TECHNICAL DATA SHEET #460 REV. 3

27120 SW 95th Avenue Wilsonville, OR 97070 800.547.0659Page 1

LOEFFLER MEDIAPRODUCT:Tubed Media:

Loeffl er Media T6820

PURPOSE:Loeffl er Media is primarily used for the isolation of Corynebacterium diphtheriae from clinical specimens. It is also used to de-termine the proteolytic activity and pigment production of various microorganisms.

PRINCIPLE:The present formula for Loeffl er Media is a modifi cation of the formula fi rst developed by Loeffl er in 18874. Beef extract, dextrose, proteose peptone, and beef serum provide the nutrients necessary for the growth of the fastidious microorganism, C. diphtheriae. It is especially useful when small numbers of the microorganisms are present in clinical specimens.1 Recovery is further enhanced when Loeffl er Media is used in combination with Potassium Tellurite Cystine Agar Media and blood agar. On Loeffl er Media, C. diphtheriae produces characteristic metachromatic granules that can be detected by staining with methylene blue; it also demonstrates the characteristic Chinese-letter formation which is due to the cells snapping off when they divide. With the above information, a presumptive identifi cation can result in 16-18 hours.

Loeffl er Media enhances the demonstration of the organisms natural morphological and physiological characteristics, i.e. pig-mentation. It can restore properties after prolonged subculturing and due to the serum content in the media, proteolytic activity, the enzymatic hydrolysis of proteins into proteoses and peptones, can be demonstrated.

FORMULA:Approximate, per 250 ml of deionized fi ltered water.

Loeffl er Media:Beef Extract ......................................................0.75 gDextrose ...........................................................1.25Sodium Chloride ...............................................1.25Proteose Micro Peptone ...................................2.50Horse Serum ................................................750.00 ml

Final pH 7.1 0.2 at 25C

PRECAUTIONS:*For in vitro diagnostic use. Observe approved biohazard precautions.

Storage: Upon receipt store at 2-8C away from direct light. Media should not be used if there are signs of contamination, deterioration (cracking, shrinking, or discoloration), or if the expiration date has passed.

Limitations: Microscopic morphology may vary with different batches/lots of Loeffl er Media.

C. diphtheriae type gravis is the least likely to be recognized on microscopic examination.3

Nasopharyngeal and throat specimens should both be obtained when C. diphtheriae is suspected since 20% of positive cultures can be missed when only one site is cultured.3

Recovery of C. diphtheriae requires culturing on selective and nonselective media.

PROCEDURE:*Specimen collection: Information on specimen collection is found in standard reference material. In general, specimens should be protected from extreme heat and cold and should be delivered to the laboratory without delay. Nasopharyngeal cultures should be obtained using an alginate swab that reaches deep into the posterior nares. For throat cultures rub vigorously over any infl ammatory lesion. Specimens can be directly plated onto a Loeffl er slant if transport time is less than 24 hours.

Method of Use: Prior to inoculation, the media should be brought to room temperature. Remove excess fl uid at the base of the slant. Inoculate the slant with the specimen swab in a fi shtail motion and incubate aerobically for 8-24 hours at 35C. Prepare smears and stain with methylene blue stain. Colonies showing metachromatic granules and Chinese-letter formation on methy-

TECHNICAL DATA SHEET #460 REV. 3

27120 SW 95th Avenue Wilsonville, OR 97070 800.547.0659Page 2

lene blue stain are defi nitively identifi ed through biochemical testings and by tests for toxigenicity.

To detect proteolytic ability, inoculate aerobic microorganisms directly on the surface of the Loeffl er slant; with anaerobes incu-bate anaerobically or fi ll the slant with thioglycollate broth before incubation. Incubate aerobically at 35C for 3-4 days, possibly longer depending on the microorganism. Examine for colonies surrounded by small craters of liquefi ed media or liquefi cation of the slant with the production of a putrefi ed odor.

Interpretation:3

Corynebacterium diphtheriae Biotypes: Metachromatic Granules: Morphology:

mitis Intensely stained Long, pleomorphic but rigid rods

intermedius Cells frequently swollen Highly pleomorphic with terminal granules with very long and Irregularly barred short rods

gravis No granules but alternate Short rods with many bands of light and dark blue cocci of pear-shaped forms

Material Required but Not Provided: Standard microbiological supplies and equipment commonly found in a microbiological laboratory are not provided.

QUALITY CONTROL:*Microorganisms Used (ATCC #): Expected Results:Corynebacterium diphtheriae (13812) GrowthCorynebacterium pseudodiphtheriticum (10700) Growth

User Quality Control: Check for signs of contamination and deterioration. Loeffl er Media should appear moderately fi rm, opaque, and white in color.

BIBLIOGRAPHY: 1. Forbes, B. A., et al., Bailey and Scotts Diagnostic Microbiology, 12th ed., C. V. Mosby, St. Louis, 2007. 2. Koneman, E. W., et al., Color Atlas and Textbook of Diagnostic Microbiology, 6th ed., J. B. Lippincott, Philadelphia, 2005. 3. Murray, P.R., et al., Manual of Clinical Microbiology, 9th ed., American Society for Microbiology, Washington, D. C., 2007. 4. Loeffl er, F., Centr. f. Bakt., 2:105, 1887.

* For more detailed information, consult appropriate references.

PML Microbiologicals, Inc.Data #460Copyright 1989 by PML Microbiologicals, Inc.Revision Date: October 2008