Germ-free organisms are multi-cellular organisms that have no microorganisms living in or on them. Such organisms are raised using various methods to control their exposure to viral, bacterial or parasitic agents. When known microbiota are introduced to a germ-free organism, it usually is referred to as a gnotobiotic organism, however technically speaking, germ-free organisms are also gnotobiotic because the status of their microbial community is known. Due to lacking a microbiome, many germ-free organisms exhibit health deficits such as defects in the immune system and difficulties with energy acquisition. Typically germ-free organisms are used in the study of a microbiome where careful control of outside contaminants is required.
Generation and cultivation
Germ-free organisms are generated by a variety of different means, but a common practice shared by many of them is some form of sterilization step followed by seclusion from the surrounding environment to prevent contamination.
Poultry
Germ-free poultry typically undergo multiple sterilization steps while still at the egg life-stage. This can involve either washing with bleach or an antibiotic solution to surface sterilize the egg. The eggs are then transferred to a sterile incubator where they are grown until hatching. Once hatched, they are provided with sterilized water and a gamma-irradiated feed. This prevents introduction of foreign microbes into their intestinal tracts. The incubators and animals' waste products are continuously monitored for possible contamination. Typically, when being used in experiments, a known microbiome is introduced to the animals at a few days of age. Contamination is still monitored and controlled for after this point, but the presence of microbes is expected.
Mice
Mice undergo a slightly different process due to lacking an egg life-stage. To create a germ-free mouse, an embryo is created through in vitro fertilization and then transplanted into a germ-free mother. If this method is not available, a mouse can be born through cesarean birth, but this comes with a higher risk of contamination. This process uses a non-germ-free mother which is sacrificed and sterilized before the pups' birth. After the cesarean birth, the pups must then be transferred to a sterile incubator with a germ-free mother for feeding and growth. These methods are only required for the generation of a germ-free mouse line. Once a line is generated, all progeny will be germ-free unless contaminated. These progeny can then be used for experimentation. Typically for experiments, each mouse is housed separately in a sterile isolator to prevent cross-contamination between mice. The mice are provided with sterilized food and water to prevent contamination. The sterilization methods can vary between experiments due to different diets or drugs the mice are exposed to. The isolators and waste products are continuously monitored for possible contamination to ensure complete sterility. As with poultry, a known microbiome can be introduced into the animals but contamination is still monitored for.
Nematodes
Nematodes can also be grown germ-free. Germ-free offspring of the nematode C. elegans, which is used in research, can be produced by rupturing adult worms to release eggs. The standard method for this is to introduce a population of adult worms to a bleach solution. This bleach solution ruptures the adult worms, breaking them down while simultaneously releasing and surface sterilizing any eggs. The sterilized eggs are washed and transferred to a plate of agar containing food for the worms. C. elegans consumes bacteria, so before the eggs can be transferred to the plate, the food must be killed by either heat or irradiation. This method for creating germ-free nematodes has the added benefit of age-synchronizing the worms, so that they are all of similar ages as they grow. Typically the worms will need to be transferred to a new plate as they consume all the food on the current plate, with each plate having been treated with heat or radiation as well. The plates can be protected from outside contamination by covering them and isolating them from possible contamination sources.
Plants
Seeds are surface sterilized with chemicals, such as ethanol or an antibiotic solution, to produce a germ-free plant. The seeds are then grown in water or other mediums until germination. After germination, the seeds are transferred to either sterile soil or soil with a specific microbiota for use in experiments. Seeds may also be transferred directly to soil and allowed to germinate. If the plants are transferred to sterile soil, typically there are two types of growth methods. The first is where the entire plant is kept sterile and in the other, only the root system is kept sterile. The method is chosen based on the requirements for the experiment. The plants are grown in isolators which are frequently checked for contamination along with the soil that the plants grow in.
Due to lacking a healthy microbiome, many germ-free organisms exhibit major health deficits. The methods used to produce germ-free organisms can also have negative side effects on the organism. Decreased hatching rates were observed in chicken eggs incubated with mercuric chloride, while treatment with peracetic acid did not cause a significant effect on hatching rates. The chickens also exhibited defects in small intestine growth and health. Germ-free mice have been shown to have defects in their immune system and energy uptake due to lacking a healthy microbiome. There is also strong evidence for interactions between the mouse microbiome and its brain development and health. Germ-free plants exhibit severe growth defects due to lacking symbionts that provide necessary nutrients to them.
Uses
Research
Germ-free organisms frequently see use in the studies of different microbiomes. Lacking any microbiome provides insights into what a microbiome contributes to the host. This is done by comparing a "normal" host to a germ-free host. Any differences between the two and can be understood to be related to the microbiome. This type of study doesn't provide much information on what the microbiome is actually doing, nor does it provide information about specific microbes in the community. To get around this, a known microbiome can be introduced into the host to see the effects of that specific microbiome. By changing the composition of the microbiome, such as switching out a single species, species-specific effects can be found. This also allows for identification of key-stone species within the microbial community.
