Protocols for Colonizing Axenic Rodents with the Altered Schaedler Flora (ASF) Using an Associated Animal Donor

Taconic utilizes the method below for association of germ-free mice with ASF.

Introduction:

This procedure assumes that one already maintains animals under gnotobiotic conditions that were previously inoculated with the ASF. This technique simply transfers the flora from one animal to another. Prior to the transfer, examinations should be performed to confirm that the donor mice harbor the ASF bacteria and that no contaminants are present. Please note that ASF360 is present in ASF-associated animals in extremely low levels and may not be easily detectable.

Day 1:

What to do: Withdraw water overnight.

How to do it: Merely remove all of the water bottles at the end of the day from the cages housing the animals that are to be associated.

Why do it: To guarantee that the following morning the animals will drink the associating "cocktail" immediately after it is presented to them.

Day 2:

What to do: Present the water-fasted axenic animals (recipients) with an aqueous suspension of feces from the donor animal.

How to do it: Early on the morning of Day 2, obtain as many freshly passed fecal pellets as possible from the donor animal by gently rubbing its abdomen with your finger. Collect the stools in an appropriately sized culture tube containing ~1 ml of sterile, untreated (i.e. no chlorine, no acid) water and securely close the tube. Immediately transfer the tube to the recipient isolator/area. Upon entering the culture tube to the recipient isolator, remove any residual sterilant, with particular attention to the culture tube cap so that no sterilant enters the inside of the tube. This may be accomplished by rinsing with water from a drinking bottle. Place the collected fecal pellets into a water drinking bottle that is ~1/4 full of autoclaved, untreated water. Place the stopper on the water bottle and shake it vigorously for one minute. This procedure, up to this point in time, should not take more than five to six minutes. Place this water bottle onto the first cage of animals to be associated and allow them to drink from it for five minutes. Then move it to the second cage for five minutes, etc., until it has been presented to the animals in every cage. Return water drinking bottles containing fresh water to all cages. This process must be completed within 1 hour after donor fecal pellet collection.

Why do it: To get the obligately anaerobic bacteria that comprise the microflora into the gastrointestinal tract of the recipient animals as quickly as possible. The majority of these bacteria will start to die as soon as the feces are passed out to the donor mouse due to oxygen toxicity.

Day 4:

What to do: The same protocol as Day 2.

How to do it: The same protocol as Day 2.

Why do it: To allow the more oxygen tolerant members of the microflora to colonize the recipient animals first. Between Day 2 and Day 4, the more oxygen-tolerant members of the microflora will colonize the gastrointestinal tract and lower the O/R potential enough so that between Day 3 and Day 4, the more oxygen-intolerant members of the microflora will be able to colonize also.