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
|Final pH ( at 25°C)||6.8±0.2|
How to Prepare Hugh Leifson Medium
- Suspend 19.35 grams in 1000 ml of distilled water.
- Heat to boil to dissolve the medium completely.
- Dispense in duplicate test tubes for aerobic and anaerobic fermentation.
- Sterilize at 15 lbs( 121 ° C) pressure for 15 minutes by autoclaving.
- 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
Type of specimen
- 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.
|Organism||Motility||Aerobic fermentation||Anaerobic fermentation|
|Enterobacter aerogenes||positive,growth away from stabline causing turbidity||acid (yellow) and gas production||acid (yellow) and gas production|
|Escherichia coli||positive, growth away from stabline causing turbidity||acid (yellow) and gas production,||acid (yellow) and gas production|
|Pseudomonas aeruginosa||positive,growth away from stabline causing turbidity||acid (yellow) production||unchanged (green) or alkaline (blue)|
|Salmonella Typhi||positive, growth away from stabline causing turbidity||acid (yellow) and gas production||acid (yellow) and gas production|
|Shigella sonnei||negative, growth along the stabline, surrounding medium||acid (yellow) production||acid (yellow) and gas production|
- 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.)