Standard Operating Procedures

SPF Mouse Serology

Last Updated: January 5, 2012

Purpose

Specific pathogen-free (SPF) mouse sentinel serology is done as part of a comprehensive preventive health care and disease surveillance program.

Responsibility

It is the responsibility of the AV to provide oversight and for the LAR staff to understand, to be proficient in, and to participate in the serology testing program.

Procedure

Examination and Sample Collection

• On a quarterly basis, collect SPF sentinel mice from each mouse room into clean shoebox cages.
  • All mouse rooms in Skaggs Building (SB), SB 066, 061B, and HSB004 mouse quarantine.
  • Take one sentinel mouse from every rack
  • Label each box with a new cage card that includes room number, rack number, and investigator.
• Move mice on a clean cart to HSB016-LAR Clinical Lab
• Fill out University of Missouri Research Animal Diagnostic Laboratory (MU RADIL) serology paperwork (print from computer or get hard copy from file cabinet).
  • The order in which the mice are listed on the submission form will be the order in which animals are necropsied. List smallest room number first and proceed to larger room numbers.
• Pre-label all eppendorf tubes that will hold samples. 
• Items needed
  • scissors                     o  cellophane tape                        o  25 gauge needles   
  • forceps                       o  1.5-mL eppendorf tubes          o  1-mL syringes
  • MB-10                         o  fecal flotation tubes                   o  sharps container
  • slides                         o  sterile scalpel blades               o  paper towels
  • sharpie® markers
• Place mouse (proper order of serology paperwork) in CO2 chamber and euthanize using 20% volume displacement per minute inside the chamber.
• Retrieve blood via cardiac puncture.
  • Use a 1 cc syringe with 25 ga needle.
  • Place blood in pre-labeled eppendorf tube.
• Label tail with the number from serology paperwork using a Sharpie® marker.
• Use dissecting microscope to check body for ectoparasites and abnormalities.
• Collect a fur tape sample and a perianal sample using cellophane tape. Press the tape firmly against the skin. For perianal samples, place tape over anus and draw a large circle around the sample area using a Sharpie® marker.
  • Place fur and perianal tapes on separate slides and label.
  • 3 samples of each specific type can fit on a slide.
  • Do not mix fur tapes and perianal tapes on the same slide.
• Incise the mouse along the ventral midline of the body.
• Check for abnormalities of internal organs (location, size, texture, color).
• Collect one fecal pellet from rectum.
  • Place in pre-labeled eppendorf tube.
  • 3 samples from different mice may go into one tube, in the order written on the submission form.
• Push contents of cecum onto a slide.
  • Label slide with mouse number.
  • Use dissecting microscope to check the sample for the presence of adult pinworms
• Double check that all procedures were finished and all necessary samples were collected before discarding each carcass.

Fecal Flotation

• Once all necropsies are completed, prepare fecal floats for parasite eggs.
• There should be a maximum of three fecal samples in one tube.
• Use a transfer pipet to add a small amount of "Vetus Feca Test  solution (a 1:18 dilution of Vetus Feca Test Dry Concentrate" diluted with water) to tube and mix well with feces.  (This 1:18 solution and concentrate are located in HSB016 metal cabinet).
• Use a transfer pipet to fill the tube to top with Vetus Feca solution, creating a meniscus.
• Place square coverslip on top of tube.
• Let stand for 10 minutes.
• Place the cover slip on a clean slide; create minimum air bubbles.
• Use compound microscope to examine the samples for eggs of internal parasites.

Separate Serum from Whole Blood

• Allow collected blood to set for at least 15 minutes in refrigerator.
• Spin blood in mini centrifuge
  • Make sure centrifuge tube holders are balanced
  • Spin 5 minutes at 10 x 1000 RPM
• Remove all serum from tube using a 200 µL Pipetman. 
  • Put serum into a clean, labeled eppidorf tube.
• Dilute serum 1:5. 
  • Take 12 µL of serum, using the 20 µL Pipetman, and place serum into a MU RADIL serology tube
  • Add 48 µL of diluant*, using the 200 µL Pipetman, to the serum in the serology tube. 
• Keep remaining undiluted serum in HS016 refrigerator until confirmation from IDEXX RADIL that samples were adequate for analysis.  

Packaging Samples for IDEXX RADIL

• Place diluted serum samples into a ziplock bag and place into a Styrofoam box
  • Add an ice pack
  • Add any necessary packing material.
  • Place the IDEXX RADIL paperwork into a ziplock bag and put inside the Styrofoam box.
  • Place lid on box and secure with tape.
• Place Styrofoam box inside a cardboard box and secure with tape.
• Mail from campus post office 
  • 2-day service (either UPS or USPS, which ever is cheaper).
  • Use IDXX RADIL address listed on paperwork.
  • Charge to UM credit card.