Oral Treponeme Enrichment Broth (OTEB) is an enriched medium for the isolation, cultivation and maintenance of Treponemes found as part of the oral flora. OTEB is a complex peptone-yeast extract medium containing volatile fatty acids and serum for a source of long chain fatty acids. Volatile fatty acids and serum are necessary nutrients for recovery of Treponemes from the oral flora.  Inoculation of the medium can be done using a gassing cannula or inside an anaerobic chamber. This medium is prepared, stored and dispensed under oxygen-free conditions to prevent the formation of oxidized products prior to use.

Formula

Proteose Peptone #2, 5.0 g

Heart Infusion Broth, 5.0 g

Yeast Extract, 5.0 g

Potassium Phosphate, dibasic, 2.0 g

Sodium Chloride, 5.0 g

Magnesium Sulfate, 0.1 g

Acetic Acid, 1.6 ml

Propionic Acid, 0.6 ml

N-Butyric Acid, 0.4 ml

N-Valeric Acid, 0.1 ml

Isovaleric Acid, 0.1 ml

Isobutyric Acid, 0.1 ml

2-Methylbutyric Acid, 0.1 ml

Vitamin K₁ (1% soln), 0.1 ml

L-Cysteine, 0.5 g

Calf Serum, inactivated, 100.0 ml

Sugar Stock Soln w/ TPP, 100.0 ml

Distilled Water, 800.0 ml

                                                                                         

Sugar Stock Solution

Glucose, 8.0 g

Fructose, 8.0 g

Sucrose, 8.0 g

Maltose, 8.0 g

Maltose, 8.0 g

Ribose, 8.0 g

Xylose, 8.0 g

Pectin, 8.0 g

Soluble Starch, 8.0 g

Sodium Pyruvate, 8.0 g

Thiamine Pyrophosphate, 750.0 mg

Distilled Water, 1000.0 ml

 

Final pH 7.0 +/- 0.2 at 25 degrees C.

Final volume 7.0 ml +/- 0.7.

Precautions

For IN VITRO DIAGNOSTIC USE only.  Approved biohazard precautions and aseptic techniques should be observed when using this product.  This product is for use only by properly-trained and qualified personnel.  Sterilize all biohazard waste prior to disposal.

 

Storage and Shelf Life

Storage:  Upon receipt, store at room temperature (13°C - 27°C) in original container until use.  Avoid overheating or freezing.  Do not use medium if there are signs of deterioration (shrinking, cracking or discoloration due to oxidation of media) or contamination.  The expiration date applies to the product in its original packaging and stored as directed.  Do not use product past the expiration date shown on the container.

 Shelf Life:  1 year from date of manufacture.

 

Procedure

Specimen Collection:  Specimens for anaerobic culture should be protected from air (oxygen) during collection, transport and processing.  Consult appropriate references for detailed instructions concerning collection and transport of anaerobes.

Methods for Use:  OTEB should be inoculated directly with clinical material.  Inoculated tubes should be immediately placed in an anaerobic atmosphere and incubated at 35-37^oC for 48 – 96 hours.  Some oral treponeme strains may require 1 – 2 weeks to achieve sufficient growth.  Detailed instructions for processing anaerobic cultures can be found in the appropriate references.

 

Materials Required But Not Provided

Standard microbiological supplies and equipment such as loops, saline blanks, slides, staining supplies, microscope, incinerator / autoclave, incubators, anaerobic chamber or anaerobic jar, disinfectant, other culture media and serological and biochemical reagents.

 

Limitations

OTEB will not provide complete information for identification of bacterial isolates.  Additional test procedures and media are required for complete identification.  Consult reference materials for additional information.

 

Quality Control

OTEB should support good growth of most Treponemes found in oral specimens.

The following organisms are routinely used for quality assurance performance testing at Anaerobe Systems. 

Organism Tested	ATCC #	Results	Time (Hours)
Treponema denticola	33521	Growth	48 – 72 h
Treponema phagedenis	27087	Growth	72 – 96 h
Bacteroides vulgatus	8482	Growth	24 h
Bacteroides fragilis	25285	Growth	24 h
Prevotella melaninogenica	25845	Growth	24 – 48 h
Fusobacterium necrophorum	25286	Growth	24 h
Fusobacterium nucleatum	25586	Growth	24 h
Clostridium perfringens	13124	Growth	24 h
Peptostreptococcus anaerobius	27337	Growth	24 h
Staphylococcus aureus	25923	Growth	24 h

 

User Quality Control: The final determination to the extent and quantity of user laboratory quality control must be determined by the end user.

If sterility testing is to be performed on this product, the appropriate percentage of the original shipment amount should be incubated anaerobically and aerobically for 48 – 96 hours.

If the nutritive capacity of this medium is to be tested for performance, it is recommended that the following ATCC organisms be evaluated for growth.

Organism	Expected Growth
T. denticola	48-72 hrs
T. phagedenis	72-96 hrs
B. fragilis	24 hrs
S. aureus	24 hrs

 

Physical Appearance:  OTEB should appear clear golden yellow in color with a slight precipitate due to the high concentration of serum in a 16mm x 100mm glass tube.

 

References

1.        Dowell, V. R., Jr. and T. M. Hawkins.  1974.  Laboratory Methods in Anaerobic Bacteriology.  CDC Laboratory Manual.  USDHEW C. D. C. Atlanta, GA 30333.

 

2.        Holdeman, L. V., F. P. Cato and W. E. C. Moore.  1977.  Anaerobe Laboratory Manual.  Virginia Polytechnic Institute and State University.  Blacksburg, VA 24061.

 

3.        Jousimies-Somer, H. R., Summanen, P., Citron, D. M., Baron, E. J., Wexler, H. M. and S. M. Finegold.  2002.  Wadsworth – KTL Anaerobic Bacteriology Manual.  Star Publishing Co., Belmont, CA 94002

 

4.        Engelkirk, P.G., Duben-Engelkirk, J., Dowell, V.R.  1992.  Principles of Practices of Clinical Anaerobic Bacteriology.  Star Publishing Co., Belmont, CA 94002.

 

5.        Isenberg, H. D.  1992.  Clinical Microbiology Procedures Handbook.  American Society for Microbiology Publishing, Washington, D.C. 20005.

 

6.        Ballows, A., et al.  1991.  Manual of Clinical Mircobiolgy.  American Society for Microbiology Publishing, Washington, D.C. 20005.

 

7.        NCCLS.  Quality Control for Commercially Prepared Microbiological Culture Media; Approved Standard- Third Edition.  (2004).  NCCLS document M22-A3.  NCCLS, 940 West Valley Road, Suite 1400, Wayne, PA 19087-1898.