HardyCHROM™ Salmonella Shigella (SS) NoPro Agar

Cat. no. G327 HardyCHROM™ SS NoPro Agar, 15x100mm Plate, 21mL 10 plates/bag

INTENDED USE

Hardy Diagnostics HardyCHROM™ SS NoPro (no-Proteus) Agar* is recommended for the selective isolation and differentiation of Salmonella and Shigella spp. from stool. HardyCHROM™ SS NoPro Agar is intended as a primary screening tool to distinguish Salmonella and Shigella spp. from non-pathogenic enteric bacteria based on colony color, while inhibiting the growth and characteristic swarming of Proteus spp. The enhanced inhibition of Proteus reduces the expense involved in working up non-pathogens that could mimic enteric pathogens. Further species confirmation of suspect colonies via conventional or automated methods is recommended.

* Manufactured and sold in the US under license from Glycosynth Limited under U.S. Patent No.’s 7,323,488 B2, 7,384,763 B2, and 7,709,223 B2.

SUMMARY

Many formulations of culture media (such as HE, SS and XLD) have been developed to isolate and differentiate Salmonella and Shigella spp. from non-pathogenic enteric bacteria. Most formulations incorporate common ingredients such as carbohydrates (especially lactose), pH indicators, and an indicator system for the detection of hydrogen sulfide. These media are made selective by the addition of bile salts and can also differentiate between Salmonella/Shigella and lactose-fermenting organisms. However, the problem has always been that colonies of non-lactose-fermenting organisms that are not pathogenic can appear similar in appearance to Salmonella and Shigella and must be subjected to further testing by using Triple Sugar Iron (TSI) Agar, Lysine Iron Agar (LIA), or Kligler Iron Agar (KIA). Screening of primary plates or secondary plating from enrichment broths often requires the inoculation of large numbers of secondary screening tubes and/or the use of costly automated identification systems in order to rule out false-positives.

The use of chromogenic substrates (chromogens) in media formulations has increased greatly in the last several years. Chromogens, when broken down by specific bacterial enzymes, will result in colored colonies. Previously, chromogenic formulations were available for Salmonella spp., but not for Shigella spp.

HardyCHROM™ SS NoPro Agar allows for the selective isolation and differentiation of Salmonella and Shigella spp. from non-pathogenic enteric bacteria. Based on colony color all species of Salmonella and Shigella can be readily distinguished from other non-lactose fermenting gram-negative bacteria. Differentiation of Salmonella and Shigella spp. from non-pathogenic bacteria is accomplished by three mechanisms: chromogenic reactions, carbohydrate fermentation, and hydrogen sulfide production. HardyCHROM™ SS NoPro Agar provides better differentiation of colonies obtained from clinical samples and enrichment procedures, resulting in less secondary screening of isolates and less false-positive results. Proteus spp. will be completely to partially inhibited, thus preventing needless identification of false positives.

Bile salts and sodium deoxycholate allow for the selective nature of HardyCHROM™ SS NoPro Agar by inhibiting gram-positive organisms. Additional selective agents are added to reduce the number of normal enteric bacteria and the formulation has been modified to prevent the growth and swarming of Proteus spp. This results in far fewer subcultures of lactose negative colonies for biochemical testing and faster detection of pathogens that may be masked by the growth of Proteus spp. Fermentable carbohydrates aid in the differentiation of enteric pathogens from delayed lactose fermenters. This reaction is visually enhanced by a pH indicator. The addition of ferric ammonium citrate and sodium thiosulfate enable the detection of H2S, noted by the production of black centered colonies. Sodium thiosulfate serves as the sulfur source and ferric ammonium citrate is added as the indicator. Peptones in the medium supply the principle source of organic nitrogen in the form of amino acids and long-chained fatty acids. Patented chromogenic substrates are incorporated to enable the production of different colored compounds when degraded by specific bacterial enzymes.

FORMULA

Ingredients per liter of deionized water:*

Carbohydrates 20.0g
Peptones 8.0g
Sodium Thiosulfate 3.5g
Sodium Deoxycholate 1.5g
Ferric Citrate 1.0g
Chromogenic Mixture 0.5g
Selective Agents 0.15g
pH Indicator 0.01g
Agar 15.0g

Final pH 6.9 +/- 0.2 at 25ºC.

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

STORAGE AND SHELF LIFE

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.

PRECAUTIONS

PROCEDURE

Specimen Collection: Consult listed references for information on specimen collection.(3-6,8) 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 the listed references for information regarding the processing of specimens.(3-6,8)

Method of Use: Allow the plates to warm to room temperature. The agar surface should be dry prior to inoculating to ensure well-isolated colonies.

Inoculate and streak the specimen as soon as possible after collection. If the specimen to be cultured is on a swab, roll the swab over a small area of the agar surface. Streak for isolation with a sterile loop. A less selective medium such as MacConkey Agar (Cat. no. G35) should also be inoculated. This increases the chance of recovery when the population of gram-negative organisms is low. It also provides indication of other organisms present in the specimen.

Incubate plates in an inverted position, protected from the light, aerobically at 35-37ºC. for 18-24 hours.

Examine plates for colonies showing typical morphology and color.

The use of enrichment procedures such as Selenite Cystine Broth (Cat. no. K69), GN Broth (Cat. no. K01 or K39), or Tetrathionate Broth (Cat. no. K65) is recommended when testing food or stool samples from food handlers.(8)

INTERPRETATION OF RESULTS

Most Salmonella serotypes will produce H2S and the colonies will have a large black center with clear perimeter. Non H2S producing Salmonella spp., including S. enterica serovar Typhi and S. enterica serovar Paratyphi and Choleraesuis, produce teal blue colored colonies.

Shigella spp. produce teal blue colored colonies; S. flexneri, S. boydii, and S. dysenteriae colonies generally have entire edges, while S. sonnei colonies may have entire or undulated edges (plasmid mediated). Note: S. dysenteriae may produce small, colorless colonies.

Note: Further species confirmation of suspect colonies via conventional or automated method is recommended. Teal color development may not be apparent at 18 hours of incubation in rare instances. Plates should be incubated a full 24 hours before being discarded as negative.

Other members of the Enterobacteriaceae will be partially to completely inhibited. However, if present, will produce:

- pink colonies, with or without purple centers (such as Escherichia spp., Klebsiella spp., Citrobacter spp., Yersinia spp., and Enterobacter spp.)

- dark blue colonies (Citrobacter koseri)

- dark blue, purple or violet colonies with clear or pink edges (such as Serratia spp., Edwardsiella spp. and Morganella morganii)

- pale pink or tan with small light to dark brown centers (Proteus spp.). Strains should be mostly inhibited.*

- small, blue colonies (such as Hafnia alvei and inactive E. coli (Alkalescens-Dispar) strains).

- brown or light pink colonies with blue/gray centers (Providencia spp.)

Organism Description Photo Color
H2S producing Salmonella spp. Colonies with large black centers with clear perimeter S. enterica growing on HardyCHROM&™ SS
Shigella spp. and non-H2S producing Salmonella spp. Teal blue colored colonies
Proteus spp.* Pale pink
Escherichia spp., Klebsiella spp., Citrobacter spp., Yersinia spp., Enterobacter spp. Pink colonies, with or without purple centers
Hafnia alvei and inactive E. coli (Alkalescens-Dispar) Small, blue colonies

LIMITATIONS

It is recommended that biochemical, immunological, molecular, or mass spectrometry testing be performed on colonies from pure culture for complete identification.

Do not interpret colors of colonies on plates that have been incubated for longer than 24 hours.

Hafnia alvei and inactive E. coli produce blue colonies similar to Salmonella and Shigella spp. H. alvei and inactive E. coli colonies are more blue (less green) and are smaller than Salmonella and Shigella spp. Further testing is recommended.

Approximately 1% of Shigella sonnei strains are sucrose positive and may exhibit pink colonies with purple centers instead of teal blue colonies.

Color-blind individuals may encounter difficulty in distinguishing the color differences on HardyCHROM™ SS NoPro Agar.

Colonies of Proteus spp. should be inhibited on HardyCHROM™ SS NoPro Agar. In the event rare patient-specific isolates grow, colonies of Proteus spp. should be small non-swarming, pale pink in color, and may or may not produce dark centers that may resemble Salmonella or Shigella spp.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, swabs, applicator sticks, other culture media such as MacConkey AgarCat. no. G35), Selenite Cystine Broth (Cat. no. K69), GN Broth (Cat. no. K01 or K39), or Tetrathionate Broth (Cat. no. K65), incinerators, incubators, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Salmonella enterica
ATCC® 14028
A 24hr 35°C Aerobic Growth; colonies with large black centers with a clear perimeter
Shigella sonnei
ATCC® 9290
A 24hr 35°C Aerobic Growth; teal blue colonies
Escherichia coli
ATCC® 25922
A 24hr 35°C Aerobic Partial to complete inhibition; pink colonies with purple centers
Pseudomonas aeruginosa
ATCC® 27853
B 24hr 35°C Aerobic Inhibited
Proteus mirabilis
ATCC® 12453
A 24hr 35°C Aerobic Inhibited

USER QUALITY CONTROL

PHYSICAL APPEARANCE

HardyCHROM™ SS NoPro Agar should appear clear, slightly opalescent, and dark pink in color

S. enterica ATCC 14028

Salmonella enterica (ATCC® 14028) colonies growing on HardyCHROM™ SS NoPro Agar (Cat. no. G327). Incubated aerobically for 24 hours at 35°C.

S. sonnei ATCC 9290

Shigella sonnei (ATCC® 9290) colonies growing on HardyCHROM™ SS NoPro Agar (Cat. no. G327). Incubated aerobically for 24 hours at 35°C.

E. coli ATCC 25922

Escherichia coli (ATCC® 25922) colonies growing on HardyCHROM™ SS NoPro Agar (Cat. no. G327). Incubated aerobically for 24 hours at 35°C.

H. alvei ATCC 29926

Hafnia alvei (ATCC® 29926) colonies growing on HardyCHROM™ SS NoPro Agar (Cat. no. G327). Incubated aerobically for 24 hours at 35°C.

P. mirabilis ATCC 43071

Proteus mirabilis (ATCC® 43071) colonies inhibited on HardyCHROM™ SS NoPro Agar (Cat. no. G327). Incubated aerobically for 24 hours at 35°C.

Uninoculated plate G327

Uninoculated plate of HardyCHROM™ SS NoPro Agar. (Cat. no. G327).

REFERENCES

1. Gruenewald, R., et al. 1991. J. Clin. Microbiol.; 29:2354-2356.

2. Farmer, J.J., et al. 1985. J. Clin. Microbiol.; 21:46-76.

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

4. Murray, P.R. Manual of Clinical Microbiology. American Society for Microbiology, Washington, D.C.

5. Forbes, B.A., et al. Bailey and Scott's Diagnostic Microbiology. C.V. Mosby Company, St. Louis, MO.

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

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

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

7. Centers for Medicare & Medicaid Services (CMS). Individualized Quality Control Plan (IQCP).


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

050616kdp