PRAS (Pre-reduced Anaerobically Sterilized) PYG-TWEEN FA/GLC is designed for determination of glucose utilization and Tween-80 stimulation by anaerobic bacteria. This biochemical determination is used to aid in the definitive identification of anaerobic bacteria (4). PYG-TWEEN FA/GLC is recommended for use in identifying gram-positive bacteria within the MIDI MIS procedures. PYG-TWEEN FA/GLC is a non-selective broth medium that was formulated by the VPI group to be used in the chromatographic analysis of cellular fatty acid within the MIDI MIS system. This type of analysis is useful in the identification of clinically significant anaerobic bacteria. A peptone yeast extract basal medium is supplemented with Tween-80 to stimulate the growth of gram-positive organisms. This media is prepared, dispensed, stored and packaged under oxygen-free conditions to prevent the formation of oxidized products prior to use.

       Anaerobe Systems PRAS Peptone Yeast
         Glucose Tween (PYG TWEEN) AS-825

Formula

Peptone, 5.0 g

Pepticase, 5.0 g

Yeast Extract, 10.0 g

L-Cysteine, 0.5 g

Hemin (0.1% Soln), 5.0 ml

Vitamin K₁ (1% Soln), 0.1 ml

Resazurin, 0.001 g

Calcium Chloride, anhydrous, 0.01 g

Magnesium Sulfate, anhydrous, 0.008 g

Potassium Phosphate, monobasic, 0.04 g

Potassium Phosphate, dibasic, 0.04 g

Sodium Chloride, 2.0 g

Sodium Bicarbonate, 0.4 g

Glucose, 10.0 g

Tween-80 (1:10 solution), 2.5 ml

Distilled Water, 1000.0 ml

 

Final pH 7.2 +/- 0.3 at 25 degrees C.

Final volume 10.0 ml +/- 1.0.

 

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), oxygen exposure, 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:  PYG-TWEEN FA/GLC should be inoculated with pure cultures of an organism.  Inoculated tubes should be immediately placed in an anaerobic atmosphere and incubated at 35 – 37^oC for 24 – 48 hours.  Detailed instructions for processing anaerobic cultures can be found in the MIDI instruction manual.

 

Materials Required But Not Provided

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

 

Limitations

PYG-TWEEN FA/GLC 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.

 

Quality Control

This medium, when cultured with appropriate control strains should show characteristic cellular fatty acid products when analyzed using the MIDI MIS system.  For interpretation of chromatographic results consult the MIDI manual.

The following organisms are routinely used for quality assurance testing, for growth and the fermentation of glucose at Anaerobe Systems.  Glucose utilization is determined on the basis of pH with the production of acid.  A strong acid will result in a pH of 5.5 or below, weak acid results in a pH of 5.5 – 6.0 and no acid production results in a pH of 6.0 or above.  Tween-80 growth stimulation is determined on comparison of growth against a control medium. 

Organism Tested	ATCC #	Results	Time (Hours)	Utilization of Glucose-Tween
Bacteroides fragilis	25285	Growth	24 h
Prevotella melaninogenica	25845	Growth	24 – 48 h
Bacteroides vulgatus	8482	Growth	24 h
Fusobacterium nucleatum	25586	Growth	24 – 48 h
Fusobacterium necrophorum	25286	Growth	24 – 48 h
Clostridium perfringens *	13124	Growth	24 h	+
Peptostreptococcus anaerobius	27337	Growth	24 h
Peptoniphilus asaccharolyticus	29743	Growth	24 – 48 h	-
Clostridium novyi t	7659	Growth	24 – 48 h	+

* Organisms specified by the MIDI MIS Manual for quality control

^t Growth stimulated when compared to Peptone Yeast Extract Broth with Glucose (PYG)

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

 

Organism	Growth in	Expected
	Hours	Reaction
C. novyi *	24-48 hrs	+
C. perfringens	24 hrs	+
B. fragilis	24 hrs
P. asaccharolyticus	48 hrs	-

* Growth stimulated when compared to Peptone Yeast Extract Broth with Glucose (PYG)

 

Physical Appearance: PYG-TWEEN FA/GLC should appear as a clear, golden-yellow 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.        Dowell, V. R., Jr. and G. L. Lombard.  1977.  Presumptive Identification of Anaerobic Non-sporeforming Gram-negative Bacilli.  USDHEW, CDC.  Atlanta, GA 30333.

 

3.        Dowell, V. R., Jr., G. L. Lombard, F. S. Thompson and A. Y. Armfield.  1977.  Media for the Isolation, Characterization and Identification of Obligately Anaerobic Bacteria.  USDHEW, CDC, Atlanta, GA 30333.

 

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

 

5.        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.

 

6.        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.

 

7.        Isenberg, H. D.  1992.  Clinical Microbiology Procedures Handbook.  American Society for Microbiology Publishing, Washington DC.

 

8.        Sherlock Microbial Identification System (MIS). Operating Manual, Version 6.  MIDI, Inc.  Newark, DE 19713.