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 all eight of the ASF anaerobes and that no contaminants are present.
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.
What to do: Present the water-fasted axenic animals (recipients) with an aqueous suspension of feces from the donor animal.
How to do it: Soon after arriving at work on the morning of Day 2, obtain as many freshly passed feces as possible from the donor animal by gently rubbing its abdomen with your finger. Place the stools in an empty water bottle and fill it half full with water, making sure that no sterilant (e.g. peracetic acid) is allowed to get into or near this water bottle. 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 remove it to the second cage for five minutes, etc., until it has been presented to the animals in every cage. Finally, after the last cage has had the water bottle for five minutes, dispense the remaining contents equally into the other water bottles in the isolator, fill them with water and then supply each cage with one such water bottle. At the end of the day, just before leaving work, withdraw all the water bottles from the recipient animals.
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.
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 3, 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.
What to do: The same protocol as Day 2 and Day 3 with one exception: use the entire contents of the caecum and the large intestine of the donor animal as the inoculum instead of just feces alone.
How to do it: Euthanize the donor animal(s) by cerebral-spinal dislocation. Excise the caecum and large intestine. Cut open the entire length of these organs and scrape out the luminal contents into the water bottle. Proceed as on Day 2 and Day 3, only do not remove the water bottles from the cages at the end of the day. Rather, continue to refill the water bottles as usual ad infinitum.
Why do it: To increase the final inoculum as much as possible.
Method II: Utilizing pure cultures of each member of the "ASF" microflora.
Before beginning this procedure, be sure to have pure cultures of the eight bacteria growing on agar plates and familiarize yourself with the cellular and colonial morphologies of each. The goal is to simply colonize most of the aerotolerant members of the ASF first (two lacotobacilli and the bacteroides), so that they will lower the oxidation/reduction potential of the gut so that the more fastidious anaerobes can then colonize. Note that all culturing must be done with pre-reduced media under strict anaerobiosis within an anaerobic glove box at all times for six of the eight members of the ASF (the lactobacilli are aerotolerant), so do it for all eight to avoid any errors.
a) At about 4 P.M or 5 P.M. inoculate each of the lactobacilli (360 and 361) and the Bacteroides (519) into separate tubes containing 8-10 ml of original formula Schaedler broth or commercially available Brucella broth. Also, heavily inoculate the spirochete-shaped anaerobe (457) into a tube of original Schaedler broth plus 2% heat-inactivated serum (either sheep or fetal calf). Brucella broth plus serum will also suffice.
a) Between 8 A.M. and 9 A.M., make wet mounts of the cultures of lactobacilli and Bacteroides to check for purity and subculture each one as a second check for purity. If the wet mounts look pure, pool the three cultures and replace the screw cap with a tight fitting sterile rubber serum vial cap that has been sterilized by steam in a glass container (e.g., petri dish) which has been pre-reduced for 2 days. The culture of the spirochete-shaped anaerobe will not be used until Day 4.
b) Spray the pooled culture test tube into the axenic isolator containing the recipient animals to be colonized, along with a glass tuberculin syringe and steel needles as follows. Autoclave (steam) the syringe plunger and barrel separately along with two sharp steel 20 gauge needles and one steel rounded, blunt "bulb-ended" 20 gauge intubating needle (do NOT rely on commercial plastic syringes and needles for sterility). The syringe plunger, barrel and needles can be autoclaved in a large screw cap bottle test tube, French Square, or similar vessel.
c) Make each recipient animal defecate as many stools as possible by gently stroking its abdomen with your finger or a forceps. One animal from each cage in the isolator up to 5 cages is plenty. Thereafter, feces from these recipient animals can be used to colonize the remaining mice or rats in the isolator.
d) Associate the syringe making sure that no sterilant has come into contact with any of its parts or the needles. If sterilant does touch any of these components, flush them liberally with water to remove the sterilant. Fit the syringe with a sharp needle. Wash any sterilant off of the rubber stopper of the pooled culture test tube, insert the sharp needle and draw up 0.5 ml of bacterial suspension. Withdraw from the tube and switch the sharp-ended needle with a blunt-ended needle.
e) Gavage the recipient animal with the 0.5 ml of bacterial suspension. If you are apprehensive or inexperienced with gavaging, just withdraw water from the animals the night before and they will drink the suspension off the end of the blunt-ended needle as you slowly release it from the syringe. Refill the syringe with another 0.5 ml of the suspension as done previously and deliver it directly into the large intestine by inserting the blunt-ended needle into the large intestine through the anus, and depressing the plunger rather rapidly (within 2 seconds) so as to deliver the suspension as far up into the large bowel as possible. Dissemble the syringe and store the barrel and plunger separately, otherwise they will stick together after drying.
a) Between 8 A.M. and 9 A.M., heavily inoculate each of the four extremely oxygen sensitive (EOS) fusiform-shaped anaerobes (356, 492, 500, 502) into separate tubes of original formula Schaedler broth plus 2% heat-inactivated serum (either sheep or fetal calf). Brucella broth plus serum will also suffice.
a) At 4 P.M., make wet mounts of all the remaining five cultures (457 from Day 1 and the four fusiform-shaped anaerobes from Day 3) and subculture them as a second purity check. Although the results of these subcultures will only be available after the fact, they will nevertheless provide documentation that the wet mounts were correct. Note that culture is far more sensitive than direct phase microscopy.
b) Aseptically deliver 1.0 ml from each of the cultures of fusiform-shaped anaerobes into the tube of the spirochete-shaped anaerobes (457) and then fit this pooled tube with a rubber serum vial cap.
c) Spray this tube of pooled anaerobes into the isolator.
d) Repeat steps outlined for Day 2, (b) through (d). Wait at least until Day 7 before monitoring for the presence of the entire flora.