Culturing Members of the Altered Schaedler Flora from Stock Vials

Taconic Biosciences does not sell vials or bacterial stocks of ASF. This protocol is provided as information only. We can provide fecal pellets from mice harboring only ASF organisms.

Introduction

The Altered Schaedler Flora (ASF) consists of eight bacteria, four of which are so-called Extremely Oxygen Sensitive (EOS). These four "fusiform-shaped" anaerobes, namely TAC:ASF numbers 356, 492 (Eubacterium plexicaudatum), 500 and 502 require strict anaerobiosis and therefore one must have an anaerobic glove box for their cultivation 1, 2. L. acidophilus (TAC:ASF 360), L. murinus (TAC:ASF 361), the Bacteroides distasonis (TAC:ASF 519) and recently named Mucispirillum schaedleri (TAC:ASF 457), the spiral-shaped anaerobe, are aerotolerant. Additional taxonomic information is available:
Dewhirst, F.E. et al. 1999. "Phylogeny of the Defined Murine Microbiota: Altered Schaedler Flora", Appl. Environ. Microb. 65(8):3287-3292
Robertson, B.R. et al. 2005. "Mucispirillum schaedleri gen. nov., sp. nov., a spiral-shaped bacterium colonizing the mucus layer of the gastrointestinal tract of laboratory rodents", Int J Syst Evol Microbiol 55:1199-1204

Culturing

Stock Cultures of the ASF are provided as frozen cell suspensions in cryopreservation ampules, which are maintained at -75 to -85°C, and are then shipped on dry ice.
It is imperative that ampules be thawed quickly in warm (~37°C) water before they are cultured. In the case of the four EOS anaerobes, it is also imperative that all media be prereduced for at least two days in an anaerobic glove box, before attempting to culture these four fastidious members of the ASF. It is also advisable to prereduce media for the Bacteroides (TAC:ASF 519) and the spiral-shaped anaerobe Mucispirillum schaedleri (TAC:ASF 457) if immediate growth is desired. Otherwise they will not start to grow until the media become reduced. Only the two lactobacilli (TAC:ASF 360 and TAC:ASF 361) will begin to start growing immediately on non-prereduced media.
After quick thawing an ampule, aseptically use a tuberculin syringe or equivalent (pre-reduced for at least a day in the case of the EOS anaerobes) to draw up 0.3 ml of the cell suspension and deliver 2-3 drops in the first 2 quadrants of a plate of Schaedler agar plus 5% sheep blood or 2.5-5.0% sheep serum. With a loop, spread the cell suspension evenly in the first 2 quadrants of the plate, flame, and then continue to triple streak into the 3rd and 4th quadrants. Incubate at 37°C for 2-6 days depending on the species. Lactobacilli will grow rapidly whereas the other members will take 3-6 days to come up.

References

Aranki, A., Syed, S. A., Kenny, E.B. and Freter, R. (1969) Isolation of anaerobic bacteria from human gingiva and mouse cecum by means of a simplified glove box procedure. Appl. Micro., 17:568-576.
Schaedler, R. W. and Orcutt, R. P. (1983) Gastrointestinal Microflora. In The Mouse in Biomedical Research, Vol. III, Foster, H.L. et al. editors. New York: Academic Press, pp. 327-345. On pages 333-334: figure 5a is #356; figures c, d and e are #492, #502 and #500 respectively.
Note that commercial formulations of so-called "Schaedler broth and Schaedler agar" bear little resemblance to true Schaedler broth/agar. See correct formulation below.

Original Formula for Schaedler’s Brotha or Agarb

Pancreatic digest of casein (trypticase) 10.0
Polypeptone (Proteose-Peptone no. 3) 5.0
Dextrose 5.0
Yeast extract 5.0
Tris (hydroxymethyl aminomethane) 3.0
Hemin 0.01c
L-Cystine 0.4
Total (gram/liter) 28.41
a J. Exp. Med. 1965, 122 (1): 59 – 66.
b 5% blood and 15 grams of agar may be added to make Schaedler Blood Agar Plates.
c Delivered by pipetting 10 ml of a stock Hemin solution containing 0.1g Hemin, 99 ml of water and 1 ml of 5N NaOH.