SODIUM CHLORIDE (NaCl) 6.5% MEDIA

Cat. no. R27 NaCl 6.5% Broth without Indicator, 13x100mm Tube, 3ml 20 tubes/box
Cat. no. L22 NaCl 6.5% Agar without Indicator, 16x100mm Tube, 5.5ml Slant 20 tubes/box
Cat. no. K49 NaCl 6.5% Broth with Indicator, 15x103mm Tube, 2ml 20 tubes/box
Cat. no. R26 NaCl 6.5% Broth with Indicator, 13x100mm Tube, 1ml 20 tubes/box
Cat. no. J165 BEA (Bile Esculin Agar) / NaCl 6.5% Agar without Indicator, 15x100mm Biplate, 10ml/10ml 10 plates/bag

INTENDED USE

Hardy Diagnostics NaCl 6.5% Media are recommended for use in salt tolerance testing to differentiate Enterococcus from non-enterococcal group D streptococci.

SUMMARY

Salt Tolerance Broth was derived from a medium modification designed by Hajna.(3) Quadri made further modifications by adding salt, dextrose and bromcresol purple to an infusion broth.(7) The medium was designed to aid in the differentiation of Enterococcus spp. from streptococci by determining the ability of enterococci to grow in the presence of 6.5% NaCl.

Salt acts as a selective agent and interferes with membrane permeability and osmotic equilibrium. Salt tolerant organisms will produce heavy growth in the broth and on solid agar within 48 hours. Organisms that are capable of growing in the presence of a high salt concentration will also ferment dextrose. Dextrose fermentation produces an acid reaction which results in the bromcresol purple indicator turning yellow. Appearance of a yellow color change in broth with indicator is indicative of a positive salt tolerance test. Growth of organisms on high salt agar is also indicative of salt tolerance, but the omission of bromcresol purple means no color reaction will occur. Organisms that do not grow on NaCl 6.5% agar are not salt tolerant. E. faecalis, E. zymogenes, E. liquifaciens, and E. durans are among the Enterococcus species that are salt tolerant.

FORMULA

Ingredients per liter of deionized water:*

Sodium Chloride 65.0gm
Brain Heart Infusion Broth 25.0gm

In addition, Cat. no. K49 and R26 contains:

Dextrose 2.0gm
Bromcresol Purple 30.0mg

Cat. no. L22 contains the above in addition to:

Agar 25.0gm

Final pH 6.9 +/- 0.3 at 25ºC.

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

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-30ºC. away from direct light. NaCl 6.5% Broth with Indicator (Cat. no. R26) and NaCl 6.5% Broth without indicator (Cat. no. R27) should be stored 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: This product is not intended for primary isolation of patient specimens. It should be used only with cultures of isolated organism. This product is used in conjunction with other biochemical tests to identify cultures of isolated organism. Information on specimen collection may be found in standard reference texts.(2,4-6)

Method of Use: Prior to inoculation, the medium should be warmed to room temperature. Using a light inoculum from an overnight culture, inoculate the medium and incubate aerobically at 35ºC. for 24-48 hours. It is necessary to use a light inoculum in order to prevent the occurrence of false-positive results on plated media, streak to obtain isolated colonies. Examine media for turbidity/growth or the presence of colonies on media without indicator. Media with indicator should be examined for a color change from purple to yellow.

INTERPRETATION OF RESULTS

A positive salt tolerance test is indicated by growth and/or turbidity in media without indicator. Media containing indicator will produce a color change from purple to yellow. A negative test is denoted by no growth and/or no color change. A negative test indicates the organism is not capable of growing in a high salt concentration.

LIMITATIONS

Infusion broth with 6.5% NaCl may produce slow reactions thereby making test interpretation difficult.

Streptococcus agalactiae (group B) will grow in the media, however it does not produce an acid reaction. Turbidity, but no color change will be present.

A light inoculum must be used when inoculating broth. Too heavy an inoculum may produce turbidity, thus resulting in a false-positive result.

MATERIALS REQUIRED BUT NOT PROVIDED

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

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
NaCl 6.5% Media without Indicator:
Enterococcus faecalis
ATCC ® 29212
E 24-48hr 35°C Aerobic Growth
Streptococcus pyogenes
ATCC ® 19615
E 24-48hr 35°C Aerobic Inhibited
Streptococcus bovis
ATCC ® 9809
E 24-48hr 35°C Aerobic Inhibited
NaCl 6.5% Media with Indicator:
Enterococcus faecalis
ATCC ® 29212
E 24-48hr 35°C Aerobic Growth; media turns yellow at 48 hours
Streptococcus pyogenes
ATCC ® 19615
E 24-48hr 35°C Aerobic Inhibited; media remains purple
Streptococcus bovis
ATCC ® 9809
E 24-48hr 35°C Aerobic Inhibited; media remains purple

User Quality Control

PHYSICAL APPEARANCE

E. faecalis growing in NaCl 6.5% Medium without Indicator

Enterococcus faecalis (ATCC® 29212) growing in NaCl 6.5% Medium without Indicator (Cat. no. R27). Incubated aerobically for 24 hours at 35ºC.

S. pyogenes inhibited in NaCl 6.5% Medium without Indicator

Streptococcus pyogenes (ATCC® 19615) inhibited in NaCl 6.5% Medium without Indicator (Cat. no. R27). Incubated aerobically for 24 hours at 35ºC.



E. faecalis growing in NaCl 6.5% Medium with Indicator

Enterococcus faecalis (ATCC® 29212) growing in NaCl 6.5% Medium with Indicator (Cat. no. R26). Incubated aerobically for 24 hours at 35ºC.

S. pyogenes inhibited in NaCl 6.5% Medium with Indicator

Streptococcus pyogenes (ATCC® 19615) inhibited in NaCl 6.5% Medium with Indicator (Cat. no. R26). Incubated aerobically for 24 hours at 35ºC.


REFERENCES

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.

3. Tille, P., et al. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.

3. Hajna, A.A. and C.A. Perry. 1943. American Journal of Public Health; 33:550-58.

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

5. MacFaddin, J.F. 1985. Media for Isolation, Cultivation, Identification, Maintenance of Bacteria, Vol. I. Williams & Wilkins, Baltimore, MD.

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

7. Quadri, S.M., et al. 1978. Journal of Clinical Microbiology; 7:238.

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

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

012617gr