Joint International Conference on Long-term Experiments ...

security. The air conditioning for this area was turned off during fumigation, and the air
supply and exhaust outlet valves were closed.
Assay of HCHO. Initially, formaldehyde vapor concentrations were measured by taking air
samples through rubber tubes protruding into the room at various sample points. Four
samples were taken per sampling time, using an evacuated liter flask. Forty milliliters of 0.5
M (NH4)2SO4 was added to each flask, which was then cooled to 4'C to dissolve the
formaldehyde. One milliliter of a suitable dilution of the resulting solution was then mixed
with 1 ml of a freshly prepared chromotropic acid reagent (1% chromotropic acid in 18 M
H2SO4), and 8 ml of 18 M H2SO4 was added. After standing for 10 min, the mixture was
diluted to a final volume of 25 ml, and the optical density was determined at 540 nm.
HCHO was estimated from a standard curve in the range 5 to 40 yg/ ml, and the mean
concentration from the four samples was determined.
Biological test systems. The spore suspension used throughout was prepared from B.
subtilis NCTC 8233 prepared by culture for 7 days on sporulation agar and suspension of
the resultant growth in water. The suspension was subjected to two cycles of heating to
65°C for 30 min followed by centrifugation and resuspension in distilled water. A sample
was then stained and examined microscopically for spores. Glass rods 5 mm in diameter and
150 mm long, with a loop at one end and a graduation mark 50 mm from the other, were
dipped into the spore suspension to the mark. They were then suspended in a laminar flow
cabinet to dry and stored in a sterile jar. Before fumigation, rods were placed in different
parts of the areas to be tested and transferred to a sterile screwcapped test tube at the end of
each procedure, immediately after evacuation of the fumigant. Twenty milliliters of saline
was added to each tube to remove the spores from the rods, and an estimate of viable spores
was obtained by plating dilutions of the suspension onto nutrient agar. This was carried out
within 2 h of removal of rods from the area. Plates were incubated at 37°C, and then
colonies were counted after 3 days and checked after 7 days.
Electric vapor generator. Initially, HCHO was vaporized on a 500-W heating plate in a
stainless-steel beaker. The apparatus used subsequently, consists of a stainless-steel 5-liter
vessel with a 1,200-W heating element. Sufficient capacity was present to allow the addition
of water for humidification and to contain the resulting foam. A copper plate was interposed
between the element and the bottom of the vessel to ensure a uniform distribution of heat at
a thermostatically controlled temperature of approximately 170°C.
Electronic formaldehyde monitor. The apparatus used was based on a Figaro 812 N-type
semiconductor device, the conductivity of which changes in the presence of adsorbed
molecules. No significant interference was observed from substances other than HCHO in
the areas fumigated or by temperature changes normally encountered. The monitor was
calibrated in an enclosed system against HCHO released by paraformaldehyde vaporization.
RESULTS
HCHO levels by the Formalin-permanganate and hot plate vaporization methods. For
Formalin-permanganate fumigations, quantities of Formalin recommended have ranged
from 12 to 59 ml/m 3 of air space , and ratios for Formalin-permanganate have ranged from
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