This document describes the production method of germ free lab animals. It discusses the definition of germ free animals and the equipment needed to produce them, such as isolators and transfer chambers. The key steps of the method are ensuring availability of germ free surrogate mothers, transferring pregnant donor mice to the isolator on day 17-20, performing caesarean sections in the transfer chamber to remove the uterine packages, and mixing the donor pups with the surrogate mother's litter in the isolator. Producing germ free animals allows their use to study reactions to diets, infectious disease pathogenesis, and other research.

1. PRODUCTION METHOD OF
GERM FREE LAB ANIMALS
Gaonkar Pankaj Prakash.
MVSc II year
pankajgaonkar12993@gmail.com
Nagpur Veterinary College,
Maharashtra Animal & Fishery Sciences University, Nagpur, India

2. DEFINITION
Germ Free animal is one which is isolated from
all demonstrable living organism like
bacteria,virus,fungus,parasite,algae,yeast.
The Aim of GF work is ISOLATION (prevent contamination from
air,water,food & environment.
Synonym- Axenic animal , Gnotobiotic.

3. Terminology
 Gnotobiotic animal:- Animal derived from aseptic caesarean or
sterile hatching of eggs i.e. restored and continuously maintained
with germ free technique under isolator condition in which flora
and fauna is clearly defined.(microbial status is known)
 Conventional animal:- Animal with uncontrolled flora, reared
under open animal room condition is association with other
animals of the same type.
 Axenic Animal :- Free from Strange or raised under sterile
condition or germ-free

4. HISTORY
 The concept of a germ-free animal was recognized more than a
century ago by Louis Pasteur (1885), although he concluded
that bacteria-free existence is impossible.
 Ten years later in 1895, Nuttle and Thierfelder at Berlin
University produced the first GF animals (guinea pigs), which
survived for as long as 13 days.
 However, owing to the lack of knowledge concerning nutrition,
it took 50 more years until the first GF rat colonies were
established in the late 1940s

5. Equipment
 Sterile isolator and set up for rearing germ free mice
 Transfer chamber compatible with the isolator
 Autoclave with validated programs for the sterilizatiion of
supplies and drinking water inside the transfer chamber
 Surgical equipment
 Luteolil Vet, 50 mg/ml, 0.1 ml, room temperature, CeVa, Holland
 VirkonS, 1% solution, room temperature, Antec Int. Ltd.

6. Isolator Technology
 Isolators provide physical barriers that allow creation of a sterile environment. These devices
have an air supply, air inlet and outlet, transfer port, and arm-length gloves, as
well as a special tank filled with disinfectant and used for the transfer of mice in and out
(Figs. 1 and 2).
 Bedding, food, water, and equipment, including cages, must first be sterilized (autoclaved) and
are then put into the isolator through the so-called sterile lock.
 Air is sterilized upon entry and exhaust by mechanical filtration under positive pressure.
 The transfer of animals in and out of the isolator is usually carried out via autoclave jars (Fig.3).
 The common practice is to separate multiple mouse strains and multiple inoculation experimental
groups in separate isolators [3], altogether increasing the cost and space for such experimentation.

8. Fig. 2. Composition of the plastic isolator

9. Fig. 2. Composition of the plastic isolator

11. Positive Pig tub Isolator

12. METHOD
Ensure availability of isolator reared, germ-free surrogate mother with newborn pups (<5 days old) at day 19 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 female(s) for the experiment.
 Day 17
Give pregnant donor female(s) a subcutaneous injection of Promon (5 mg/0.1 ml).
 Day 18
Carefully following the protocol for isolator entry procedures, transfer the sterile instruments and
supplies required for surgery into the isolator in which the surrogate female(s) are housed.
Prepare the hysterectomy suite/surgical transfer chamber: fill up the reservoir with 1%
VirkonS, sterilize the surgical compartment and ventilate it overnight.
 Day 20
Transfer water, paper towels and surgical instruments from the isolator to the sterilized compartment
of the transfer chamber.

13. Method….continued…
Working in the Non-sterile compartment of the surgical transfer
chamber;
 sacrifice the donor female by cervical dislocation and submerge the
whole animal in the 1% VirkonS solution for 1 minute.
 Use sterile scissors to open the abdomen, wear sterile gloves.
 Clamp the top of each uterine horn and the base of the uterus close
to the cervix.
 Cut out the ‘uterine package’ and place it in the transfer chamber
reservoir filled with 1% VirkonS for 1 min.

14. Fig. 4. Establishment of GF mice
by Caesarean section.
(A) The uterine sac is removed
and clamped together at the
top of each horn and at the
base close to the cervix.
(B) (B) The uterine sac is placed in
a glass jar containing
disinfectant.
(C) The uterine sac is transferred
to the isolator, where it is
opened, and the pups are
removed, cleaned, and
stimulated to breath.
(D) The pups are introduced to
the GF foster mother.

15. Method….continued…
Inside the sterile compartment of the transfer chamber;
 rinse the ‘uterine package’ with sterile water to remove the VirkonS.
 Open the ‘uterine package’ with scissors and take out the pups.
 Stimulate breathing of the pups while cleaning them with dry paper towel.
 When pups are breathing normally and have gained a ‘healthy’ skin color transfer them to the
isolator housing the surrogate mother.
 Take out her own litter and mix pups from both litters outside the cage.
 Remove some of the original pups so that the surrogate mother has the same number of pups to
feed. Gently rub the pups with bedding material from the surrogate mother’s cage soaked in water.
Put the mixed litter together with the foster mother.
 Check for adoption not earlier than 24 hours after transfer.
 Monitor a microbiological status of the isolator and the animals it houses 3 weeks after transfer.

16. USE of GERM FREE Animals
 To study reactions on the diet and its development
on the diet.
 ACTS AS SOURCE OF STERILE ORGAN,TISSUE.
 Study defence mechanism
 Study aetiology of infectious diseases
 Study process of physiological ageing.
 Study wound healing process

17. REFERENCES
 EMMA protocol - production of germ-free mice
Protocol for generating germ-free (axenic) mice using caesarean
section rederivation.
 Use of Germ-Free Animal Models in Microbiota-Related Research
Maha Al-Asmakh1,2* and Fahad Zadjali3
 Book:- Care & Management of Laboratory & Pet Animals –
Y.B.Rajeshwari