Hugh Leifson Medium : Principle, Preparation and Colony Morphology

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Hugh Leifson Medium : Principle, Preparation and Colony Morphology

Hugh Leifson Medium (Oxidative-Fermentative Medium ) is used to distinguish between anaerobic and aerobic breakdown of carbohydrate( glucose).Hugh Leifson Medium was developed by Hugh and Leifson.They described the taxonomic significance of fermentation and oxidative metabolism of carbohydrates in gram- negative intestinal bacteria.There are two ways of using carbohydrates by microorganisms, namely fermentation and oxidation.This property can often be used to differentiate some bacteria.

Principle of Hugh Leifson Medium

  • Oxidative-Fermentative Medium contains a high concentration of carbohydrate and a low concentration of peptone to avoid the possibility of an aerobic organism using peptone and producing an alkaline condition that neutralizes the slight acidity produced by an oxidative organism.
  • Dipotassium phosphate promotes fermentation and acts as a pH control buffer. Agar concentration enables the determination of motility and aids in the distribution of acid throughout the tube produced on the surface of the medium.
  • The tubes for aerobic and anaerobic fermentation are inoculated and the agar surface of a duplicate tube is covered with a layer of sterile paraffin oil, approximately 25 mm thick and incubated at 37 ° C.
  • Oxidative organisms produce acid in unsealed tubes with little or no growth and no acid formation in sealed tubes, while fermentative organisms produce acid in both sealed and unsealed tubes.
  • When acid is produced, it is indicated by a change in color from greenish blue to yellow throughout the medium. For 2 hours, the liquid paraffin tube used should be sterilized dry at 160- 170 ° C. Wet sterilization with high pressure is not sufficient for this purpose.

Composition of Oxidative-Fermentative Medium

Sodium chloride5.00
Dipotassium phosphate0.300
Bromothymol blue0.050
Final pH ( at 25°C)6.8±0.2
Also read  Biochemical catalase test principle and interpretations (All catalase positive organism)

How to Prepare Hugh Leifson Medium

  1. Suspend 19.35 grams in 1000 ml of distilled water.
  2. Heat to boil to dissolve the medium completely.
  3. Dispense in duplicate test tubes for aerobic and anaerobic fermentation.
  4. Sterilize at 15 lbs( 121 ° C) pressure for 15 minutes by autoclaving.
  5. Cool the tubed medium in a straight position

Appearance of Hugh Leifson Medium

It appears Greenish blue coloured, clear to slightly opalescent gel forms in tubes as butts

Inoculated hugh leifson medium
Inoculated hugh leifson medium

Type of specimen

  • Blood
  • Food and dairy samples
  • Water samples

Cultural Response  and readings of Hugh Leifson Medium

Cultural characteristics observed after incubation for 18- 48 hours at 35- 37 ° C.

OrganismMotility Aerobic fermentationAnaerobic fermentation
Enterobacter aerogenespositive,growth away from stabline causing turbidityacid (yellow) and gas production acid (yellow) and gas production
Escherichia colipositive, growth away from stabline causing turbidityacid (yellow) and gas production, acid (yellow) and gas production
Pseudomonas aeruginosapositive,growth away from stabline causing turbidityacid (yellow) production unchanged (green) or alkaline (blue)
Salmonella Typhi positive, growth away from stabline causing turbidityacid (yellow) and gas production acid (yellow) and gas production
Shigella sonnei negative, growth along the stabline, surrounding mediumacid (yellow) production acid (yellow) and gas production
Cultural responses on Hugh leifson media
Cultural responses on Hugh leifson media


  • Hugh and Leifson, 1953, J. Bacteriol., 66:24.
  • MacFaddin J.F., 1985, Media for Isolation-Cultivation-Identification- Maintenance of Medical Bacteria,Vol. I, Williams and Wilkins, Baltimore.
  • Finegold S. M., Martin W. J., and Scott E. G., 1978, Bailey and Scott’s Diagnostic Microbiology, 5th Ed., The C.V. Mosby Co., St. Louis.
  • Downes F. P. and Ito K., (Ed.), 2001, Compendium of Methods for the Microbiological Examination of Foods, 4th Ed., American Public Health Association, Washington, D.C.
  • American Public Health Association, Standard Methods for the Examination of Dairy Products, 1978, 14th Ed., Washington D.C. 6. Greenberg A. E., Clesceri L. S. and Eaton A. D., (Eds.)
Also read  PYR Broth Test and Disc method For Steptococcus pyogenes

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About the Author: Arthur Westmann

DEFFE ARTHUR (AMOEBAMANN) is the founder and author of MLTGEEKS and MLTEXPO.He’s from Cameroon and is currently a Final year State Medical Laboratory Technician (MLT MA). Beyond lab works, he’s a passionate internet user with a keen interest in web design and blogging. Furthermore He likes traveling, hanging around with friends and social networking to do in his spare time.

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