the test. 1/ An atomizer capable of providing 15,000 +3,000,spOres per square ;eitimeter. D 508.1-3 }lETHOD508.1