Czapek Dox Agar : Introduction, Principle, Composition, Test Procedure, Colony Characteristics and Uses

Czapek Dox Agar is a semisynthetic medium used for the cultivation of fungi, containing sodium nitrate as the sole source of nitrogen. Sucrose serves as the sole source of carbon while sodium nitrate serves as the sole source of nitrogen. Dipotassium phosphate buffers the medium. Magnesium sulphate, potassium chloride, ferrous sulphate serves as sources of essential ions. Agar acts as solidifying agent. In modified  Czapek Dox Agar,  magnesium glycerophosphate and potassium sulphate replace the magnesium sulphate and potassium phosphate of the original which prevents the precipitation of magnesium phosphate and it is also a highly satisfactory substrate for chlamydospore production by Candida albicans.

Introduction of Czapek Dox Agar

Czapek medium, also called Czapek’s agar (CZA) from the surname of inventor Czech botanist Friedrich Johann Franz Czapek (1902-1903) . Later CZA modified by another American inventor, chemist Arthur Wayland Dox  (1910) and thus its name became’ Czapek-Dox medium’ and  formula prepared according to Thom and Church. It is recommended for use in qualitative procedures for the cultivation of saprophytic fungi and  soil bacteria. The medium contains sucrose as the sole source of carbon and nitrate as the only inorganic source of nitrogen. Czapek Dox Agar (Modified) it is an oxoid modification called Oxoid Czapek Dox Agar which contains magnesium glycerophosphate and potassium sulphate replace the magnesium sulphate and potassium phosphate of the original. This modification prevents the precipitation of magnesium phosphate. The medium is also a highly satisfactory substrate for chlamydospore production by Candida albicans.

Czapek’s  agar

Czapek-Dox Agar

Czapek Dox Agar (Modified)

Principle of Czapek Dox Agar

Czapek Dox Agar is a semisynthetic medium used for the cultivation of fungi, containing sodium nitrate as the sole source of nitrogen. Sucrose serves as the sole source of carbon while sodium nitrate serves as the sole source of nitrogen. Dipotassium phosphate buffers the medium. Magnesium sulphate, potassium chloride, ferrous sulphate serves as sources of essential ions. Agar acts as solidifying agent. In modified  Czapek Dox Agar,  magnesium glycerophosphate and potassium sulphate replace the magnesium sulphate and potassium phosphate of the original which prevents the precipitation of magnesium phosphate and it is also a highly satisfactory substrate for chlamydospore production by Candida albicans.

Composition of Czapek  Agar ( original)

  • Cane sugar: 30 gm
  • Monopotassium phosphate: 1 gm
  • magnesium sulfate: 0.5 gm
  • Potassium chloride: 0.5 gm
  • Iron sulfate : 0.01 gm
  • Distilled water (D/W): 1000 ml

Composition of Czapek Dox Agar

  • Sucrose 30.0 gm
  • Sodium Nitrate 2.0 gm
  • Dipotassium Phosphate 1.0 gm
  • Magnesium Sulfate 0.5 gm
  • Potassium Chloride 0.5 gm
  • Ferrous Sulfate 0.01 gm
  • Agar 15.0 gm
  • Distilled Water(D/W): 1000 ml

Final pH 7.3 +/- 0.3 at 25ºC.

Composition of Czapek Dox Agar ( modified)

                   (Oxoid Czapek Dox Agar )

  • Sodium nitrate: 2.0 gm
  • Potassium chloride: 0.5 gm
  • Magnesium glycerophosphate: 0.5 gm
  • Ferrous sulphate: 0.01 gm
  • Potassium sulphate: 0.35 gm
  • Sucrose: 30.0 gm
  • Agar: 12.0 gm
  • Distilled Water (D/W): 1000 ml

pH 6.8 ± 0.2 @ 25°C

Preparation of Czapek Dox Agar

  1. Suspend 49.01 grams Czapek Dox Agar in 1 liter purified / distilled or deionized water.
  2. Heat to boiling to dissolve the medium completely.
  3. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
  4. After autoclaving,  leave for cooling to 45-50°C.
  5. Mix well before dispensing.
  6. Pour Czapek Dox Agar into each plate and leave plates on the sterile surface until the agar has solidified.
  7. Store the plates in a refrigerator at 2-8°C.

Storage and Shelf life of Czapek Dox Agar

  • Store at 2-8ºC  and away from direct light.
  • Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), contamination.
  • Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.

Test Requirements

  • Test specimens ( samples or fungal growth)
  • Inoculating loop
  • Bunsen burner
  • Incubator
  • Control strains (For positive control: Candida albicans ATCC 10231 and Aspergillus niger ATCC 9642, For negative control: Uninoculated medium)
  • Biological Safety cabinet (BSC)

Test procedure

  1. Allow the plates to warm at 37°C or  to room temperature, and the agar surface to dry before inoculating.
  2. Take plates.
  3. Inoculate by spreading the sample material thinly on the surface of the culture medium.
  4. Incubate for up to 1 week at 28ºC. Optimal incubation temperatures are as follows: Penicillium species at 20-25ºC, Aspergillus species at 30ºC and Candida species at 28ºC.

Result Interpretation  of Czapek Dox Agar

Presence of yeast or mold or both: Fungal growth

Control strains-

Candida albicans: Luxuriant growth with cream-colored colonies

Aspergillus niger: Luxuriant growth with White/yellow mycelium, black spores

Uninoculated medium: No growth

Colony Characteristics of various organisms in Czapek Dox Agar

Aspergillus brasiliensis: Good growth

Candida albicans:  Good growth; cream-colored colonies

Saccharomyces cerevisiae: Good growth

Aspergillus niger: White/yellow mycelium, black spores

Aspergillus flavus: Macroscopic features: Colonies on Czapek Dox agar are granular, flat, often with radial grooves and yellow where as microscopic features in LPCB preparation are globose to subglose conidia ( 3-6 µm in size),  septate hyphae, stipe, condiophore, vesicle and phialides as shown above picture.

Uses of Czapek Dox Agar

The application of Czapek Dox Agar are as follows-

  1. Czapek Dox Agar is  APHA( American Public Health Association)  recommended  fungal medium for isolation of Aspergillus, Penicillium, Paecilomyces and some other fungi with similar physiological requirements.
  2. It is also recommended for the general cultivation of fungi from water samples.
  3. The medium is widely used for taxonomic studies of Aspergillus, Penicillium and actinomycetes.
  4. Czapek-Dox Agar produces luxuriant growth of almost all saprophytic Aspergilli causing the organisms to produce characteristic mycelia and conidia.
  5. The acidity of the medium may be increased for the cultivation of acidophilic organisms such as yeasts.
  6. The medium is useful in a variety of microbiological procedures, including fungi and mildew resistance tests and soil microbiology testing.
  7. Oxoid Czapek Dox is the most satisfactory medium comparatively than rice infusion agar and corn meal agar for chlamydospore production by Candida albicans.

Key Notes on Czapek Dox Agar

  • Czapek-Dox Agar is recommended for use in cultivating fungi and bacteria capable of using inorganic nitrogen.
  • Czapek and Dox did not add agar but many recipes add 15 g to make a solid medium.
  • Most Penicillium species have an optimum growth temperature between 20° and 25°C, while many Aspergillus species grow best at about 30°C. However, different fungi grow over a wide range of temperatures; Aspergillus fumigatus grows well at 50°C and Cladosporium herbarum will grow on meat at -6°C.

Limitations of Czapek Dox Agar

  • This medium is a general purpose medium and may not support the growth of fastidious organisms.
  • Further tests need colonies from pure culture for complete identification like biochemical, immunological, molecular, or mass spectrometry.
  • Modification preferred  for two reasons, 1. due to prevention of  precipitation of magnesium phosphate and 2.  chlamydospore production by Candida albicans.

References

  1. Medical Mycology. Editors:  Emmons and Binford, 2nd ed 1970, Publisher Lea and Febiger, Philadelphia.
  2. Rippon’s JW: Medical Microbiology. The pathogenic fungi and the pathogenic Actinomycetes. 3rd ed 1988 Publisher WB saunder co, Philadelphia.
  3. Clinical Microbiology Procedure Hand book , Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
  4. A Text Book of Medical Mycology. Editor: Jagdish Chandar.  Publication Mehata, India.
  5.  Practical Laboratory Mycology. Editors: Koneman E.W. and G.D. Roberts, 3rd ed 1985, Publisher Williams and Wilkins, Baltimore.
  6. https://catalog.hardydiagnostics.com/cp_prod/Content/hugo/Czapek-DoxAgar.html
  7. http://www.oxoid.com/uk/blue/prod_detail/prod_detail.asp?pr=CM0097
  8. https://en.wikipedia.org/wiki/Czapek_medium
  9. http://www.himedialabs.com/TD/M075.pdf
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