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EVERY STEP OF THE WAY

C57BL/6NCrl (B6N) Germ-Free MiceBackgroundSoon after birth, the gastrointestinal tract and other

body surfaces of mammals are colonized by complex

communities of microorganisms, traditionally termed

microflora; more recently, they have also been called

microbiota and microbiomes, which some differentiate as

referring to microbial taxa and genomes, respectively.

The “normal” autochthonous (i.e., indigenous) mammalian

gut microbiota consist largely of beneficial, or commensal,

bacteria that synthesize vitamins essential to host nutrition

and provide a barrier to infection by pathogens. Gut flora

also include significant numbers of archaea, eukaryotes,

and viruses (including bacteriophages).1 Microbes

are by far most numerous in the large intestines, with

concentrations that can reach trillions of microbial cells per

gram of feces in the colon and represent 1,000 different

species.2

In humans, the number of cells that compose the

microbiota reportedly are equivalent to or 10-fold greater

than the number of human somatic cells, depending on

whether nonnucleated erythrocytes are counted.3 Therefore,

it is not surprising that the gut and other microbiota have

been found to play a key role in the development and

homeostasis of host anatomy, physiology, metabolism,

and immunity, as evidenced by the many abnormalities,

such as an underdeveloped immune system and a markedly

enlarged cecum, that characterize axenic (germ-free)

rodents demonstrably free of all foreign bacteria as well as

fungi, protozoa, parasites, and viruses.4

Research into the role of microbiota in health and disease

has increased exponentially during the past decade,

encouraged by advances in molecular genetics that

have led to the development of numerous genetically

engineered mutant animal models, as well as sophisticated,

culture-independent molecular tools for analyzing the

microbiome, notably massively parallel “next-generation”

DNA sequencing.5 This research has demonstrated

that the constituents of the gut microflora can abrogate

or accentuate the phenotypes of mutant models.6,7,8

Clinical studies have linked dysbiosis, or imbalances of

microbiota, and the loss of microbial diversity (in part

caused by the overuse of antibiotics in agriculture and

medicine) to spikes in the incidence of an array of human

diseases, ranging from juvenile diabetes to autism.9,10

RESEARCH MODELS AND SERVICES

SummaryGerm-free rodents have been

essential to microbiome research

and the production of specific

pathogen-free (SPF) rodent models.

This document describes the

background, uses, production,

shipment, and microbiological

monitoring of Charles River’s

C57BL/6NCrl (B6N) germ-free mice.

C57BL/6NCrl (B6N) Germ-Free Mice

Furthermore, the composition of patients’ microflora has

recently been reported to influence the efficacy of cancer

immunotherapy.11,12 Thus, studying and explicating the

interaction between hosts and their microbiota is of critical

importance to public health as well as animal research.

Charles River’s experience with germ-free technology

goes back to the 1950s, when the veterinarian who

founded Charles River Laboratories, Dr. Henry Foster, and

his colleagues incorporated germ-free rederivation into

the “cesarean-originated barrier-sustained” process they

pioneered for the large-scale production of SPF mice and

rats.13 In this process, germ-free rodents are associated

(i.e., colonized) with a defined cocktail of commensal

bacteria to normalize their physiology and prime their

immune systems. The cocktail most often used for this

purpose is the altered Schaedler flora (ASF) developed by

Roger Orcutt and colleagues at Charles River in the 1970s

(Table 1).14,15 In contrast to the original Schaedler flora16 on

which it was based, the ASF is fully anaerobic; moreover,

half of the eight species of bacteria in the ASF are extremely

oxygen-sensitive (EOS) fusiform anaerobes highly

representative of the autochthonous microbiota. Germ-

free and defined flora-associated animals are classified as

gnotobiotic, from the Greek roots gnostos (“known”) and

bios (“life”). By contrast, barrier-maintained SPF rodents

develop a complex microbiota that is defined only to the

extent that it does not include a limited list of pathogens.

Table 1. Compositive of Charles River Altered Schaedler Flora (ASF)*

Designation In Original Schaedler Taxonomy Genbank Accession

ASF 356 X Clostridium species AQFQ00000000.1

ASF 360 X Lactobacillus intestinalis AQFR00000000.1

ASF 361 X Lactobacillus murinus AQFs00000000.1

ASF 457 Mucispirillum schaedleri AYGZ00000000.1

ASF 492 Eubacterium plexicaudatum AQFT00000000.1

ASF 500 Pseudoflavonifactor species AYJP00000000.1

ASF 502 Clostridium species AQFU00000000.1

ASF 519 X Parabacteroides goldsteinii AQFV00000000.1

* The four ASF bacteria from the original Schaedler flora were isolated from the stomach and intestines of NCS mice in the 1960s by Russell W. Schaedler at Rockefeller University. The other ASF organisms were originally isolated from the large intestine of CD-1 mice in the 1960s at Charles River by Roger P. Orcutt (a graduate student of Schaedler’s).

Research ApplicationsB6N germ-free mice may be used as embryo transfer

recipients or foster dams for germ-free rederivation of

mutant mouse models. In addition, they may be compared

to SPF or Elite (opportunistic pathogen-free) B6N mice

to generally assess the relationship between microbiota

and phenotypes. Alternatively, the germ-free B6N mice

may be associated with a single microbial species (mono-

associated), defined microbiota like the ASF, or complex

polymicrobial mixtures to measure and understand the

effects of microbiota on phenotypes and experimental

responses.17,18,19 Germ-free mice have also been engrafted

with human microbiota by fecal transfer or inoculation of

defined microflora in order to investigate the contribution of

microbe-host relationships to human diseases.20

Production

Rederivation

The B6N strain was obtained by Charles River from the

National Institutes of Health in 1974. The current colonies of

germ-free B6N mice were rederived by sterile hysterectomy

followed by fostering on germ-free dams provided by the

Gnotobiotics and Microbiology Core at Boston Children’s

Hospital. Extensive testing by culture and culture-

independent methods described below has verified the

germ-free status of the rederived B6N colonies.

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Husbandry

An example of the plastic isolators in which the germ-free

mice are housed is shown in Figure 1. Before being used,

an isolator is tested for leaks, chemically sterilized with 2%

peracetic acid, and ventilated. Sterilization of the ventilated

isolator is confirmed by culturing swabs of surfaces,

caging, and supplies collected from the isolator over several

weeks.

Once in use, an isolator is kept sterile by being ventilated

with HEPA-filtered air under positive pressure. Supplies,

such as food, bedding, water, and caging, are autoclaved

in transfer cylinders. To assure sterilization, supplies are

arranged in cylinders following standard configurations

(Figure 2). Self-contained bioindicators containing heat-

stable bacterial spores (e.g., EZTest® Steam, Mesa Labs)

and temperature indicators are placed throughout each

autoclave run, including inside a test cylinder. Autoclave

printouts are examined to confirm that the appropriate

cycle was chosen and ran without error. Temperature

indicators are evaluated and the bioindicators, including

those retrieved from inside the test cylinder, are incubated

(at 55-60 oC). Cylinders are released for use only if all

bioindicators read negative.

Figure 1. Plastic isolator for germ-free husbandry

View of a drum filled with cages, the inside of the drum showing underlying perforations in the steel structure required for

sterilizing steam to reach internal materials during the autoclave cycle.

To antiseptically import the supplies, the cylinder is attached to the isolator port with a plastic sleeve (Figure 3); the supplies

are transferred into the isolator through a double-door lock system that has been disinfected by spraying with 2% peracetic

acid or a sterilizing level of chlorine dioxide (e.g., CLIDOX-S® diluted 1:3:1). The double-door lock system is also used to

transfer animals, samples, and other materials in and out of the isolator. All manipulations of mice and supplies inside the

isolator are through gloves and sleeves attached to the isolator walls.

Figure 2. Cylinder for autoclaving supplies

Figure 3. Autoclaved cylinder attached to isolator

C57BL/6NCrl (B6N) Germ-Free Mice

Shipping

To preserve their germ-free status during shipment,

mice are transferred from the isolator in which they are

housed through the disinfected double-door lock system

into cages within a germ-free shipper sterilized with

ethylene oxide gas. Upon receipt of a lightweight shipper,

the shipper should be attached to the port of the isolator

and the mice antiseptically transferred.

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Microbiological MonitoringGerm-free colonies are monitored for extraneous bacteria

and fungi, and for pathogens. Table 2 shows the test

methods, samples, and frequencies. Testing for extraneous

microbes is conducted frequently, based on the potentially

high incidence of this type of contamination and its

significant consequences to customers. Weekly, a slurry

from each isolator (consisting of feces and environmental

swabs in animal drinking water) is inoculated onto various

culture media, then incubated aerobically and anaerobically

in a dedicated anaerobic workstation. Because microbial

contaminants may be fastidious or non-cultivable on

cell-free media (like much of the indigenous microbiota),

culture-independent methods are employed. Wet mounts

of the slurries collected each week are examined by

phase microscopy for motile organisms. In addition, feces

collected quarterly from mice in each isolator are assayed

by PCR for the bacterial 16S ribosomal RNA (rRNA) gene.

Table 2. Microbiological Surveillance of Germ-Free Mouse Isolators

Methodology

Sample Type: Tests Frequency:

Slurry Weekly

Feces Quarterly

Animals Annually

MicrobiologyCulture Phase microscopy

X X

X X

PCRBacterial 16S rRNA Rodent Pathogens

X X

Gross and Microscopic ExamsNecropsy Parasitology

X X

Serology MFIA/IFA X

Comprehensive health monitoring for pathogens is

performed annually on mice from each isolator. This

comparatively low testing frequency is justified by the

historically negligible incidence of pathogens infecting

isolator-housed colonies. Animal organs and tissues

are grossly examined, and histopathology is carried out

if lesions suggesting an infectious disease process are

observed. Specimens from the gut and skin are examined

microscopically for endo- and ectoparasites. Blood

samples are screened for pathogen-specific antibodies by

the multiplexed fluorometric immunoassay (MFIA®), with

corroboration of unexpected (or indeterminate) findings

mostly by indirect immunofluorescence assays (IFA).

For cultural isolation of bacteria and fungi, various

microbiologic culture media are inoculated with

respiratory and gut samples and incubated aerobically

and anaerobically. Samples for anaerobic culture are

collected from euthanized mice dissected in an anaerobic

workstation. Isolates are identified both according to their

colonial and cellular morphology and by MALDI-TOF mass

spectrometry, and, if necessary, by PCR. In addition, swabs

of the skin, oral cavity, and feces are tested by PCR for

pathogens of all types.

The confirmed detection of bacterial, viral, parasitic, or

fungal agents in germ-free mice or isolators would result

in immediate cessation of shipment from the isolator and

immediate elimination of the isolator colony. Charles River

considers each isolator to be a microbiologic unit and

will not test and cull individual cages within an isolator.

It remains our policy to inform our customers in a timely

manner of any breaches in animal health or genetic integrity,

providing urgent colony health information via email or other

method. For assistance regarding specific information on

Charles River monitoring procedures, additional data on

animals, or interpretation of the monitoring information,

please direct inquiries to Charles River Technical Services

(877-274-8371) or email AskCharlesRiver@crl.com.

askcharlesriver@criver.com • www.criver.com © 2018, Charles River Laboratories International, Inc.

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