Peptone Iron Agar

Cat. no. Q46 Peptone Iron Agar, 16x125mm Tube, 10ml Deep 20 tubes/box


Hardy Diagnostics Peptone Iron Agar is recommended for the detection of hydrogen sulfide production by coliform bacteria.

This product is not intended to be used for the diagnosis of human disease.


In 1877, Gayon demonstrated the production of hydrogen sulfide by bacteria using a medium containing lead acetate. (3) Positive results using Lead Acetate Agar exhibited a browning or brownish-blackening of the medium, which proved difficult to interpret with some strains of Enterobacteriaceae .

In the 1930s, Levine et al. developed a more definitive solid medium for detecting hydrogen sulfide production by using proteose peptone, with ferric citrate as the indicator, as a suitable alternative to Lead Acetate Agar. (5,6,10) Research by Levine et al. and Tittsler and Sandholzer showed that the rate of hydrogen sulfide production using Peptone Iron Agar was markedly increased compared to Lead Acetate Agar, and hydrogen sulfide production was more clearly evidenced by an intense blackening of the medium. (9,10) Peptone Iron Agar has since become a useful medium for differentiating Voges-Proskauer negative, methyl-red positive, and citrate positive coliforms from other members of the Enterobacteriaceae .

Peptone Iron Agar is a modification of the original formulation by Levine et al. and incorporates peptone along with proteose peptone as a source of organic nitrogen. The more soluble ferric ammonium citrate replaces ferric citrate and is used in conjunction with sodium thiosulfate as an indicator of hydrogen sulfide production. Sodium glycerophosphate is used as a buffering compound and agar is the solidifying agent.


Ingredients per liter of deionized water:*

Peptone 15.0gm
Proteose Peptone 5.0gm
Sodium Glycerophosphate 1.0gm
Ferric Ammonium Citrate 0.5gm
Sodium Thiosulfate 0.08gm
Agar 15.0gm

Final pH 6.7 +/- 0.2 at 25ºC.

* Adjusted and/or supplemented as required to meet performance criteria.


Storage: Upon receipt store at 2-8ºC. away from direct light. Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.



Specimen Collection: Specimen collection is not applicable since this medium is not intended for primary isolation from clinical specimens. As a general rule, infectious material should be submitted directly to the laboratory without delay and protected from excessive heat and cold. If there is to be a delay in processing, the specimen should be inoculated onto an appropriate transport media and refrigerated until inoculation. Consult listed references for information on specimen collection. (1,4,7,8)

Method of Use:

1. Using a well isolated colony from an 18-24 hour culture, pick the center of a colony with an inoculating needle. Inoculate the medium by stabbing the center of the agar to a depth of 1/4 to 1/2 an inch from the bottom of the tube.

2. Incubate the inoculated medium with a loose cap, aerobically at 35ºC. for 18 to 48 hours.

3. Observe inoculated tubes for growth and a blackening of the medium along the inoculation line, which is indicative of hydrogen sulfide production.


Positive results are exhibited by any blackening of the medium along the line of inoculum or throughout the bottom of the tube indicating hydrogen sulfide production.

For further identification of possible coliform bacteria, refer to the appropriate references. (2,4,7)



Standard microbiological supplies and equipment such as loops, inoculation needles, swabs, applicator sticks, other culture media, incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.


Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Proteus vulgaris
ATCC ® 6380**
D 18-48hr 35°C Aerobic Growth; blackening of the medium indicative of H 2 S production
Salmonella enteritidis
ATCC ® 13076
D 18-48hr 35°C Aerobic Growth; blackening of the medium indicative of H 2 S production
Escherichia coli
ATCC ® 25922**
D 18-48hr 35°C Aerobic Growth; no H 2 S production
Enterobacter aerogenes
ATCC ® 13048
D 18-48hr 35°C Aerobic Growth; no H 2 S production

** Recommended QC strains for User Quality Control according to the CLSI document M22 when applicable.



1. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.

2. APHA Technical Committee on Microbiological Methods for Foods. Compendium of Methods for the Microbiological Examination of Foods, APHA, Washington, D.C.

3. Gayon, U. 1877. Note de M.U. Gayon présentée par M. Pasteur. Comp. rend. Acad. Sci.; 85:1074-1076

4. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.

5. Levine M., S.S. Epstein, and R.H. Vaughn. 1934. Differential reactions in the colon group of bacteria. Am. J. Public Health; 24:505-510.

6. Levine M., R. Vaughn, S.S. Epstein, and D.Q. Anderson. 1932. Some differential reactions in the colon-aerogenes group of bacteria. Proc. Soc. Exp. Biol. Med.; 29:1022-1024.

7. Jorgensen., et al. Manual of Clinical Microbiology, American Society for Microbiology, Washington, D.C.

8. Quality Assurance for Commercially Prepared Microbiological Culture Media, M22. Clinical and Laboratory Standards Institute (CLSI - formerly NCCLS), Wayne, PA.

9. Tittsler, R.P. and L.A. Sandholzer. 1937. Advantages of peptide iron agar for the routine detection of hydrogen sulphide production. Am. J. Public Health; 27:1240-1242.

10. Vaughn, R, and M. Levine. 1936. Hydrogen sulfide production as a differential test in the colon group. J. Bact.; 31:24.

ATCC is a registered trademark of the American Type Culture Collection.