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Ventilation Design Handbook on Animal Research Facilities Using Static Microisolators 
4. 1.2.4 Experimental Procedure 
There were two environmental relative humidity treatments (low and high relative humidity) and 
three replications per treatment so there were six experimental units. Since there were only three 
calorimeters, this experiment was divided into two time periods. During test 1 (Oct. 18, 1997 to 
Oct. 27, 1997), two experimental units were at the 35 percent relative humidity treatment and 
one experimental unit was at the 75 percent relative humidity treatment. During test 2 (Dec. 13, 
1997 to Dec. 22, 1997), two experimental units were at the 75 percent relative humidity 
treatment and one at the 35 percent treatment. When the mice first arrived, they were randomly 
assigned to 15 cages, then some adjustments were made to equalize average mouse weight 
among the cages. Only 12 cages were used in the tests, but extra mice were ordered to replace 
experimental animals if problems occurred. None of the extra mice were used. During test 1, 10 
cages were randomly assigned to the 35 percent relative humidity treatment and five cages to the 
75 percent relative humidity treatment. During test 2, 10 cages were assigned to the 75 percent 
and 5 cages to the 35 percent relative humidity treatments. The assignment of cages is shown in 
appendix I: sections 3.2.9 and 3.2.10. 
After the 3-day acclimation period, there was a 10-day test period when the mice were placed in 
the calorimeters for 10 hours each day where the measurements were taken (see standard 
operating procedures in appendix I: section 3.3). During the rest of the day, the mice were kept in 
their respective environmental chambers. The same four cages were always randomly assigned 
to a different calorimeter each day and were an experimental unit (the randomized assignments 
are in appendix I: section 3.2.9 and 3.2.10). At the morning of every day of the tests, the mice, 
feed, water, and litter were weighed separately. Four cages with five mice each were placed in 
each calorimeter for a total of 20 mice in each calorimeter. The three calorimeters were operated 
at the same temperature (24.0±1.5 °C (75.2±2.7 °F). Data were collected three times during the 
photophase (approximately at 10:40, 11:20 and 12:00 a.m.) and three times during the 
scotophase (approximately at 3:15, 4:00 and 4:40 p.m.). Since the lights were shut off at 1:00 
p.m., half of the data were obtained during the daily photophase and half during the scotophase, 
so effects of light could be determined. 
The calorimeters were in the horizontal position so airflow approached the front of the cages (see 
figure 4.34). The four cages were positioned on two levels (as in a cage rack). The calorimeter 
static pressure was kept negative. The fresh air exchange rates for the calorimeters varied from 5 
to 9.3 L/min. Fresh airflow rates were increased over the 10-day test period to keep ammonia 
levels low. After the mice were placed in the calorimeter and also after the lights were turned off, 
a dehumidification system was manually turned on for approximately one hour for the low 
humidity calorimeters to reduce the humidity. The dehumidification system was only operated 
for about one hour then the gas levels were allowed to stabilize for around two hours before 
readings were taken. Weights of the desiccant cylinders were determined at the start and end of 
each daily experiment so water balances could be calculated. At the beginning of each daily 
experiment, the cylinders were emptied and refilled with recharged desiccant. Water production 
was measured based on water added to calorimeter, different weights of desiccant cylinders in 
