Loeffler Medium is a modified form of the original culture medium formulated by Loeffler in 1887.
It is specifically designed to enhance the primary as well as secondary isolation and cultivation of fastidious pathogenic microorganisms.
The medium is particularly useful for isolating organisms from clinical specimens obtained from the nose and throat.
Loeffler Medium helps restore the virulence of microorganisms that may be reduced or lost due to prolonged incubation or repeated subculturing.
It also aids in the recovery of important identifying characteristics of microorganisms, including their microscopic appearance and colonial morphology.
The high serum content of the medium supports microbial growth and assists in the detection of proteolytic activity of organisms.
In addition, the medium is used for the demonstration of pigment production by certain microorganisms.
Loeffler Medium is also employed for observing the formation of ascospores in specific fungal species.
Composition of Loeffler Medium
Proteose peptone is included at a concentration of 2.5 g per liter, serving as a source of nitrogen, amino acids, and growth factors required for bacterial growth.
Dextrose is added at 2.5 g per liter as a readily fermentable carbohydrate that provides energy for microorganisms.
Sodium chloride is present at 1.25 g per liter to maintain osmotic balance and support proper cellular function.
Beef extract is incorporated at 2.5 g per liter, supplying essential vitamins, minerals, and additional growth-promoting substances.
Horse serum constitutes 750.0 mL per liter, providing rich nutrients and serum proteins that enhance the growth of fastidious organisms and help restore virulence and characteristic features.
The final pH of the medium is adjusted to 7.6 ± 0.3 at 25 °C, creating an optimal environment for the cultivation of target microorganisms.
Principle of Loeffler Medium
Loeffler Medium contains horse serum, beef extract, dextrose, and proteose peptones, which collectively provide complex nitrogenous substances and essential nutrients required for microbial growth.
These components specifically support the growth of Corynebacterium diphtheriae by supplying proteins, amino acids, and growth factors.
Sodium chloride is included to provide essential ions and to maintain osmotic balance within the medium.
Dextrose serves as a source of fermentable carbohydrates, supplying energy for the metabolic activities of the organisms.
During the sterilization process, the horse serum causes the medium to coagulate, forming a firm serum-based medium.
The serum also acts as a rich protein source that is utilized in the metabolism of corynebacteria and other microorganisms.
The medium enhances the development of metachromatic granules in Corynebacterium diphtheriae, which can be clearly observed using methylene blue staining.
The presence and formation of these metachromatic granules demonstrate the characteristic cellular morphology of C. diphtheriae, aiding in its identification.
Preparation and Method of Use of Loeffler Medium
Suspend 8.75 g of the dehydrated medium in 250 mL of distilled water.
Heat gently and dissolve the medium completely.
Sterilize the dissolved medium by autoclaving at 10 lbs pressure (115 °C) for 20 minutes.
Allow the medium to cool to 50–55 °C.
Aseptically add 750 mL of sterile horse serum to the cooled medium.
Mix thoroughly to ensure uniform distribution of the serum.
Aseptically dispense the prepared medium into sterile test tubes.
Sterilize the medium by inspissation at 80–85 °C for 2 hours in free-flowing steam for at least three consecutive days.
Before inoculation, allow the prepared medium to equilibrate to room temperature.
Using a fishtail motion, directly inoculate the specimen swab onto the surface of the medium.
Incubate the inoculated medium aerobically at 35 °C for up to 4 days.
Observe the cultures daily for typical colonial morphology suggestive of growth.
Perform a methylene blue stain to detect the presence of metachromatic granules and to observe cellular morphology resembling Chinese-letter arrangement.
Make definitive identification of Corynebacterium diphtheriae by conducting biochemical tests and toxigenicity testing.
For Detection of Proteolysis of Aerobic Microorganisms
For detection of proteolysis by aerobic microorganisms, inoculate the medium with isolated colonies of the test organism.
Incubate aerobically at 35 °C for 3–4 days.
Observe for typical colonial morphology and evidence of proteolysis.
For Detection of Proteolysis of Anaerobic Microorganisms
For detection of proteolysis by anaerobic microorganisms, inoculate the medium with isolated colonies of the organism.
Incubate anaerobically at 35 °C for 3–4 days, or alternatively overlay the inoculated slant with Thioglycollate Broth just before incubation, tighten the cap, and incubate aerobically.
Observe the cultures for typical colonial morphology and proteolytic activity.
Result Interpretation on Loeffler Medium
On Loeffler Medium, growth of Corynebacterium species is characterized by the development of typical colonies, and on methylene blue staining the organisms show metachromatic granules with a cellular arrangement suggestive of Chinese-letter formation.
Proteolysis on Loeffler Medium is indicated by the presence of colonies surrounded by a small crater of liquefied medium or by liquefaction of the slant, often accompanied by the production of a putrid odor.
Corynebacterium diphtheriae shows growth in the form of minute, cream-colored colonies with slightly raised centers, and methylene blue staining reveals prominent metachromatic granules.
Corynebacterium pseudodiphtheriticum produces minute, cream-colored colonies, but does not show the distinctive features associated with toxigenic C. diphtheriae.
Pseudomonas aeruginosa exhibits good growth, forming green-colored colonies along with proteolytic activity on the medium.
Staphylococcus aureus demonstrates good growth with the formation of yellow to golden colonies.
Streptococcus pyogenes shows fair to good growth on Loeffler Medium and is non-proteolytic.
Uses of Loeffler Medium
The primary use of Loeffler Medium is for the growth and morphological characterization of microorganisms belonging to the genus Corynebacterium.
The formulation specifically enhances the formation of metachromatic granules within the cells, aiding in the identification of Corynebacterium species.
Owing to its high serum content, Loeffler Medium is useful for the determination of proteolytic activity of various microorganisms.
The gray-white surface of the medium provides an excellent contrast, making it easier to detect and observe colonial pigmentation.
When all extraneous moisture is aseptically removed from the slants and the upper portion of the slant is heated until rupture occurs, the medium can also be used for the detection of ascospores.
Limitations of Loeffler Medium
Loeffler Medium alone is not sufficient for complete identification, and colonies obtained from pure culture should be further analyzed using biochemical, immunological, molecular, or mass spectrometry–based tests.
For optimal isolation of Corynebacterium diphtheriae, it is recommended that both selective and non-selective media be inoculated alongside Loeffler Medium, with Potassium Tellurite Cystine Agar and Blood Agar suggested for enhanced recovery.
To maximize the recovery of C. diphtheriae, both nasopharyngeal and throat specimens should be collected at the time of specimen acquisition.
Microscopic morphology may vary depending on the lot of Loeffler Medium used, which can affect consistency in observations.
Gram-positive microorganisms other than Corynebacterium may also produce metachromatic granules when grown on Loeffler Medium, potentially leading to misinterpretation.
Detection of proteolytic activity in some microorganisms may require incubation periods longer than the recommended four days, which can delay results.
References
HiMedia Laboratories. Loeffler Medium – Technical Data Sheet (M537).
Quelab Laboratories. Loeffler Medium: Product Information and Instructions for Use.
Thermo Fisher Scientific. Instructions for Use: Loeffler Medium.
Becton, Dickinson and Company (BD). Loeffler Medium: Technical Insert and Product Documentation.
