Specific Pathogen Free (SPF) Training
NOTE: This PDF has been created from an online course format, so some of the navigation information is
not applicable. The links and references are also not functional. Please contact [email protected]
if you need any of the link or reference information.
Lesson 1. Introduction
Page 1. Instructions
New SPF Users:
If you require access to a housing facility, there are two pre-requisites that you’ll need to
complete prior to requesting a facility orientation.
1. Completion of the SPF Course and passing the exam with a 100%
2. IACUC approval on your PI’s protocol (if you have not done so yet, you will need to submit
a New Personnel Form to the IACUC. Instructions for submission are on the form).
Once the course is completed, please print out your Certificate of Completion and keep it for your
records. You are NOT required to submit an “SPF Access Renewal Form”. If you need instructions
on how to register for a facility orientation, please contact the Training Coordinator.
SPF Renewals:
If you are renewing your SPF training you MUST submit the SPF Access Renewal Form to the
Training Coordinator immediately upon completion of the course and passing the exam with a
100% to ensure that your proxy card is renewed for the facility to which you need access.
In addition, once the course is completed, print out your Certificate of Completion and keep it for
your records. Please do NOT send it to the Training Coordinator.
NOTE: This course is required annually in order to maintain facility access via your proxy card. If
you do not renew the course on an annual basis, your proxy card will expire. If this happens, the
SPF on-line course as well as the facility orientation must then be completed in order to regain
access. You will be sent a courtesy SPF expiration reminder email; however, individuals are
responsible for keeping track of their training and updating it annually.
Page 2. Course Introduction
Specific Pathogen Free (SPF) facilities are designed to maintain rodents in an environment that is
free of certain infectious organisms that are pathogenic and/or capable of interfering with
research objectives. The personnel working in these facilities must adhere to strict guidelines and
procedures to avoid introducing pathogens into the environment. A working knowledge of SPF
techniques is essential in order for personnel to successfully function within the facility.
This web course is designed to provide the knowledge necessary to work within University of
Washington SPF animal facilities, including Harborview Research & Training (HR&T), Brotman,
Roosevelt, CHDD, Foege, and the Health Sciences Building K-wing and T-wing. Facility information,
pathogen concerns, requirements, and proper procedures are all covered in this session. All
personnel working with rodents are required to complete this session on an annual basis in order
to keep their SPF knowledge current.
Please note that for individuals new to the UW SPF facilities, this course is the 1st of a 2-part
series that must be completed in order to obtain access to an animal housing facility. After
completion of this SPF procedures course, new personnel, or personnel whose access has lapsed,
must next complete an in-person facility orientation to the particular facility in which they will
work.
Page 3. Course Goals
The goals of this course are to:
Provide a definition and description of Specific Pathogen Free.
Provide background information on the common microbes excluded from the UW SPF facilities.
Provide detailed instruction on procedures that must be used when working within a Specific
Pathogen Free facility.
Function as a resource of information and contacts for working within Specific Pathogen Free
facilities at the University of Washington.
Page 4. Course Requirements
All personnel working with SPF rodents must complete this course and pass the exam.
Upon completion of this course, personnel will be allowed to register for a Facility Orientation.
You must attend an orientation to gain access to the specific facility in which you will work. NOTE:
Individuals renewing their SPF training need not attend the facility orientation annually.
All personnel must complete this course on an annual basis (every 12 months) in order to
maintain their access to UW SPF facilities. If your SPF training expires (more than 12 months old),
your access to the animal facilities will be denied, and you will be required to retake the SPF
online course as well as attend a facility orientation for the areas that you need access to.
Personnel completing this course for the first time should review all the chapters and must pass
the exam. Individuals renewing their training only need to pass the exam.
Page 5. Course Exam
To take the exam covering this course at any time, click on the Specific Pathogen Free (SPF)
Training Exam link on the previous webpage.
You must score 100% to pass the final 40-question exam. If you do not pass, you may take the
exam again as many times as you wish.
Once you pass the exam, you will receive a confirmation email.
Lesson 2. What is SPF?
Page 1. Defining SPF
SPF is an acronym for Specific Pathogen Free. It is a general term used to indicate that certain
pathogens (disease causing microbes) have been excluded from an animal or a colony of animals.
It does not necessarily indicate that all pathogens or microbes that can affect the species are
excluded. It is important to define the specific pathogens excluded. The UW maintains a list of
viruses, bacteria and parasites that are excluded from SPF rodent facilities. The list for mice
includes pathogens such as mouse hepatitis virus (MHV), but does not include Helicobacter spp.,
except in a few selected rooms. The list also includes:
1. Organisms that may not cause clinical disease in normal animals but may cause illness in
immune deficient animals or may affect research results in normal or immune deficient animals.
2. Zoonotic Microbes - Pathogens which can be passed from animals to humans.
Page 2. How Pathogen Free are the Rodents?
Please note that, although the animals may be classified as SPF, they may not be free of all
potential pathogens. Specific Pathogen Free status indicates that the rodents have been tested
and determined free of designated pathogens, but not necessarily free of all pathogens or
microbes.
Lesson 3. Infectious Agents
Page 1. Infectious Agents - Introduction
Like all animals, rodents are susceptible to a variety of viruses, bacteria and parasites. Some of
these agents are capable of inducing disease outright, while others may significantly alter host
responses to experimental conditions without causing overt signs of disease (i.e., sub clinical
infections). For all these reasons, it is important to keep these agents out of our SPF vivaria.
The Rodent Health Monitoring Program (RHMP) is the division of the UW’s Department of
Comparative Medicine that is responsible for disease surveillance of rodent colonies. Currently,
they monitor for a long list of infectious agents.
If you have any questions regarding RHMP, please email [email protected].
Page 2. Excluded Agents
These infectious agents are excluded from the SPF facilities at the University of Washington:
 Mouse hepatitis virus (MHV)
 Mouse parvovirus (MPV)
 Minute virus of mice (MVM)
 Reovirus-3 (Reo-3)
 Pneumonia virus of mice (PVM)
 Epizootic diarrhea of infant mice (EDIM)
 Theiler’s murine encephalomyelitis virus (TMEV)
 Lymphocytic choriomeningitis virus (LCMV)
 Ectromelia (mouse pox)
 Sendai virus
 Sialodacryoadenitis virus (SDAV)
 Rat parvoviruses
 Mycoplasma pulmonis
 Pinworms
 Fur mites
 Helicobacter spp. are excluded from selected animal rooms.
Many of these agents are of contemporary significance in laboratory rodent colonies worldwide,
and several have affected rodents at the University of Washington (e.g., MHV, pinworms,
parvoviruses and fur mites). If detected, excluded agents are eradicated using various methods
(see Lesson 5, Page 3 Outbreaks).
For further information on these infectious agents please click here.
Page 3. Mouse Hepatitis Virus
Mouse hepatitis virus (MHV), a coronavirus, is the infectious agent that historically has been
involved in the most outbreaks in the UW SPF mouse facilities. It is highly contagious between
mice, and is prevalent in laboratory and wild mouse populations throughout the world. Some
animals may clear the infection, but others will continue to shed the organism. Recovered
animals can still be susceptible to infection with a different strain of the virus.
MHV only infects mice. It is shed in the feces and mice can become infected by licking or sniffing
infected mice or bedding. In addition, MHV can be a contaminant of biological materials such as
cell cultures and transplantable tumors, and thereby infect recipient mice.
Numerous strains of MHV exist and more may be created as the virus spontaneously mutates.
The relationships between virus strain and host age, immune status, and genotype are complex
and not always predictable. Depending on the virus strain and the immune status of the mouse,
infection may be clinically silent or result in overt disease. Depending on the strain of the virus, a
mouse without a competent immune system, such as a SCID or nude mouse, may become very ill
and die, or can harbor MHV and shed the virus intermittently for an extended period of time. For
these reasons, excluding MHV is a priority.
Disease, when it occurs, may be limited to the intestine and draining lymph nodes, or spread
throughout the body. The name “hepatitis” is somewhat misleading, as other organs besides the
liver may be affected. For example, the central nervous system (brain and spinal cord) is a target
for some of the more pathogenic strains of MHV.
Whether or not MHV causes disease, the virus is notorious for interfering with research by
altering the biological responses of mice. Infections modulate the immune response. Liver
metabolism can also be affected.
There is no treatment or preventive vaccination. The two most successful methods for
elimination of an outbreak are 1) identification and euthanasia of infected mice, and 2) embryo
rederivation. Far less reliable methods include caesarian rederivation (neonatal transfer) and the
so-called "burnout" (temporary cessation of breeding), and should only be attempted in fully
immunocompetent mice under carefully selected and managed situations.
Page 4. Sialodacryoadenitis Virus
Rats are susceptible to another highly contagious coronavirus, Sialodacryoadenitis virus (SDAV).
The primary targets of the virus are the salivary and lacrimal (tear-producing) glands.
Transmission is by aerosol exposure or direct contact with infected animals.
SDAV infections may be subclinical (show no clinical signs) or cause clinical signs such as sniffling,
swelling under the jaw, dark red encrustations around the eyes and nostrils, swollen eyes and
corneal ulceration. Respiratory infection with SDAV can increase susceptibility to other
pathogens. Pulmonary macrophage function can be altered. Other consequences of SDAV
infection include reduced food intake, behavioral changes and reproductive disorders, for
example neonatal death. Athymic nude rats are unable to clear SDAV infections and develop
chronic wasting disease.
Mortality due to SDAV is unusual in adult rats, except for an increased likelihood of death under
anesthesia. Anesthetic deaths are attributable to transient infection of the respiratory tract with
this virus. Like MHV, there is no treatment or preventive vaccination for SDAV. Eliminating the
virus from a colony is best accomplished by testing animals and euthanizing those found to be
infected, or rederivation by embryo transfer.
Page 5. Parvoviruses
There are several parvoviruses that infect rodents, including:
Mouse Parvovirus (MPV; formerly called mouse orphan parvovirus) causes no overt signs of
disease in mice. However, it is problematic for research because the virus can persist in lymphoid
tissues of adult, immunocompetent mice, resulting in significant perturbations of the immune
response. Intermittent shedding of the virus makes it particularly difficult to detect. MPV appears
to be on the increase in mouse colonies nationwide. It was first discovered as a contaminant of Tcell lines.
Minute virus of mice (MVM) is also a common contaminant of biological materials. It can cause
changes in tumor growth kinetics. It has not been associated with overt signs of disease under
natural conditions of infection. Control can be complicated by the fact that it has been shown to
survive cesarean rederivation.
Rat Virus (RV; formerly called Kilham rat virus) can cause significant pathology in adult rats,
especially under immunosuppressive conditions. It is transmitted by respiratory, fecal-oral, and
transplacental modes. It can contaminate transplantable tumors and tissue culture cell lines. It is
reported to suppress cytotoxic leukocyte activity and stimulate interferon production. It has been
reported to influence the development of diabetes in BB rats.
Rat Parvovirus (RPV; formerly rat orphan parvovirus) seems to be nonpathogenic but has a strong
affinity for lymphoid tissue.
Page 6. Pinworms
Pinworms in mice and rats belong to two major genera, Syphacia and Aspicularis. All rodents can
be infected with one or more species of pinworms, and the young are particularly susceptible.
Mice and rats may be affected with the same species of pinworm and transmit them to each
other. These pinworms are not a zoonotic problem: rodents and humans do not share pinworms.
(Humans have their own pinworm, Enterobius vermicularis).
Light pinworm infections are usually subclinical, while heavy infections may result in rectal
prolapse, intussusception (intestine telescoping upon itself), fecal impaction and diarrhea.
Pinworms may also impact research by decreasing weight gain, changing behavior, and altering
drug absorption and immune responses. They have also been reported to enhance or suppress
tumor growth.
Transmission of pinworms occurs easily when eggs passed in feces are ingested by another
rodent. Excluding pinworms from a colony is challenging because the eggs are so resistant to
environmental degradation and most chemical disinfectants. Infective eggs can survive for
months outside the host. The microscopic eggs also stick to people (e.g., skin and hair) and things
(e.g., shoes, clothing, papers, instruments), and drift along in the air and dust.
Pinworms are treated with medicated feed or water, but their total elimination from a colony
once it is contaminated can be difficult to achieve with certainty. Treatment is expensive. For
these reasons, it is preferable to prevent pinworms from entering a colony in the first place.
Page 7. Fur Mites
There are several genera and species of fur mites that infest rodents. Mice may have mixed
infestations of Myobia, Radfordia and/or Myocoptes; whereas, rats are usually infected with
Radfordia.
Because transmission is usually by direct contact between animals, fur mites in rodents are not
easily transferred when the animals are maintained in filter top cages. However, if one mouse in a
cage has mites, all of its cagemates will have them as well. Moving animals between cages, as for
mating, is a common way to transmit mites.
Clinical signs of mite infestation are quite varied and depend on the species of mite as well as the
strain of the host. C57/BL6 mice are especially sensitive to the effects of Myobia infestation,
developing hypersensitivity reactions that can lead to pruritis (itching), alopecia (hair loss),
ulceration and secondary bacterial skin infections. This type of allergic reaction can adversely
impact immunological and other types of research. Infestations can also decrease fertility and
reduce the growth rates of young animals.
Mites are treatable with medication, usually given in the water. However, the treatment is
expensive and can interfere with research protocols in some instances. It is preferable to keep
animals with fur mites out of the facility.
Lesson 4. Mechanisms of Pathogen Transfer
Page 1. Mechanisms of Transfer - Introduction
There are several ways pathogens can enter the animal environment. The SPF procedures
described in this course are designed to prevent this entry.
There are four basic routes of pathogen introduction
 Contact
 Fomites
 Airborne/Aerosol
 Materials of biological origin
Page 2. Contact
Any time new animals are brought into a colony there is a risk that they could bring a pathogen
with them.
The UW has two measures in place to prevent pathogen introduction via this route:
Approved Vendors: Rodents coming from these vendors will be housed directly in an existing
colony room as long as they arrive in a stable, intact animal-shipping container and pass a health
inspection.
Incoming Quarantine: Rodents coming from non-approved commercial vendors, or from other
educational institutions, must go through a quarantine period while the pathogen status of those
animals is checked via the sentinel system.
For more information on Approved Vendors and Incoming Quarantine please see Lesson 14.
Page 3. Fomites
Fomites are non-living, inanimate objects (e.g., dust, clothing, equipment, etc.) that carry
pathogens. A pathogen can "hitch a ride" on these objects and be introduced into the
environment.
Sterilizing equipment prevents fomite transmission. To decrease the chances of people entering
the SPF areas with fomites, personnel are required to wear specialized garments in order to enter
SPF areas.
Page 4. Airborne/Aerosol
Pathogen particles (e.g., viruses, bacteria, etc.), respiratory droplets, and small fomites which can
float on air currents can introduce disease via the air.
At the UW, rodents housed in SPF rooms are protected by the filtered caging systems. While the
cage remains closed (cage top in place with proper fit) airborne contaminants cannot enter.
In order to protect the animals when the cage is open, the animal cage is opened only inside a
Laminar Flow Hood. When properly used, the hood provides a clean air environment and
prevents airborne contaminants from entering the cages.
Page 5. Materials of Biological Origin
Materials of biological origin are living materials such as cells, tissues, cultures, transplantable
tumors and even protozoa, as well as other materials such as serum. If these materials are
introduced into a live animal, that animal can become infected with any pathogen that is carried
by the material. Therefore, materials of biological origin must be tested to make sure they don't
harbor unwanted infectious agents.
Unless materials of biological origin are harvested de novo from SPF rodents here at University of
Washington, they must be tested before they are inoculated, implanted or otherwise introduced
into animals here. As an example, the ATCC (American Type Culture Collection) does not regularly
test for the presence of adventitious infectious agents, and recommends that their customers do
so.
Fortunately, materials of biological origin can be tested relatively quickly and inexpensively. PCR
testing is currently recommended as a more rapid and sensitive alternate to the slower and more
expensive antibody production tests, and costs only a few hundred dollars to rule out a battery of
agents. Results are usually available within a few weeks.
Please contact the Rodent Health Monitoring Program for instructions on how to arrange
testing.
Lesson 5. Monitoring for Pathogens
Page 1. Rodent Health Monitoring Program
The Rodent Health Monitoring Program (RHMP) performs an essential research support service
by tracking the pathogen status of individual rodent racks, room by room. This is accomplished
via a sentinel monitoring system, which the RHMP coordinates and oversees.
The RHMP generates room and facility health reports based on the findings of the sentinel
monitoring system. These health reports are provided upon request to investigators wishing to
ship rodents to other facilities.
Once a month the RHMP notifies the Specific Pathogen Free Operations Committee (SPFO)
regarding any SPF issues. The role of SPFO is to address outbreaks and violations of SPF
Procedures. If investigators have concerns regarding SPF procedures or outbreaks, they are
welcome to bring it to the attention of the SPFO committee for resolution. Please contact the
manager of RHMP to have items put on the SPF agenda.
The RHMP reviews requests to receive rodents from other institutions ("Non-Approved
Vendors"), and either approves or disapproves these requests based on the pathogen status of
the sending institution. Rodents must be classified as SPF by the vendor in order to be considered
for approval by RHMP. Rodents from Non-Approved Vendors go through Incoming Quarantine
before being housed in vivarium rooms. The length of stay in quarantine (usually 5-9 weeks,
including testing time) is determined by the RHMP, again depending primarily on the pathogen
status of the sending institution, and the results of pathogen testing via the sentinel system.
Test results for materials of biological origin are also reviewed by the RHMP prior to the use of
such materials in University of Washington rodents.
Requests to transfer rodents between facilities (and sometimes rooms) within the University of
Washington vivaria system are routed through the RHMP for approval based on pathogen status.
Last but not least: should a pathogen be detected, the RHMP is responsible for its elimination.
Briefly, the entire room is placed under quarantine until the infected rodents are identified and
removed (or otherwise cleared of their infection) and successive rounds of sentinel testing
demonstrate that the pathogen is no longer present.
Page 2. Sentinels
Sentinel rodents monitor the pathogen status of investigator's rodents. Every rack in the room is
monitored. As you are facing a rack, the cage containing the two sentinels usually resides in the
lower right-hand position.
Every time an investigator's rodent cage is changed, about a tablespoon of soiled bedding from
the cage is transferred to the sentinel cage. In this way, sentinels are exposed to whatever
pathogens may be present in the urine, feces, fur, saliva, dander, etc., from 100% of the cages on
the rack. To prevent disruptions in the sentinel program, investigative personnel must not handle
or move sentinels or sentinel cages.
At the end of the monitoring period (usually one quarter, or twelve weeks) the sentinels are
sampled and tested for several infectious agents. Sentinels may be tested more or less frequently
for selected pathogens.
Page 3. Outbreaks
If a pathogen is detected, the entire room is placed under quarantine. The RHMP notifies
investigators who have rodents in the room. The RHMP will give groups further instruction on
how to work in a quarantined room.
What happens next depends on the type of pathogen that was detected. In the case of a parasite
like pinworms or fur mites, treatment is begun by Veterinary Services. Treatment is generally
effective, but can be lengthy and expensive. For example, pinworm treatment takes
approximately 3 months.
In the case of a virus like MHV, or certain bacteria for which no effective treatment exists,
infected rodents are identified through testing conducted by the RHMP. Usually, rodents found to
be infected are euthanized. If an entire line of rodents is infected however, and cannot be found
at another SPF facility, then there may be other options to save the line from extinction. Embryo
rederivation is one of those options.
Once virus-or bacteria-infected rodents have been identified and removed, new sentinels are
brought into the room and receive soiled bedding from the remaining animal cages. Successive
rounds of sentinel testing are conducted until it is determined that the pathogen is no longer
present in the room. Usually, this takes four consecutive negative tests. The frequency of this
testing varies depending on the type of pathogen, therefore, RHMP will notify all groups in the
room of the scheduling of testing when the room is placed under quarantine.
How long quarantine lasts depends on the pathogen involved, and how well it was contained
before anyone knew it was there. This last statement cannot be overemphasized. As long as SPF
procedures are rigorously followed, it's quite possible for a pathogen to be limited to one or a few
cages! This is remarkable, considering the extremely contagious nature of pathogens like MHV,
and demonstrates how well SPF equipment and procedures can perform when used properly and
consistently.
Page 4. Materials of Biological Origin
The RHMP is responsible for approving the use of materials of biological origin in UW SPF rodents.
For details, see Lesson 4: Mechanisms of Pathogen Transfer.
Lesson 6. SPF Facilities at the University of Washington
Page 1. What kinds of SPF facilities does the UW have?
The University of Washington maintains two types of SPF facilities:
Barrier facilities and Non-Barrier facilities.
Page 2. Barrier Facility
The K-wing Barrier facility is the strictest level of rodent housing that the UW maintains and
requires that rodents leaving the facility may only return by first going through quarantine. This
facility requires that personnel wear dedicated facility garments as part of the entry procedures.
Page 3. Non-Barrier Facility
The Non-Barrier facilities maintain animals in a clean environment, but allow rodents to leave the
facility and return without going through a quarantine period. Personnel entering non-barrier
facilities may be required to put on dedicated scrubs and shoes (check with facility supervisor). A
non-barrier facility may house other species besides rodents.
The procedures required to return rodents to a non-barrier facility are:
1. Paper transport boxes are not allowed to re-enter the facility. Make sure to transport
your animals in a filter-top cage.
2. The cage being returned must be sprayed with Clidox® at the entrance to the facility (if
applicable), at the door to the animal room, and again before being placed in the hood.
3. The animal must then be housed in a "new" sterile cage. You must give the animal new
food, water, nestlet, wire rack, and cage top.
Please see the facility supervisor if you have questions regarding this process.
Lesson 7. Facility Entry Procedures
Page 1. Introduction to Barrier and Non-Barrier Facilities
If you have duties in both barrier and non-barrier facilities, you should perform your work in the
barrier facility before going to the non-barrier facility. Adherence to the procedures outlined in
this chapter is vital to maintaining the pathogen-free status of the animals housed in these
facilities.
Page 2. Entering a Barrier or Non-Barrier Facility
Follow these steps for preparing to enter a Barrier or Non-Barrier Facility:
1. Enter the facility locker room.
2. Remove street clothing, except for undergarments, and place it into a locker.
3. Remove any jewelry that will be exposed while wearing your scrubs (including wrist
jewelry, necklaces, and earrings).
4. Put on dedicated facility garments (scrubs, dedicated shoes, and bonnets).
Check with the facility supervisor to find out which areas of the non-barrier facility you are
required to wear scrubs in and which areas are okay for street clothes.
Lesson 8. Animal Housing and Procedure Room Entry Procedures
Page 1. Entering Dedicated Clothing Animal Housing and Procedure Rooms
Follow these steps when entering an animal housing or procedure room in a dedicated clothing
facility:
1. Put on a pair of gloves.
2. If you will be working under the hood, put on a pair of sleeves.
Page 2. Entering Non-Dedicated Clothing Animal Housing and Procedure Rooms
Follow these steps when entering an animal housing or procedure room in a non-dedicated
clothing facility:
1. Put on shoe covers over your footwear as you step across the threshold of the doorway
into the room.
2. Put on a hair bonnet (be sure to cover all of your hair).
3. Put on a pair of gloves.
4. Put on a lab coat (if required).
5. Put on a pair of sleeves if you will be working in the hood.
Lesson 9. Introducing Supplies and Equipment into SPF Rooms
Page 1. Introduction to Supplies and Equipment
Often a large variety of equipment and supplies are required to maintain and work with animals
in an SPF environment. While these items are necessary, it is very important to ensure that they
do not introduce contaminants into the environment.
Research groups are allowed to keep necessary experimental equipment in SPF animal rooms;
however, items must be prepared and introduced according to the procedures outlined in this
chapter. For large equipment, please contact facility supervisor.
NOTE: Cell phones or other devices capable of taking photographs are not allowed in Animal
Facilities unless prior arrangement and approval has been made with the Dept of Comparative
Medicine.
Page 2. Experimental Supplies
Experimental equipment entering an SPF facility must be sterilized prior to entry. The only
exception to this rule is for materials of biological origin (see below). If you do not have access to
an autoclave or gas sterilizer through your lab, this service is offered free of charge through
Central Processing at the UW Medical Center.
If it is impossible to sterilize a piece of equipment by the two methods listed above, you should
contact the supervisor of your facility to gain assistance in determining whether disinfection is an
acceptable alternative.
Items should be double-wrapped (e.g., two autoclave bags, two surgical wraps, etc.) and then
sterilized.
Bringing items into an animal room: Upon arriving at the facility, remove the outer wrap from
the item, discard the wrap in the trash outside the facility entrance door, and then take the item
inside. When you reach the door to the animal room, spray the inner wrap with disinfectant, and
place the item into the room. When you are ready to use the item in the hood, remove the inner
wrapper and place the item in the hood.
For new items, place into a plastic bag and spray bag into facility. Once in the facility, remove bag
prior to entering the animal or procedure room. Store all supplies in your personal plastic storage
container.
Page 3. Materials of Biological Origin
Materials of biological origin such as cells, tissues, serum, and cultures will be damaged or
destroyed by autoclaving or gas sterilization techniques. Therefore, they must be tested before
they are introduced into animals. Please contact Rodent Health Monitoring Program at
[email protected] for instructions.
Once found to be free of pathogens:
1. Place materials inside a sterile transport container (such as a centrifuge tube). The
container should be securely sealed and then placed inside a closed plastic bag.
2. At the facility entrance door remove the outer bag and discard.
3. Spray the transport container thoroughly with the disinfectant provided at the facility
entrance.
4. Spray the transport container into the animal room.
5. Spray the transport container into the hood.
Page 4. Paper Documents
To facilitate use of documents in SPF facilities, there are specific procedures which must be
followed.
Documents must be either faxed into the facility or encased in plastic. If encased in plastic,
documents must remain in plastic and be sprayed into facility and animal rooms.
For additional suggestions, please contact facility supervisor.
Page 5. Carts
Please check with the facility supervisor to see if carts are allowed in the facility. You may be
required to leave your carts outside the entry doors.
Lesson 10. Transportation of Rodents
Page 1. Transporting Rodents
Terminal procedures: Rodents may be transported in filter-top cages or paper transport boxes
provided by facilities.
Survival procedures: Rodents must be transported to approved procedure locations in filter-top
cages, not in paper transport boxes (paper boxes cannot re-enter the facility). Re-house in new
cage upon returning. (Animals may not be returned to the K-wing facility without first going
through quarantine).
All cages must be covered or draped when traveling through public corridors. This includes
empty cages.
Please avoid highly populated public areas.
Lesson 11. Hoods
Page 1. Introduction
Make sure the hood is TURNED ON before beginning your work!
Page 2. Types of Hoods
When working in UW SPF Animal Facilities you will typically work with one of the following types
of hoods:
Horizontal Laminar Flow Hood: Protects what’s inside the hood, not the user, from microbial
contamination. The user may be exposed to allergens.
Vertical Laminar Flow Hood: Protects both what’s inside the hood and the user from microbial
contamination and allergens.
Certified Biosafety Cabinet: Protects both what’s inside the hood and the user from microbial
contamination and allergens, including agents infectious to humans. Infectious agents can only
be used in biosafety cabinets. Please consult with facility supervisor before using chemical
hazards and infectious agents.
Certified Fume Hood: Protects user from volatile chemicals or anesthetics. Fume hoods DO NOT
protect the rodents from microbial contamination. These hoods are often found in many
investigator labs and are in some procedure rooms.
Lesson 12. Working in an Animal Room Hood
Page 1. Introduction
One of the most important aspects of working within an SPF facility is the procedure used for
working in the animal room hood. The steps outlined in this chapter must be followed closely in
order to avoid contamination to the SPF environment and potential contamination to your
research animals.
Overview of steps:
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
Put on gloves and sleeves.
Turn on the hood. (Verify that blower is on).
Spray inside of hood except perforated surfaces with Clidox.
Wipe with paper towel.
Spray work surface and gloves. Leave wet.
Collect your cage and place on wet work surface.
Spray sides of cage (not filter top).
Open cage top, spray hands, work with animals.
Close cage and return to rack.
Spray work surface and wipe dry.
Repeat steps 5-10 to work with additional cages.
When done, spray inside of hood except perforated surfaces and wipe dry.
Turn off hood.
When exiting the room, remove all garments that were put on when you entered.
Page 2. Dressing to Work in the Animal Room Hood
When working in a hood, you MUST wear sleeves and gloves at all times, in addition to your gown
or dedicated facility scrubs.
Pull the sleeves on so your arm is covered from below your wrist to just above your elbow. Pull
cuff of gloves up over sleeves. Make sure no skin is exposed. There should be no gap between
gloves and sleeves.
The cuff of the sleeve should remain under the gloves and the top of the sleeve should stay above
your elbow at all times while working in the hood.
Page 3. Preparing the Hood
To begin working in a hood, check to make sure the power switches for the lights and blower are
on.
Disinfect your gloves by spraying them with Clidox.
Disinfect the work area within the hood by spraying all flat surfaces (work surface, sides,
faceshield) with Clidox. Wipe with paper towel. Then re-spray the work surface leaving it wet. DO
NOT spray the light fixture or the perforated air supply surface. Instead, wipe the perforated air
supply with a wet paper towel.
Page 4. Transferring Cages
When transferring animal cages from the housing rack to the hood, make sure that the cage top
stays securely in place. Opening the animal cage outside of the hood can expose your animals to
pathogens.
Page 5. Placing Cages and Equipment into the Hood
All items (cages, supply boxes, your gloves) entering the hood must be disinfected with Clidox.
Equipment: Spray and place inside the hood.
Cages: Set the cage on the wet work surface. Then spray the side of the cage. If the cage has a
solid cage top then you can also spray it. If your cage has a filter top then do not spray the filter as
this is a liquid pathway for pathogens to enter the cage.
Page 6. Opening a Cage in the Hood
The cage can be opened once it is completely inside the hood.
Remove the cage top and flip it over as you set it on the work surface, so the interior surface
points upward. If using a filter top, make sure to set it on a clean, dry portion of the work surface,
being careful to prevent the filter from getting wet.
To access the animals inside the cage, turn the wire rack sideways (90 degrees) and slide it to the
rear of the cage. If you need to remove the wire rack to access your animals do not place the rack
on the filter, it can damage the filter top. Clean an area on the work surface and place the wire
rack on the freshly cleaned area. If you are working with a system using a water bottle, be sure to
turn the bottle so the valve points upward.
Page 7. Closing a Cage
Once you have completed your work with the animals, replace the wire rack and return the water
bottle to its original position with the valve pointing down.
Return the top to the cage, making sure it is securely in place. The cage top should sit evenly and
securely on all sides of the cage.
Page 8. Returning a Cage to a Rack
Remove the cage from the hood and return it to the housing rack. Be sure the cage seats
completely into the rack, especially when using a ventilated or automatic watering rack system.
Please see Lesson 13 for further information on water devices.
It is important to return the cage to the space that it came from. If you put a cage into a new
space (e.g., weaning, separating fighting mice, setting up breeding cages) and you are using a
ventilated rack where the lixits (water ports) remain on the rack, then the lixit should be sprayed
with disinfectant and wiped clean prior to putting a cage into a new space.
In the event that you drop a cage or an animal follow these steps:
1. Gather up the animals immediately, recruit help for this if necessary.
2. Go to the hood. Following SPF procedures, place the animal(s) in a new cage with a new
wire rack, new water bottle and food.
3. Fill out a separate cage card and label the cage with a Vet Check card.
4. Place the cage on the lowest shelf.
5. Fill out a Sick Animal Report, and contact Vet Services immediately. Vet Services will
contact RHMP who will contact the investigative group to determine what action to take
regarding the animals.
6. Sweep the floor where cage was dropped, if any bedding spilled. Take the dirty cage to
the cage wash.
Setting up new cages of mice:
1. Be sure the food hopper is full of food
2. Place nestlet in cage
3. If the caging system has automatic watering system put gel into the bottom of the cage
and toggle the lixit. Place a toggle or gel/toggle sign on the cage to help the animal
technician spot newly weaned mice who need to be taught how to use the automatic
watering system.
4. If the caging system uses water bottles be sure the cage has a water bottle. Be sure the
sipper tube is put on tightly.
5. Place a cage card on the cage indicating the primary investigator, budget number, IACUC
protocol, contact information, species, receipt date or date of birth, and room number.
6. Each new cage needs to be accounted for on the per diem sheet.
Page 9. Working with Multiple Cages Sequentially
If you intend to work with more than one cage during your session, the following procedures
MUST be followed:
 Disinfect the work surface in the hood between each cage. Wipe wet work surface with
paper towel. Then, spray work surface with Clidox and leave wet.
 If working with cages from the same row in the rack (horizontal movement), you must
disinfect your gloves (spray with disinfectant) between each cage.
 If you move from one row to a different row (vertical movement), you must change
your gloves.
 If you move from one side of a rack to a different side of a rack, you must change your
sleeves and your gloves.
 If your gloves becomes damaged or sticky, you must change them.
 When working with different groups of animals you should change your gloves.
 Any equipment being used with animals must be disinfected between each cage.
NOTE: It is important NOT to work with more than one cage at a time in the hood, except when
weaning litters or setting up breeding cages.
Page 10. Working with Experimental Supplies
You may store experimental supplies in the room as long as they are stored in closed, non-porous
supply boxes (cardboard and Styrofoam are unacceptable). The box must be handled in the same
fashion as an animal cage.
Page 11. Completing Work in the Hood
When you have completed your work session, the inside of the hood must be completely
disinfected and wiped dry. Be sure the towel and food boxes are securely closed.
Shut down the hood by turning off the lights and the blowers.
When exiting the animal room, remove all the garments you put on as you entered, and dispose
of them in the room. If you are wearing shoe covers you MUST be certain to remove your shoe
covers as you step across the doorway.
Lesson 13. SPF Caging Systems at the UW
Page 1. Introduction to SPF Caging Systems
The caging systems used for housing SPF rodents are designed to provide the animals with
environments isolated from outside contamination. The caging systems are designed so that each
cage has its own food, water, and filtered-air supply.
The University of Washington uses two primary types of caging systems:
 Micro-isolator caging
 Individually Ventilated caging
The different caging systems and watering devices are discussed on the following pages. It is
important to understand the basic aspects of the caging systems under your care as a rodent may
die from dehydration in as little as 24-36 hours.
NOTE: The facility supervisor needs to be notified using a Special Service Request if animals are on
treated water.
Page 2. Micro-isolator Caging
Micro-isolator (MI) cages are closed, static cage systems that use a high efficiency filter within the
cage lid. This filter removes particulates from the air as the air passes through the filter although
gaseous contaminates can still pass through. The food and water supply are maintained within
the cage.
These cages utilize no special rack system. The cages may be placed onto the shelves of standard
racks.
Page 3. Individually Ventilated Caging
The ventilated cage rack uses a built-in HEPA filter system, thus providing a forced clean air
supply and exhaust for each individual cage. These systems may have a rack-mounted automatic
watering system.
The UW currently uses individually ventilated rack systems manufactured by three different
companies:
 Allentown Caging Equipment
 Thoren Caging Systems
 BioZone®
Page 4. Allentown Ventilated Racks
The Allentown ventilated racks house cages that are individually ventilated and may or may not
be equipped with cage tops that have filters. The racks may be outfitted with automatic watering
systems with the lixits residing in each cage. Once the cage is engaged (air/water connected) into
the rack, the lixit will provide water when toggled. If the rack does not have an automatic
watering system they must have a water bottle placed inside of the cage.
The racks are equipped with an easy-connect lock that displays the status of the air/water
connection. When a cage is engaged, there will not be a red circle present. When a cage is
disengaged, there will be a red circle present.
If using a water bottle within a cage (i.e. for treated water) in an Allentown rack with an
automatic watering system, a micro-isolator filtered cage top must be used. If you do not want
your animals to have access to untreated water from the lixit, ensure that the cage is not engaged
into the rack. This is accomplished by turning the bottom of the cage in the opposite direction
(lixit facing out). Please see facility supervisor for a supply of filter tops.
Page 5. Thoren Ventilated Racks
The Thoren ventilated racks house cages that have filter tops and the racks are equipped with an
automatic watering system. The lixits on these racks are connected to the rack (unlike the
Allentown system) and fit into the cages once the cages are in place.
If using a water bottle (i.e. for treated water) while utilizing the Thoren caging system you must
request a wire top that allows for a water bottle to be used (see the facility supervisor’s office). If
you do not want your animals to have access to untreated water from the lixit, ensure that the
cage is not engaged into the rack. This is accomplished by not pushing the cage all the way in. Do
NOT replace the cage backwards as this could cause a flood if the cage comes in contact with the
lixit.
Page 6. BioZone Ventilated Racks
The BioZone ventilated racks contain cages with filter tops; however they do not have an
automatic watering system. All cages have a water bottle attached to the outside of the cage.
Page 7. Biosafety Room Water
All biosafety room water is provided through a water bottle. Please follow the above guidelines
for water bottle use in these rooms depending on the rack system utilized.
Lesson 14. SPF Housing, Rodent Weaning, Rodent Ordering, Transfers, and
Shipping
Page 1. Space Requirements
When housing your rodents, it is very important to follow all the required guidelines so that they
will be healthy and happy. There should never be more than five adult mice in a standard size
cage. The Institutional Animal Care and Use Committee (IACUC) must approve any variance to this
policy.
Page 2. Space Requirements for Breeding
There are additional requirements for cages that are set up for breeding. The UW IACUC policy
allows:
 1 adult male mouse, 2 adult female mice, and 2 litters up to 21 days of age
 1 adult female mouse, 1 litter up to 28 days of age and 1 litter under 21 days of age
See the IACUC Policies for additional information.
Page 3. Arranging Housing Space
Prior to ordering new cages of rodents, you should contact the facility supervisor to insure that
your investigative group has space available on the rack.
Page 4. Weaning of Rodents
Rodent pups need to be weaned or separated at 21 days of age unless approval was received
from the IACUC for a weaning variance in the applicable protocol. Weaning variances should be
posted on the cage card or rack. Ensure that each new cage has a cage card with the mother’s
identification and cage location in addition to the normally required information. Each new cage
produced needs to be accounted for on the per diem sheet.
When rodents are weaned, they need to be shown how to obtain water out of the lixit or water
bottle. Toggling (lightly touching the end of the lixit) produces drops of water that teach the mice
where the water is. When using Allentown cages, you must use the charging station located in the
hood to toggle the lixit. When using Thoren cages, toggle the lixit located on the rack. When using
water bottles, gently shake the bottle to ensure water will drip from the sipper tube.
Gel (Napa Nectar) is added to the cages of weaning animals to ensure moisture intake until the
animals learn to use the lixit. Additional options for nutritionally challenged pups are to place
food in an easy to reach location or to soften food pellets with water (this option requires more
frequent food changes in order to prevent contamination by urine or feces).
Page 5. Importing Rodents
Rodents may be imported into UW animal facilities from either of two general classes of vendors:
Approved and Non-Approved Vendors.
Approved Vendors
Approved Vendors have been approved by RHMP and are generally the larger commercial
companies such as Charles River, Jackson Laboratories, Taconic Farms, and Harlan Laboratories.
These companies perform frequent testing and guarantee that their rodents are free from a long
list of potential pathogens, including zoonotic agents (pathogens transmissible to humans).
Rodents purchased from Approved Vendors may be housed directly in vivarium rodent rooms. An
Approved Vendor Request Form can be obtained from the Department of Comparative Medicine
Website in the 'Animal Transfer & Procurement Forms' section. A current list of Approved
Vendors can be obtained by contacting Animal Purchasing [email protected]
Non-Approved Vendors
Orders from Non-Approved Vendors are approved by RHMP on an individual basis. Examples of
Non-Approved Vendors include smaller commercial vendors, other educational institutions,
biotechnology firms, and non-profit research organizations like Fred Hutchinson Cancer Research
Center. The difference between an Approved and a Non-Approved Vendor reflects frequency of
pathogen testing, quality and security of facilities and transport methods, in-and-out traffic of
animals (and people), history of outbreaks, etc. For example, other institutions would consider
the UW a Non-Approved Vendor.
A Non-Approved Vendor Request Packet can be obtained from the Department of Comparative
Medicine Website in the 'Animal Transfer & Procurement Forms' section. The packet includes an
Ordering Guide and a Non-Approved Vendor Request Form.
The final step in the process is approval by the RHMP. It requires that the RHMP has the
completed Non-Approved Vendor request form from the investigator requesting the animals,
together with current (within the last three months) health reports from the sending institution.
The investigator requesting the rodents is responsible for obtaining the health reports and having
them sent to Animal Purchasing ([email protected], fax 206-543-7706).
Most Non-Approved Vendor requests receive prompt approval, because health reports from
sending institutions are usually complete, up-to-date, and indicate no evidence of infection.
However, sending institutions are sometimes under their own quarantine for an outbreak, and
approval must be delayed until the institution has the infection under control. One of the
functions of the RHMP is to work with sending institutions to resolve such issues.
Once a shipment is approved, the investigator will be notified of the shipping date. When the
rodents arrive, they will be housed in Incoming Quarantine for a period of time (see Incoming
Quarantine).
In rare instances, approval may be denied. This primarily happens when the sending institution is
unable or unwilling to eradicate an infection(s). The investigator will be notified of the
disapproval, and various options will be suggested by the RHMP. Embryo rederivation, possibly by
an Approved Vendor, is usually recommended as the most reliable way to eliminate an
infection(s).
Page 6. Incoming Quarantine
Rodents arriving from a Non-Approved Vendor must go through a period of Incoming Quarantine
before being housed in a general vivarium animal room. The length of stay in Incoming
Quarantine is determined by the RHMP, and depends primarily on the overall pathogen status
and outbreak history of the sending institution. Most Incoming Quarantine stays last 5-9 weeks,
including testing.
During quarantine, sentinels are assigned to monitor each shipment. At the end of the quarantine
period, the pathogen status of the investigator’s rodents is assessed via testing these sentinels.
The investigator will be notified when his or her rodents “pass” quarantine and are released by
the RHMP to their final, pre-approved room destination.
Breeding is typically permitted in Incoming Quarantine two weeks prior to the scheduled release
date so that pups are not born in quarantine. In special cases (eg. fragile lines with a narrow
breeding window), breeding may be permitted earlier. Please contact the RHMP if you want to
set up breeders.
In specific situations rodents may be removed from Incoming Quarantine for terminal
procedures. Please contact the RHMP if you wish to retrieve your rodents from quarantine for
this purpose. The procedures may be performed in labs or procedure rooms, as long as the area is
thoroughly disinfected afterwards.
Page 7. Shipping Rodents from the UW
Investigators wishing to send their rodents to collaborators elsewhere will need to ask the RHMP
for current health reports. A Request for Rodent Health Monitoring Report form can be obtained
from the Department of Comparative Medicine Website in the 'Animal Transfer & Procurement
Forms'. Please submit the completed form to [email protected]. The RHMP will fax the
appropriate health report to the receiving institution. For additional information, please
reference the Rodent Health Monitoring Program’s website.
NOTE: Whether or not to accept the rodents is entirely up to the receiving institution. The RHMP
is not involved beyond providing the health reports. However, the RHMP is happy to help clarify
any issues the receiving institution may have regarding the pathogen status of the animals or our
facilities in general.
A health certificate must accompany rodents destined for interstate or international travel. This is
different from a rodent health monitoring report. Health certificates are available through
Veterinary Services and can be requested at the Veterinary Services’ website. Please allow three
(3) business days to obtain a health certificate, as the rodents must be physically examined by an
authorized veterinarian. Health certificates should not be requested until you have received
approval to ship from the receiving institution. International shipments may have additional
requirements depending on the receiving country.
It is imperative that investigators wait to ship until after approval is granted (in writing) by the
receiving institution. Once approval is granted, the investigator is responsible for packing the
rodents safely for their journey, making the shipping arrangements with the appropriate
commercial carrier, and actually delivering the rodents into the hands of the commercial carrier’s
delivery person. The RHMP and Veterinary Services are pleased to offer advice on these matters,
but they can not ship the animals.
Page 8. Transferring Rodents Between Rooms and Facilities
In order to transfer rodents between UW facilities or rooms you must complete a UW Facility to
UW Facility Transfer Request Form. In order to transfer rodents between budgets and/or
protocols, you must complete a Budget and/or Protocol Transfer Request form. These forms can
be found at the Department of Comparative Medicine website.
E-mail the completed form to the Rodent Health Monitoring Program. Rodent Health Monitoring
will check the health status of the rooms and will notify you by email when the transfer has been
approved. Before you move your animals, contact the new facility supervisor to arrange for
caging.
In general, if you are transferring rodents within buildings you are responsible for the physical
transfer. Please see your facility supervisor for instructions and supplies. Off site transfers (such
as HR&T, Brotman and Roosevelt) require the use of Department of Comparative Medicine
transport vehicles. You may not transfer animals in personal vehicles. Please contact the facility
supervisor to make arrangements.
IMPORTANT:
1. Please transfer your cages following SPF guidelines.
2. Unless you are using your own cage cards, pick up new cage cards at the receiving facility
supervisor's office.
3. Update room per diem sheets.
To avoid unnecessary charges, please replace the cage cards and update room per diem sheets
within 7 days.
Lesson 15. Veterinary Services
Page 1. Introduction to Veterinary Services
Veterinary Services in the Department of Comparative Medicine provides veterinary clinical care
to the research animals housed and used at the UW.
The Veterinary Services staff includes faculty veterinarians, post-doctoral veterinarians,
veterinary technologists and veterinary technicians.
In addition to providing clinical care for all animals (except non-human primates) at the UW,
Veterinary Services personnel provide consulting services for investigators and their technicians.
Federal regulations require consultation with veterinary staff when planning any study that
involves more than momentary pain. Staff members assist researchers in numerous ways
including advice on the appropriate use of anesthetics, analgesics and antibiotics, as well as preoperative and post-operative management of animals. Veterinary Services is equipped with
facilities for animal exams, treatment, surgery, and radiology. They also serve as the liaison to the
Pathology Division of the Department of Comparative Medicine.
NOTE: Veterinary Services is not a dispensary. You may not order or purchase drugs, medication,
or supplies through Veterinary Services. However, staff members will provide information on
product ordering through a variety of veterinary supply and research companies.
Page 2. Location and Contacts
Veterinary Services office hours are 8am – 5pm, Monday through Friday. An on-call veterinarian is
available by pager (206-583-1853) 24 hours a day. Contact numbers and Email addresses for
Veterinary Services are as follows:
Location
Phone
Email
Fax
6th Floor (F608) (206) 543-6257
[email protected]
(206) 685-9433
K-wing (K024C)
(206) 616-8716
[email protected]
(206) 221-6988
HR&T (SB006B)
(206) 341-5065
[email protected]
(206) 341-5062
Brotman (B49)
(206) 897-1508 [email protected] (206) 897-1517
Foege (N033)
(206) 221-4803
[email protected]
(206) 221-4856
Page 3. Reporting Sick or Injured Animals
If one of your animals is sick, whether the condition is experimentally induced or naturally
occurring, it must be reported to Veterinary Services. It is the responsibility of the person who
finds the sick animal to report it; this means the report may be submitted by the animal tech,
research tech, PI or anyone else who has access to the room. Veterinary Services is here to help
with experimental problems as well as care for animals with naturally occurring diseases.
What to do if you have or see a sick animal:
1. Place a "vet check" sign on the animal's cage (available in animal rooms, animal facility
hallways, and Veterinary Services).
2. Fill out a sick animal report (available in animal rooms, Veterinary Services, and on line via
this link: Vet Services Sick Animal Report).
3. Bring or fax report to the Veterinary Services office that cares for the animals in question.
For all emergencies, call Veterinary Services immediately. If there is no answer, or if it is after
hours, page the on-duty veterinarian at 206-583-1853.
Lesson 16. Important Reminders
Page 1. Reminders
New SPF Users:
If you require access to a housing facility, there are two pre-requisites that you’ll need to
complete prior to requesting a facility orientation.
1. Completion of the SPF Course and passing the exam with a 100%
2. IACUC approval on your PI’s protocol (if you have not done so yet, you will need to submit
a New Personnel Form to the IACUC. Instructions for submission are on the form).
Once the course is completed, please print out your Certificate of Completion and keep it for your
records. You are NOT required to submit an “SPF Access Renewal Form”. If you need instructions
on how to register for a facility orientation, please contact the Training Coordinator.
SPF Renewals:
If you are renewing your SPF training you MUST submit the SPF Access Renewal Form to the
Training Coordinator immediately upon completion of the course and passing the exam with a
100% to ensure that your proxy card is renewed for the facility to which you need access.
In addition, once the course is completed, print out your Certificate of Completion and keep it for
your records. Please do NOT send it to the Training Coordinator.
NOTE: This course is required annually in order to maintain facility access via your proxy card. If
you do not renew the course on an annual basis, your proxy card will expire. If this happens, the
SPF on-line course as well as the facility orientation must then be completed in order to regain
access. You will be sent a courtesy SPF expiration reminder email; however, individuals are
responsible for keeping track of their training and updating it annually.