Czapek Agar: Foundation, Preparation, Uses And Limitations

Czapek Agar (CZA) is a selective solid culture medium specially designed for the cultivation of saprophytic bacteria and fungi. It was created by the botanist Friedrich Johann Franz Czapek, of Polish origin, and the American chemist Arthur Wayland Dox; for this reason, this agar is also known as Czapek-Dox medium.

The original medium was liquid, but agar was later added to make it a solid medium. Czapek medium is composed of sodium nitrate, dipotassium phosphate, magnesium sulfate, potassium chloride, ferrous sulfate, sucrose, agar, and distilled water.

Czapek agar

Czapek agar A. Aspergillus parasiticus B. Aspergillus fumigatus Source: A. Medmyco [CC BY-SA 4.0 (https://creativecommons.org/licenses/by-sa/4.0))/ B. Photo: Dr. David Midgley. Wikipedia.com

The pH of Czapek agar is 7.3, but there is a variant to which 10% lactic acid is added, the pH of which remains at 3.5. This acid medium is used for the cultivation of acidophilic microorganisms.

On the other hand, some commercial houses modified the composition of Czapek agar by changing magnesium sulfate and bipotassium phosphate by glycerophosphate; This modification prevents the formation of the magnesium phosphate precipitate observed with the conventional formula. 
Likewise, there is another variant to which yeast extract is added.

Finally, Czapek agar is recommended by the American Public Health Association APHA for the study of Actinomycetes sp, Aspergillus sp, Paecilomyces sp and Penicillium sp. These species are characterized by being environmental microorganisms and opportunistic pathogens.

Article index

  • one

    Basis

  • two

    Preparation

    • 2.1

      Medium Czapek-Dox

    • 2.2

      Czapek medium at acidic pH

    • 23

      Modified Czapek Medium

    • 2.4

      Czapek-yeast medium

  • 3

    Use

    • 3.1

      Sown

    • 3.2

      Incubation temperature and time

    • 3.3

      Characteristics of some colonies on this medium

  • 4

    QA

  • 5

    Limitations

  • 6

    References

Basis

This medium contains sucrose; this carbohydrate serves as the carbon source, while sodium nitrate is the nitrogen source, but unlike other media the nitrogen provided is inorganic.

For this reason, the Czapek medium is considered selective, since only microorganisms capable of using inorganic compounds as the sole nitrogen source will be able to develop.

As a regulator of the osmolarity of the medium is bipotassium phosphate. For their part, magnesium sulfate, potassium chloride and ferrous sulfate provide essential minerals for the growth of saprophytic (free-living) microorganisms. This formula forms a precipitate of magnesium phosphate.

Finally, the agar-agar is the compound that provides the solid consistency to the medium and water is the solvent for all the components.

With the modified Czapek agar formula there is no precipitate formation and it improves the growth performance of some fungi.

The extreme acidity of Acid Czapek Agar makes it a more selective medium, allowing only the growth of acidophilic microorganisms, capable of utilizing inorganic nitrogen. 
Meanwhile, Czapek-yeast agar improves the performance of certain mushrooms.

Preparation

Medium Czapek-Dox

Weigh out 49 g of the dehydrated culture medium and dissolve in distilled water. Apply heat until boiling. Shake the mixture frequently until completely dissolved.

-Czapek Agar Wedges

Distribute the dissolved mixture into tubes and autoclave at 121 ° C for 15 minutes. When removing from the autoclave, homogenize before laying down the tubes and allow to solidify.

-Czapek agar plates

Autoclave the dissolved mixture at 121 ° C for 15 minutes and leave it to cool to 50 ° C, homogenize and pour 20 ml into sterile Petri dishes. Allow to solidify, invert and store in the refrigerator until use.

The pH should be 7.3 ± 0.2. The color of the dehydrated medium is white and the prepared medium is light amber, slightly cloudy due to the presence of a flocculating precipitate.

Czapek medium at acidic pH

Prepare as already described, but adding 10 ml of sterile 10% lactic acid for each liter of medium prepared. The modified Czapek medium should remain at pH 3.5 ± 0.2.

Modified Czapek Medium

Weigh out 45.4 g of the dehydrated medium and dissolve in 1 liter of water. The rest of the preparation is the same as described above. The color of the dehydrated medium is white and the prepared medium is whitish. The final pH of the medium is 6.8 ± 0.2.

Czapek-yeast medium

Also called Czapek Yeast Extract Agar (CYA). Contains the elements of the classic Czapek agar plus yeast extract.

Use

The Czapek medium is used to search for saprophytic microorganisms in samples of soil, vegetables, grains, air, insects, among others.

Czapek agar at acidic pH is used for the growth of acidophilic microorganisms, while modified Czapek agar is especially useful for the formation of chlamydospores in species of the Candida albicans complex .

Finally, Czapek-yeast agar is very useful for the isolation of Aspergillus and Penicillium.

Sown

-Agar Czapek

To search for Actinomycetes in the soil, proceed as follows: the sample is treated with phenolized water for 30 min and then 1 mL of the suspension is sown on Czapek agar. Incubate at 28 ° C for 5 to 7 days.

For vegetable, grain and insect samples they are placed directly on the agar. You can also make dilutions and seed 0.1 ml on the surface of the agar.

-Modified Czapek agar

For the seeding, a straight loop or needle is used, it is impregnated with the swab and the agar is inoculated by making a puncture until it touches the base of the plate. Incubate for 24 hours at 28 ° C and examine under a microscope above and below the plate to see if chlamydospores have formed.

Incubation temperature and time

The incubation temperature and time vary depending on the type of microorganism to be isolated. 
Plates are generally incubated for one to two weeks in a temperature range of 20 to 25 ° C.

However, there are specific conditions for some strains. For example, most species of the genus Aspergillus have optimal growth at 30 ° C; however A. fumigatus grows at 50 ° C.

Regarding the incubation time, one or two weeks is normal, however Candida albicans can develop in 24 to 48 hours at 25 ° C.

Characteristics of some colonies on this medium

-Penicillium atrovenetum

It develops in 12 days of incubation at 27 ° C. Their colonies are small (25 mm).

The colonies have a velvety appearance, with a defined white border and a blue-green hue in the center that turns gray as it ages. The reverse side of the colony is orange, making the middle the same color due to pigment production.

-Aspergillus niger

It develops with white to yellow mycelium and black spores.

-Candida albicans

Creamy light beige colonies develop. In modified Czapek agar it forms chlamydospores.

-Aspergillus parasiticus

A dark green mycelium develops in 7 days at 25 ° C.

QA

To evaluate the quality of the culture medium, certified control strains can be sown, such as: Aspergillus niger MKTA 16404, Candida albicans MKTA 10231.

Aspergillus niger  develops white to yellow mycelium and black spores in approximately 5 days, while Candida albicans will have satisfactory growth in 24 hours of incubation at 25 ° C; colonies are cream colored.

Limitations

Some studies have revealed that for the recovery of Actinomycetes the agar with the best yield is potato dextrose agar, with Czapek agar in second place, but for the recovery of Aspergillus and Penicillium, Czapek agar is the one that provides the highest recovery rate.

References

  1. Dávila M, et al. Antagonistic actinomycetes against phytopathogenic fungi of agricultural importance.  Rev. Mex. Science. Agríc  2013, 4 (8): 1187-1196. Available at: scielo.org.
  2. Luna M, Lozada Y, Trigos Á. Isolation of strains of Aspergillus niger , producing ochratoxin A, in stored green coffee (Coffea arabica). Rev. Mex. Mic. 2010; 32: 63-68. Available at.scielo.org.
  3. Wikipedia contributors. Czapek medium. Wikipedia, The Free Encyclopedia. January 13, 2019, 20:30 UTC. Available at: en.wikipedia.org
  4. Conda Pronadisa Laboratories. Modified Czapek-Dox Agar. Available at: condalab.com
  5. Culture Media Microkit. Czapek-Dox Agar. Diosponible in: Medioscultivo.com

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