Establishing Colony Sterility

During the installation of a germ-free vivarium, it is important to establish an effective sterility monitoring program to demonstrate that all aspects of a germ-free vivarium are free of pathogens. Specific tests can be run to confirm sterility as needed, such as after protocol changes or when using a new reagent lot.

Sampling and Testing to Assess Sterility

Please consult LTM™ for specific reagent testing volume and instructions.

Sample SourceTest(s)Sample Requirements
FeedRodent feed sterility test10 – 50 grams following sterilization, collected aseptically 
WaterGerm-free water testing:

Microbial bioburden water test with counts
(bacterial identification upon request)		Two 100 mL containers containing chlorine neutralization

BeddingBedding sterility20 – 25 grams following sterilization, collected aseptically
Toys
(enrichment )		Environmental swab (culture)E-swabs run across surfaces of any enrichment item post-sterilization
Dietary supplements (enrichment)Sterility test
(broth cultures)		10 – 50 grams following sterilization, collected aseptically
Isolators
  • Environmental swab (culture)

  • RODAC plate with bacterial identification (upon request)

  • Surveillance plus PRIA®
  • E-swabs run across surfaces that may contain bacteria or fungi

  • RODAC plates^



  • Fecal pellet, body swab, and oral swab (pooled by sample type, up to 10 of each), one swab of the isolator floor, one swab of the exhaust ports EAD™ sample (for PRIA®)
Germ-free animalsSurveillance plus PRIA®
  • Up to 10 fecal pellets pooled in a microcentrifuge tube
  • Fecal pellet and body swab and oral swab (pooled by sample type, up to 10 of each)


*   If contaminants are present, MALDI-TOF can be ordered for microbe identification
^   RODAC plates not included – follow manufacturers' guidance for sampling


Maintaining Colony Sterility

Once a germ-free vivarium is established, routine diagnostic testing provides early detection in case of a barrier breach. Testing enables investigators to discover breach locations and identify the offending microorganisms. Each of the following test methodologies plays an important, unique role in the monitoring process. Because they complement each other, joint sample submissions are strongly recommended.    


3 Types of Testing for Routine Animal Health Monitoring  

 

Culture-based

Detects viable anaerobic and aerobic bacteria or fungi.

 

Germ-free 16S PCR

Determines the presence of microbial DNA but does not differentiate between viable organisms and free-floating agent DNA. Designed to minimize the risk of false positives.
 

PRIA

Detects targeted rodent pathogens, including viruses, bacteria, fungi, and parasites that may be disruptive to research or consequential to animal health.

 

Test(s)Recommended FrequencyTests Included and Sample Requirements
Germ-free culture – standard
WeeklyOne e-swab with up to three fecal pellets may be submitted for all culture tests (aerobic, anaerobic, fungal, and wet mount)

Wet mount

Fungal culture
Germ-free culture – plus (culture
and molecular testing)		Monthly

One e-swab with up to three fecal pellets may be submitted for all culture tests (aerobic, anaerobic, fungal, and wet mount)

Germ-free 16S PCR – 10 fecal pellets are pooled in a microcentrifuge tube from a common source (e.g., colony or isolator)

Fungal culture

Wet mount

Germ-free 16S PCR
MonthlyGerm-free 16S PCR – 10 fecal pellets are pooled in a microcentrifuge tube from a common source (e.g., colony or isolator)
Surveillance plus PRIA®QuarterlySurveillance plus PRIA® – Up to 10 fecal pellets per microcentrifuge tube

Oral/body swab (one swab per cage for up to five animals or swab each animal with individual swabs. Pool the swab tips up to 10:1). EAD™ sample appropriate for housing type*.

*Follow the sampling protocol based on your institution's caging method, ensuring that all required sample types are submitted. Contact us with any questions.


Frequently Asked Questions About Health Monitoring for Germ-Free Animals 

  • What contaminants should germ-free colonies be tested for?

    Germ-free colonies should be monitored for extraneous bacteria and fungi as well as pathogens.

  • How do I send in my samples to be tested?

    Complimentary sample shipping supplies (e.g., swabs, vials) may be requested via Laboratory Testing Management® (LTM™), our online order management platform. LTM™ may also be used to generate submission forms and schedule samples for submission. 

    By creating orders via LTM™, you can track your samples online throughout the entire process, and our laboratory will know when to expect your samples for testing.

  • What should I do if I get a positive result?

    We offer guidance on confirmation testing and next steps when infectious agents are detected in germ-free mice or isolators.

  • How often should extraneous microbes be tested?

    Testing for extraneous microbes should be conducted frequently.  We recommend that germ-free culture should be conducted weekly, germ-free 16S PCR monthly, and surveillance plus PRIA® quarterly.