How to culture on buffered charcoal yeast extract agar

Buffered charcoal yeast extract agar base (BCYE) with added supplements is used for the selective culture of Legionella species and legionella isolation from clinical and other samples.Legionella species do not form spores and are narrow gram negative rods.Legionella causes pneumonia( Legionnaires disease)  or a milk, febrile disease( pontiac fever).They do not oxidise or ferment carbohydrates in conventional media or grow on sheep’s blood agar.Growth on Buffered Charcoal Yeast Extract Agar is much better and faster.Amino acids are key energy sources for Legionella.The amino acid L- cystine is an absolute requirement as it plays a major role in Legionella ‘s growth metabolism.This amino acid and ferrous pyrophosphate contribute to Legionella ‘s growth.

Principle of Buffered charcoal yeast extract agar

  • The media contains charcoal, a detoxifier.
  • The extract of yeast is a rich source of both vitamins, nitrogen and carbon.
  • ACES Buffer maintains optimum pH for growth while L- cysteine hydrochloride stimulates the growth of Legionella species with ferric pyrophosphate and α-ketoglutarate.
  • Antibiotic supplements may be used to eliminate contaminating microorganisms for selective isolation.Legionella Selective Supplement II which contains cephalothin, colistin,cefsulodin,cephalaxin, vancomycin and cycloheximide or Legionella Selective Supplement IV containing glycine, polymyxin B, anisomycin, vancomycin, blue bromothymol and purple bromocresol are often used to increase selectivity
  • Always wear gowns, masks and gloves while dealing with Legionella cultures.

Composition of Buffered charcoal yeast extract agar

IngredientsGrams / Litre
Yeast extract 10.000
Charcoal activated 2.000
ACES buffer10.000
a-Ketoglutarate monopotassium salt1.000
Agar17.000
Final pH at temperature 25°C6.9±0.2

How to Prepare Buffered charcoal yeast extract agar

  1. Start by suspending 20 grams in 500 ml distilled water.
  2. Add 2.4 g KOH pellets and mix to dissolve
  3. Heat to boil to completely dissolve the medium.
  4. Sterilize by autoclaving for 15 minutes at 121 ° C( 15 lbs) pressure then cool to 50°C.
  5. Add the sterile rehydrated content of 1 vial to each Legionella supplement aseptically
  6. Mix well and pour with constant stirring to ensure that charcoal particles are distributed evenly.
  7. For additional selectivity, Legionella Selective Supplements can be added to the molten medium according to your choice.
Also read  Robertson's Cooked Meat (RCM) Medium ideal for the culture of fastidious anaerobic bacteria

Appearance of Buffered charcoal yeast extract agar after preparation

Buffered charcoal yeast extract appears grey-black coloured opalescent gel forms when poured and solidified in petri plates

Uninoculated Buffered charcoal yeast agar
Uninoculated Buffered charcoal yeast agar

Plate reading and Colonies morphology on Buffered charcoal yeast extract agar

The following cultural characteristics are observed in 90% humid atmosphere with added Legionella Supplement for legionella culture after an incubation at  temperature 35-37°C for 3-4 days.

OrganismGrowthRecoveryColour of colony
Escherichia colinone-poor<=10%
Legionella dumoffiiluxuriant>50%light blue-grey
Legionella pneumophila (pnemonia)luxuriant >50%white grey to blue grey
Staphylococcus epidermidis and staphylococcal pneumonianone-poor<10%
Legionella spp colonies in Buffered charcoal yeast extract MLTGEEKS
Legionella spp colonies in Buffered charcoal yeast extract

References

  • Broome C. V., Fraser D. W., 1979, Epidemiol. Rev 1:1-16.
  • George J. R. et al, 1980, J. Clin. Microbiol., 11:286
  • Feeley J. C., Gorman G. W., Weaver R. E. et al, 1978, J. Clin. Microbiol., 8 : 320-325.
  • Jones G. T., Hebert G. A., (Eds.), 1979, US Department of Health, Education and Welfare Publication No. (CDC) 79-8375, Atlanta, Centers for Disease Control.
  • Feeley J. C., Gibson R. J., Gorman G. W. et al, 1979, J. Clin. Microbiol., 10:437.
  • Paseulle, Feely et al, 1980, J. Infect. Dis., 191:727.
  • Edelstein P. H., 1981, J. Clin. Microbiol., 14:298.
  • Bopp C. A., Sumner J. W., Morris G. K. and Wells J. G., 1981, J. Clin. Microbiol., 13:714.
  • Vicker R., Brown and Garrity, 1981, J. Clin. Microbiol., 13:380.

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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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