A Brief History of the Use of Microfloras in Gnotobiotic Rodents

When one cesarean derives a mammal into the axenic or "germ-free" state, one removes all bacteria from this animal host. The results are very dramatic. Vitamin K, which is supplied by intestinal bacteria in "normal" mice, must now be added to the diet. The cecum, which contains 100 billion bacteria/g in "normal " mice, suddenly contains no bacteria at all. The cecal swelling that results in this bacterial-free condition represents the most pronounced anomaly of the germ-free state. In a small percentage of animals, the cecum becomes so large that it twists in the peritoneal cavity and wraps the small intestine around itself, causing death referred to as "intestinal strangulation" or "cecal volvulus". Likewise, the enlarged cecum has adverse consequences to animal breeders because it competes with impregnated uterine horns for abdominal space, resulting in lower production indices.

The second most pronounced effect of the germ-free state is the increased susceptibility to infectious disease agents. The LD50 for Salmonella in "normal" mice is a million organisms whereas it is only 1-10 in germ-free mice! This was certainly no surprise since infectious disease organisms do not have to compete with any other bacteria (the so-called "normal" flora) for nutrients in germ-free animals. If a new barrier room is stocked with germ-free animals, death can occur even due to just "opportunistic" pathogens (bacteria which do not cause any disease in non-stressed "normal" mice due to the presence of the "normal" microflora).

It is therefore desirable to colonize germ-free mice with a bacterial microflora before removing them from their isolators and introducing them into a barrier room. In the mid-1960s, Dr. Russell W. Schaedler of the Rockefeller University was the first to colonize germ-free mice with selected bacteria isolated from "normal" mice1,2. He then started supplying animal breeders with a series of floras, the first of which was published by Dennis Baker of Carworth Farms in 19663. Note, however, that these early microfloras consisted of the easier-to-grow aerobic members of the microflora along with some of the less oxygen sensitive anaerobes. Anaerobic glove box technology was not available at that time to allow for the isolation and cultivation of the so-called EOS (extremely oxygen sensitive) "fusiform-shaped" bacteria which make up the vast majority of the "normal flora" of rodents.

Prior to leaving The Rockefeller University to chair the Department of Microbiology at Thomas Jefferson University, Dr. Schaedler provided additional bacteria to breeders including one of the EOS fusiform-shaped bacteria. At one major animal supplier, this flora had been finalized to contain the following eight bacteria out of a possible 100+ species:

  1. E. coli var. mutabilis (grows aerobically)
  2. Streptococcus fecalis (grows aerobically)
  3. Lactobacillus acidophilus (grows anaerobically, but some "breakthrough" is noted on aerobic plates)
  4. Lactobacillus salivarius (grows anaerobically, but some "breakthrough" is noted on aerobic plates)
  5. Group N Streptococcus (grows anaerobically only)
  6. Bacteroides distasonis (grows anaerobically only)
  7. An unidentified Clostridium species (grows anaerobically only)
  8. An unidentified Fusiform-shaped bacterium, #356 (grows anaerobically only and is EOS)
Although this flora did not totally "normalize" the cecum, it did provide protection from opportunistic bacteria when these gnotobiotes were used to stock barrier rooms (to date, the "cocktail" containing the lowest number of bacteria that has been able to completely reduce the cecal size to normal, has consisted of 59 strains).

In 1978, the National Cancer Institute sought to revise this "Schaedler Flora" or "cocktail" of eight bacteria in order to standardize the microflora to be used in colonizing axenic (germ-free) rodents at all of the NCI contract and in-house facilities. The contractors argued that aerobic contaminants (e.g., the common staphylococci and streptococci) were difficult to monitor, because the aerobic bacteria in the flora overgrew on the aerobic plates. Researchers at NIH argued that the presence of any coliform was undesirable and that the so-called Schaedler coli (E. coli var. mutabilis) was especially troublesome because it was a very slow lactose fermenter. It appeared as a non-lactose fermenter on primary isolation, making it virtually indistinguishable from a Salmonella, etc., thereby making the monitoring for these types of contaminants even more difficult.

The new microflora was devised by not only eliminating all aerobic bacteria (although a small percentage of the anaerobic lactobacilli are microaerophilic and gradually "break through" on aerobic plates), but also by excluding all cocci and blunt-ended spore forming rods which represent the vast majority of contaminants in gnotobiotic isolators. Since it was desirable to keep the total number of bacteria in the cocktail to a maximum of eight, four bacteria from the original flora (E. coli, S. fecalis, anaerobic group N Streptococcus and an unidentified Clostridium) were replaced with three species of fusiform-shaped anaerobes and one anaerobic spirochete, all of which were isolated from mice4, as follows:

Altered Schaedler Flora Composition

ASF #Updated taxonomy4-5,14-15Previously identified asSensitivity to oxygen
ASF356Clostridium sp.Fusiform #356EOS
ASF360Lactobacillus intestinalisLactobacillus sp., Lactobacillus acidophilusnone
ASF361Ligilactobacillus (Lactobacillus) murinusLactobacillus salivariusnone
ASF457Mucispirillum schaedleriSpirochete #457none
ASF492Eubacterium plexicaudatumFusiform #492EOS
ASF500Pseudoflavonifractor sp.Firmicutes bacterium, Fusiform #500EOS
ASF502Schaedlerella arabinosiphilaClostridium sp., Fusiform #502EOS
ASF519Parabacteroides goldsteiniiParabacteroides sp., Bacteroides distasonisnone
EOS = extremely oxygen sensitive
Historically all commercial suppliers have used a version of ASF to associate rederived mice and rats. By 1980, all NCI suppliers, A.R.S. Sprague Dawley, Charles River, Harlan (now Envigo), Leo Goodwin and Simonsen had adopted this microflora for all of their isolator animals (not just their NCI rodents). Consequently, gnotobiotes colonized with this flora have been used to stock barrier rooms at all major suppliers for over the past ten years, making it the international microflora for rodents. However, one should recognize the fact that following removal from their isolators and introduction into barrier rooms, these animals soon become colonized with numerous other bacteria, some undoubtedly from caretakers and others undoubtedly from the ingredients in the pasteurized animal feed which survive autoclaving. There are several review articles on the normal flora of rodents6-12. The draft sequences of the ASF organisms were published in 201413.

At Taconic Biosciences, newly derived animals are associated with ASF in a gnotobiotic setting. The Foundation Colonies for many popular lines are maintained in our gnotobiotic center and provide breeder stock to the various sites at which each line is produced worldwide. Taconic can provide limited quantities of ASF donor animals to investigators who want to associate animals with this flora.
1. Schaedler, Russell W. et al. 1965. The Development of the Bacterial Flora in the Gastrointestinal Tract of Mice. J. Exp. Med., 122(1):59-66.
2. Schaedler, Russell W. et al., 1965. Association of Germ-free Mice with Bacteria Isolated from Normal Mice. J. Exp. Med., 122(1):77-82.
3. Baker, Dennis E.J. 1966. The Commercial Production of Mice with a Specified Flora. In "Viruses of Laboratory Rodents", Robert Holdenried (ed.), U.S. Government Printing Office, Cancer Monograph 20:161-166.
4. Dewhirst, Floyd E., et al. 1999. Phylogeny of the Defined Murine Microbiota: Altered Schaedler Flora. Appl. Environ. Micro., 65(8):3287-3292.
5. Wymore, Brand M, et al. 2015. The Altered Schaedler Flora: Continued Applications of a Defined Murine Microbial Community. ILAR J., 56(2):169-78.
6. Savage, Dwayne C. 1977. Microbial Ecology of the Gastrointestinal Tract. Ann. Rev. Micro., 31:107-133.
7. Schaedler, Russell W. and Orcutt, Roger P. 1983. Gastrointestinal Microflora. In "The Mouse in Biomedical Research", H.L. Foster et al (ed), Academic Press, N.Y., N.Y., pp 327-345.
8. Lee, Adrian 1985. Neglected Niches: The Microbial Ecology of the Gastrointestinal Tract. In "Advances in Microbial Ecology", Vol. 8, K.C. Marshall (ed.), Plenum Press, N.Y., N.Y., pp. 115.
9. Savage, Dwayne C. 1986. Gastrointestinal Microflora of Rodents. In "Laboratory Animals: Laboratory animal models for domestic animal production", World Animal Science, C2, Chapter 3, E.J. Ruitenburg and P.W.J Peters (editors), Elsevier, N.Y., N.Y., pp: 85-117.
10. Wilson, K. 1995. The Gastrointestinal Microflora. In, T. Yamada (ed.), "Textbook of Gastroenterology", J.P. Lippincott, Philadelphia, Pa., pp. 607-615.
11. Falk, P. et al. 1998. Creating and maintaining the gastrointestinal ecosystem: What we know and need to know from gnotobiology. Microbiol. Mol. Biol. Rev. 62:1157-1170.
12. Neish, Andrew S. 2002. The gut microflora and intestinal epithelial cells: a continuing dialogue. Microbes and Infection, 4:309-317.
13. Wannemuehler, M. et al. 2014. Draft Genome Sequences of the Altered Schaedler Flora, a Defined Bacterial Community from Gnotobiotic Mice. Genome Announc. 10;2(2).
14. Zheng, Jinshui, et al. 2020. A taxonomic note on the genus Lactobacillus: Description of 23 novel genera, emended description of the genus Lactobacillus Beijerinck 1901, and union of Lactobacillaceae and Leuconostocaceae. Int J Syst Evol Microbiol, 70(4):2782-2858.
15. Soh, Melissa, et al. 2019. Schaedlerella arabinosiphila gen. nov., sp. nov., a D-arabinose-utilizing bacterium isolated from faeces of C57BL/6J mice that is a close relative of Clostridium species ASF 502. Int J Syst Evol Microbiol, 69(11):3616-3622.

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