Loeffler Medium Composition, Principle, Preparation, Results, Uses

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Loeffler Medium, a modified version of the 1887 Loeffler formula, is now called Loeffler Medium. Loeffler medium is a modified formula that Loeffler developed in 1887. It enhances primary and secondary isolation, and cultivates fastidious pathogenic microorganisms. After prolonged subculturing or prolonged incubation, Loeffler medium restores virulence as well as other identifying properties (microscopic/colonial). High serum levels are useful in determining organisms’ proteolytic activity. It can also be used to demonstrate pigmentation and ascospores.

Composition of Loeffler Medium

Proteose Peptone2.5gm
Sodium Chloride1.25gm
Beef Extract2.5gm
Horse Serum750.0 ml

Final pH (at 25°C): 7.6 +/- 0.3 

Principle of Loeffler Medium

The medium is made up of horse serum, beef extract, proteose peptones and dextrose. These nutrients provide the complex nitrogenous substances as well as the nutrients needed to support the growth Corynebacterium Diphtheriae. To supply essential ions, sodium chloride is also added. Dextrose is an important source of fermentable carbohydrate. The medium coagulates during sterilization due to the serum. This is why it is the main source of protein for metabolism in corynebacteria as well as other organisms. Medium enhances the formation of metachromatic crystals, as shown in methyleneblue stains. The granules formed by C. diphtheriae demonstrates its cellular morphology.


Intended to Use

Recommended for the cultivation of Corynebacterium Diphtheriae using clinical specimens or pure cultures. This allows for detection of chromogenesis and proteolysis, as well as the production of ascospores.

Preparation and Method of Use of Loeffler Medium

  1. 8.75 grams should be taken in 250 ml of distilled water.
  2. Mix the medium well and sterilize it by placing in an autoclave at 10 lbs pressure (115degC), for 20 minutes.
  3. Cool the mixture to 50-55°C, then add 750ml of sterile horse serum.
  4. Mix well, and then aseptically dispense into sterilized tubes.
  5. Inspissate the medium at 80-85degC for two hours, followed by free-flowing steam for at most three consecutive days.
  6. Allow the medium to reach room temperature before inoculation.
  7. Use a fishtail motion to inoculate the specimen by swabbing onto the medium.
  8. At 35oC, incubate aerobically. For up to 4 days
  9. Keep an eye out for colonial morphology
  10. To check for metachromatic granules or appearances that suggest Chinese-letter formation of cells, use methylene blue stain.
  11. Toxigenicity and biochemical tests are used to confirm the identification of C. diphtheriae.

For Detection of Proteolysis of Aerobic Microorganisms

  1. Use isolated colonies of the organism to inoculate medium.
  2. For 3-4 days, incubate aerobically at 35oC.
  3. For typical colonial morphology, observe.

For Detection of Proteolysis of Anaerobic Microorganisms

  1. Use isolated colonies of the organism to inoculate medium.
  2. At 35oC, incubate anaerobically. For 3-4 days, you can either incubate the inoculated slant in anaerobically at 35oC for three to four days or add Thioglycollate Broth to the surface just before incubation. Once the cap is tightened, incubate aerobically.
  3. For typical colonial morphology, observe.

Result Interpretation on Loeffler Medium

  • Corynebacterium spores grow on Loeffler Medium. Corynebacterium species show metachromatic granules that are suggestive of Chinese-letter formation in the methylene blue stain.
  • Proteolysis can be identified by colonies that are surrounded by small craters of liquefied media or liquefaction at the slant, which produce a pungent odor.
Corynebacterium diphtheriae Growth; minute, and cream-colored colonies with slightly raised centers; metachromatic granules seen in methylene blue stain
Corynebacterium pseudodiptheriticumGrowth; minute, and cream-colored colonies
Pseudomonas aeruginosaGood growth; green colonies with proteolysis
Staphylococcus aureusGood growth; yellow to gold colonies
Streptococcus pyogenesFair to good growth; non-proteolytic

Uses of Loeffler Medium

  • Loeffler medium’s primary purpose is to grow and characterize members of the genus Corynebacterium. This formulation increases the formation of metachromatic aggregates in the cells of these organisms.
  • Loeffler medium, which is high in serum, can be used to determine the proteolytic activity of microorganisms.
  • The medium’s gray-white exterior makes it an ideal background for observation and detection of colonial pigmentation.
  • This medium can be used to detect ascospores if all moisture has been removed aseptically from the Slants.

Limitations of Loeffler Medium

  • For complete identification, it is recommended that colonies grown from pure culture be subject to biochemical, immunological and molecular testing.
  • For C. diphtheriae isolating C. dickseriae, it is recommended that both selective and non-selective media are inoculated simultaneously with Loeffler Medium. Potassium Tellurite Cistine Cystine Agar or Blood Agar can be used for enhanced recovery.
  • A specimen of C. diphtheriae should be collected immediately after specimen collection to maximize its recovery.
  • Variation in microscopic structure may be a problem for Loeffler Medium.
  • Other Gram-positive microorganisms than Corynebacterium can produce metachromatic Granules when grown in Loeffler Medium.
  • Some microorganisms might require longer incubation times to detect proteolysis.
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