Anaerobe Systems PRAS
         Indole-Nitrate Broth AS-861

Formula

Pancreatic Digest of Casein, 20.0 g

Tryptophan, 0.2 g

Disodium Phosphate, 2.0 g

Agar, 1.0 g

Hemin (0.1% soln), 5.0 ml

Vitamin K₁ (1% soln), 0.1 ml

Dextrose, 1.0 g

Potassium Nitrate, 1.0 g

Distilled Water, 1000.0 ml

 

Final pH 7.2 +/- 0.2 at 25^oC.

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 packaging until use.  Avoid overheating or freezing.  Do not use medium if there are signs of deterioration (discoloration or evaporation) 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 of specimens.  Consult appropriate references for detailed instructions concerning collection and transport of anaerobes.

Methods for Use:  INDOLE-NITRATE should be inoculated with pure cultures of the organism being tested.  Inoculated tubes should be immediately placed in an anaerobic atmosphere and incubated at 35 – 37^oC for 18 – 48 hours.  Before performing each of the following tests, divide the volume into two equal parts.  Perform each test separately, do not mix test reagents.  Additional periods of incubation may be necessary.  Detailed instructions for processing anaerobic cultures can be found in the appropriate references.

Indole Test:  Once sufficient growth is achieved, usually with in 18 – 48 hours of incubation, perform one of the following test procedures.

Ehrlich:  Add 1.0 ml of xylene to a 48 hour broth culture, shake vigorously for 20 seconds and let stand for 1 to 2 minutes.  This will allow the xylene extract to come to the top of the broth.  Next, gently add 0.5 ml of the Ehrlich reagent down the side of the tube.  A red ring at the interface between the media and reagent within 5 minutes represents a positive result.

Kovacs:  Add 5 drops of Kovacs reagent to a 48 hour broth culture.  Do not shake after the                    addition of reagent.  A red color at the surface of the medium represents a positive result.

DMACA:  Add 5 drops of p-Dimethylaminocinnamaldehyde (1% in 1N HCl) to a 48 hour broth culture.  Do not shake after the addition of reagent.  A blue color at the surface of the medium represents a positive result.  No change or a pinkish color is negative.

Nitrate Test:  Once sufficient growth is achieved, add 5 drops each of N,N-dimethyl-1-naphthylamine and sulfanilic acid directly to the broth culture.  Do not shake the tube.  A red color will develop within 1 to 2 minutes, which indicates a positive result.  The nitrate has been reduced to nitrite.  If no color has developed, this indicates a negative result.  To confirm this result, a pinch of zinc dust is added to the culture if within 5 –10 minutes a red color develops nitrate has not been reduced to nitrite.  After the addition of zinc, the culture remains clear this indicates a positive result due to nitrate has been completely reduced to nitrogen gas.

 

Materials Required But Not Provided

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

 

Limitations

INDOLE-NITRATE will not provide complete information for the identification of bacterial isolates.  Additional test procedures and media are required for complete identification.  Consult reference materials for additional information.  The indole test is not recommended for testing on organisms that are capable of reducing nitrate to nitrite.  Nitrites can interfere with the detection of indole.

 

Quality Control

If used properly, this medium should support good growth of aerobes, microaerophiles, facultative and obligate anaerobes.

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

Organism Tested	ATCC #	Results	Time (Hours)	Expected Results Indole          Nitrate
Bacteroides ureolyticus	33481	Growth	24 – 48 h		+
Bacteroides vulgatus	8482	Growth	24 – 48 h
Bacteroides fragilis	25285	Growth	24 h	-	-
Fusobacterium necrophorum	25286	Growth	24 – 48 h	+
Fusobacterium nucleatum	25586	Growth	24 – 48 h	+
Clostridium perfringens	13124	Growth	24 h
Peptostreptococcus anaerobius	27337	Growth	24 – 48 h
Propionibacterium acnes	6919	Growth	24 – 48 h
Prevotella melaninogenica	25845	Growth	48 h
Escherichia coli	25922	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/chemical reactivity capacity of this medium is to be tested for performance, it is recommended that the following ATCC organisms be evaluated for growth/inhibition.

 

Organism	Growth in	Expected Results
	Hours	Indole	Nitrate
B. ureolyticus	24-48 hrs		+
B. fragilis	24 hrs	-	-
F. necrophorum	24-48 hrs	+
F. nucleatum	24-48 hrs	+

 

Physical Appearance:  INDOLE-NITRATE should appear as a clear, yellowish liquid within the 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.        Engelkirk, P. G., Duben-Engelkirk, J. and Dowell, V. R.  1992.  Principles and Practices of Clinical Anaerobic Bacteriology.  Star Publishing Co., Belmont, CA 94002.

 

4.        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.

 

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 Microbiology.  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.