EMMA SOPs - Production of Germ-Free Mice
SOP for Generating Germ Free (Axenic) Mice Using Caesarean Section Rederivation
1. Purpose
To describe the process used to generate germ free mice using caesarean section rederivation.
2. Scope
All personnel working with germ free conversion
3. Content:
Equipment
- Sterile isolator and set up for rearing germ free mice
- Autoclave; 121 degrees ºC for 20 minutes
- Container for the transfer of material in and out of the isolator
- Promon Vet; 50 mg/ml, 0,1 ml; Room temperature, Boehringer Ingelheim Denmark A/S
- Virkon; 1% solution, 10 grams to 1 liter of water; Room temperature, Antec Int. Ltd
- Hyamin 1622; 1 kg in 3 liters of alcohol; Room temperature, VWR International
- Surgical equipment
Method
Ensure availability of isolator reared, germ free recipient surrogate mother with newborn pups (<1 week old) at day 20 of procedure (see below)
Day 0
Set up the relevant mating of mouse strain to be converted to germ free status.
Day 1
Check for mating plug and identify the donor females for the experiment.
Day 17
Inject Promon, 0.1 ml sc to the pregnant donor female(s).
Day 19
Following the SOP for isolator entry procedures, transfer sterile instruments and materials required for surgery. Prepare Virkon (1g/100 ml) and store a container with fresh Virkon in Hyamin solution overnight (to sterilize the outside of the container).
Day 20
Sacrifice the donor female(s) (neck dislocation) and submerge the whole animal in the 1% Virkon solution for 1 minute. Use sterile scissors to open the abdomen (wear sterile gloves) and locate the uterus. Using sterile surgical tread ligate the top of each uterine horn and the base of the uterus close to the cervix. Cut the sealed ‘uterine package’ and place it in the container of 1% Virkon prepared on day 19.
Following the SOP for isolator entry procedures, transfer the Virkon container holding the ‘uterine package’ into the isolator.
Inside the isolator
Rinse the ‘uterine package’ with sterile water to remove the Virkon. Open the sealed ‘uterine package’ using sharp scissors and take out the pups.
Stimulate breathing of the pups with gentle massage.
When pups are breathing normally and have gained a ‘healthy’ skin color, gently rub the pups with bedding material soaked in water, taken from the cage housing the recipient surrogate mother.
Add the new pups to her litter. If necessary, remove some of the existing pups so that the surrogate mother has a manageable number of pups to nurse.
Periodically monitor the microbiological status of the isolator and the animals it houses.