Monitoring of Humidity, Cage and

Bedding Temperatures in Static

Mouse Caging Following Steam

Sterilization

Gina M Ward, MS, LAT

Husbandry and Facility Coordinator

April 1, 2009

Background

At our institution, many rodent facilities are barrier

maintained where supplies, including caging, are

sterilized before use in rooms.

Due to equipment failure, holidays, and staffing issues,

there may be time constraints as to how quickly clean

supplies can be provided.

BackgroundMajor time constraint may be prolonged cooling time

of cages before use in animal rooms

Sterilized cage set-ups were cool to the touch after

approximately 45 minutes following removal from the

autoclave

Scenario Over the course of 4 months, received reports of unexplained

rodent deaths in 2 cages in isolated facilities

No recent experimental or husbandry manipulations were

described

Only commonality between two isolated events – animals

recently changed into newly sterilized cages

Question / Concern

Although cages felt cool to the touch and can be

handled in less than an hour after autoclaving

…what was the environment like INSIDE the

cages?

…and could this have impacted the mice?

Mice and temperature regulation

Neonates are ectothermic and do not have well-developed temperature control before 20 days of age

Despite various adaptations such as highly vascularized ears and unhaired tails, which increase the surface area for heat loss, thermolysisis very difficult for mice

Mice do not pant, but do possess footpad sweat glands

Mice have low tolerance for extremes in heat.

Normative values range from 70-76°F

Animals exposed to > 85°F without prior acclimation may exhibit clinical effects

According to the Guide:

Mice can tolerate environmental temperatures of 64-79°F

Acceptable room humidity range is 30-70%

Mice and hyperthermia

Working hypothesis

We believed that cage set-ups autoclaved

routinely in our facility were retaining heat

for prolonged periods of time

Internal cage temp

Bedding temp

Cage humidity

Relevant Literature Cage environment post-sterilization NOT previously reported.

Related research:

Differences in temperature among bedding type differed among breeds and housing density

Mice exposed to temperatures ≥ 98.6°F for a period of 60 minutes were shown to exhibit escape behavior

Increase in body weight loss and rectal temperature seen when environmental temperature ≥ 98.6°F for 60 minutes

Cages with filter tops have been shown to impede airflow

The cage microenvironment has been shown to be unaffected by room environmental changes

Facility Background All rodent caging, bedding, food, etc. must be autoclaved

prior to use within barrier facilities

Autoclave conditions:

Sterilizing Temp – 121° Celsius

Sterilizing Time – 16 minutes

Drying Time – 5 minutes

Cage Description Full Cage Set-Up includes:

Polycarbonate cage bottom, wire lid and low profile micro isolator lid.

Cages filled with ¼ inch corncob bedding.

Wire lid filled to capacity with autoclavable rodent chow.

Cages then stacked in two rows,

front and back, ten high and

seven across and placed on

three-sided bulk truck and

covered

Experimental Design Immediately following sterilization,

hygrothermometers placed in cages

Timex TX5170 Indoor/Outdoor Thermometer with Indoor Hygrometer: measures temperature inside cage, humidity inside cage, and temperature of bedding using sensor

Experimental Design Temperature and humidity readings were recorded at 10-

minute intervals following the first reading

Average time from removal of truck from autoclave to first temperature reading = 23 minutes

This includes 10 minutes of acclimation time for hygrometer once placed.

Monitoring sessions lasted for 150 minutes

Performed three trials for the front of the truck, and three trials for the back

Experimental Design For each trial, 6 cages selected from center column and either

side column

Recorded room temp and humidity, as well as cage temp, humidity, and bedding temp in a cool mouse cage prior to experiment

Side Center

1 Top Top

2

3

4

5 Middle Middle

6

7

8

9

10 Bottom Bottom

Experimental Design Measurements taken in 6 cages

occupied with 5 adult mice, in am

and pm, every 10 minutes for one

hour, on two different days

Measurements taken in empty

cage at the beginning of each

sterilizing trial. Cage cooled for

at least 24 hours

ResultsOccupied Cage

(5 mice)

Unoccupied

Cage

Cage Temperature (oF) 72.3 ± 2.57 72.5 ± 1.2

Cage Humidity (%) 81.8 ± 4.52 49.2 ± 4.5

Bedding Temperature (oF) 70.7 ± 0.38 71.0 ± 1.2

Results No significant difference between front and back rows,

thus data combined for a total of 6 data points for each

spot: top side, middle side, bottom side, top center,

middle center, bottom center

Range

At time 0 At 150 minutes

Cage Temperature 84.8-107.6 oF 71.8-80.6oF

Bedding

Temperature

96.7-124.9oF 71.6-86.2oF

Cage Humidity* 63-91% 52-91%

* Hygrometer could not measure humidity over 91%

Results Took on average 13 minutes to move trucks out of

autoclave into cooling room, unpack truck, place

hygrometers, and repack truck

Allowed 10 minute acclimation for hygrometer before

time 0 recorded

Temperature Inside of Cage

Difference across spots at 60 minutes (p=0.014).

At 60 minutes, top side cooler (p=0.037) and middle center hotter

(p=0.0017) than average.

Bottom side cooled more quickly (p=0.0044).

70

80

90

100

110

120

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150

Deg

rees

Fa

hre

nh

eit

Top Side

Middle Side

Bottom Side

Top Center

Middle Center

Bottom Center

Temperature of Bedding

70

80

90

100

110

120

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150

Deg

rees

Fa

hre

nh

eit

Top Side

Middle Side

Bottom Side

Top Center

Middle Center

Bottom Center

Difference across spots at 50 minutes (p=0.017).

At 50 minutes, top side cooler (p=0.025) and middle center hotter

(p=0.0032), than average.

Bottom side cooled more quickly (p=0.0002).

Humidity Inside of Cage

50

60

70

80

90

100

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150

% H

um

idit

y

Top Side

Middle Side

Bottom Side

Top Center

Middle Center

Bottom Center

Difference across spots at 20 minutes (p=0.0041).

At 20 minutes, top side lower (p=0.032) and middle center higher

(p=0.0032) humidity than average.

Both cages on top decreased humidity more quickly (p > 0.001).

Conclusions

Cages across the top had fastest internal humidity drop

Middle center cage had consistently higher cage and bedding temperatures

Internal cage and bedding temperature cooled more quickly for the bottom side cage

Despite being cool to the touch after 45 minutes, the inside environment of rodent cages and bedding remains extremely hot, average 86oF and 92oF respectively

Conclusions Mice can be maintained at environmental conditions of up

to 79oF. On average the inside of the cage lowered to this temperature by 100 minutes and the bedding did so by130 minutes

Recall it took on average 23 minutes to start collecting data from the time the cages left the autoclave

Given these findings, trucks must be allowed to cool a minimum of 2.5 to 3 hours, with autoclavable cover removed, before use

Optimal conditions would allow for overnight cooling

Acknowledgements: Dr. F. Claire Hankenson, Senior Associate Director, ULAR,

University of Pennsylvania

Dr. Kurt Hankenson and Jason Combs, University of Pennsylvania Veterinary School of Medicine

Kelly Cole, The Ohio State University College of Veterinary Medicine

Jamal Edwards and Troy Hillanbrand, ULAR, University of Pennsylvania

Financial support for this project was graciously provided by University Laboratory Animal Resources. Ms. Cole was partially supported by funds from the American College of Laboratory Animal Medicine.

References: American Association for Laboratory Animal Science. 2006. Laboratory

Mouse Handbook. AALAS, Memphis.

Institute of Laboratory Animal Resources, N. R. C. 1996. Guide for the Care and Use of Laboratory Animals. National Academy Press, Washington, D.C.

Smith, E., J. D. Stockwell, I. Schweitzer, S. H. Langley, and A. L. Smith. 2004. Evaluation of cage micro-environment of mice housed on various types of bedding materials. Contemp Top Lab Anim Sci 43:12-7.

Harikai, N., T. Sugawara, K. Tomogane, K. Mizuno, and S. Tashiro. 2004. Acute heat stress induces jumping escape behavior in mice. Physiol Behav83:373-6.

Harikai, N., K. Tomogane, M. Miyamoto, K. Shimada, S. Onodera, and S. Tashiro. 2003. Dynamic responses to acute heat stress between 34 degrees C and 38.5 degrees C, and characteristics of heat stress response in mice. BiolPharm Bull 26:701-8.

Reeb, C.K. Jones, R.B., Bearg, D.W., Bedigan, H., Paigen, B.1997. Impact of Room Ventilation rates on mouse cage ventilation and microenvironment. Cont. Top. Lab Anim Sci 36 (1): 74-79.