LOEFFLER MEDIUM

Cat. no. L28 Loeffler Medium, 16x100mm Tube, 5.5ml Slant 20 or 100 tubes/box

INTENDED USE

Hardy Diagnostics Loeffler Medium is recommended for the cultivation of Corynebacterium diphtheriae.

This medium is also useful in demonstrating proteolysis and pigment production of various microorganisms.

SUMMARY

Hardy Diagnostics Loeffler Medium is a modification of the original formula developed by Loeffler in 1887.(6-8) The medium contains horse serum, beef extract, dextrose and proteose peptones which together supply the complex nitrogenous substances and nutrients necessary to support the growth of Corynebacterium diphtheriae. Sodium chloride is added to supply essential ions.

The medium enhances the development of metachromatic granules as seen in methylene blue stains. Formation of the granules demonstrates the characteristic cellular morphology of C. diphtheriae.

Loeffler Medium is also useful for demonstrating pigment production and in determining the proteolytic activity of various microorganisms.

FORMULA

Ingredients per liter of deionized water:*

Proteose Peptone 1.5gm
Dextrose 1.25gm
Sodium Chloride 1.25gm
Beef Extract 0.75gm
Horse Serum 750.0ml

Final pH 7.6 +/- 0.3 at 25ºC.

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

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-8 degrees C. away from direct light. Media should not be used if there are any signs of deterioration, 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: 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. (1-5)

For Isolation of Corynebacterium diphtheria:

Method of Use:

1. Prior to inoculation, allow the medium to equilibrate to room temperature.

2. Using a fishtail motion, directly inoculate specimen swab onto the medium.

3. Incubate aerobically at 35ºC. for up to 4 days.

4. Observe daily for typical colonial morphology of corynebacteria.

5. Perform methylene blue stain to check for the presence of metachromatic granules and appearance suggestive of chinese-letter formation of cells.

6. Definitive identification of C. diphtheriae is made by performing biochemical and toxigenicity tests.

For Detection of Proteolysis of Aerobic Microorganisms

1. Inoculate medium with isolated colonies of the organism in question.

2. Incubate aerobically at 35ºC. for 3-4 days.

3. Observe for typical colonial morphology.

F or Detection of Proteolysis of Anaerobic Microorganisms

1. Inoculate medium with isolated colonies of the organism in question.

2. Incubate anaerobically at 35ºC. for 3-4 days or overlay the inoculated slant with Thioglycollate Broth just prior to incubation, tighten cap and incubate aerobically.

3. Observe for typical colonial morphology.

INTERPRETATION OF RESULTS

Growth of Cornyebacterium species on Loeffler Medium appear as minute, and cream colored colonies with slightly raised centers.

Cornyebacterium species reveal metachromatic granules and appearance suggestive of chinese-letter formation in methylene blue stain.

Proteolysis is indicated by the appearance of colonies surrounded by a small crater of liquefied medium or liquefaction of the slant with the production of a putrid odor.

LIMITATIONS

It is recommended that selective and non-selective media be inoculated in parallel to Loeffler Medium for isolating C. diphtheriae; Potassium Tellurite Cystine Agar and Blood Agar are recommend for enhanced recovery.(5)

To optimize recovery of C. diphtheriae, a nasopharyngeal and throat specimen should be obtained upon specimen collection.

Variation in microscopic morphology may vary from lot to lot of Loeffler Medium.

Gram-positive microorganisms other than Corynebacterium may produce metachromatic granules when grown on Loeffler Medium.

Detection of proteolysis by some microorganisms may require incubation periods beyond the recommended four days.

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
Corynebacterium diphtheriae
ATCC ® 13812
A 72-96hr 35°C Aerobic Growth; metachromatic granules seen in methylene blue stain

User Quality Control

PHYSICAL APPEARANCE

Loeffler Medium should appear opaque, and gray-white in color.

C. diphtheriae growing on Loeffler Medium

Corynebacterium diphtheriae (ATCC ® 13812) colonies growing on Loeffler Medium (Cat. no. L28). Incubated aerobically for 72 hours at 35ºC.

Loeffler Medium

Uninoculated tube of Loeffler Medium (Cat. no. L28).

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.

2. Jorgensen., et al. Manual of Clinical Microbiology, 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.

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

5. Koneman, E.W., et al. Color Atlas and Textbook of Diagnostic Microbiology, J.B. Lippincott Company, Philadelphia, PA.

6. Loeffler, F. 1897. Zentralb. Bakteriol. Parasitenkd; 2:102-106.

7. Petran, E.L. and Perry, C.A. 1929. J. Lab. Clin. Med.; 25:71-78.

8. Buck, T.C. 1949. J. Lab. Clin. Med.; 34:582-582.


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

031016gr