The Sabouraud agar, also known as Sabouraud dextrose agar, it is a solid culture medium, specially enriched for the isolation and development of fungi, such as yeasts, molds and dermatophytes.
Therefore, this medium cannot be lacking in a microbiology laboratory to investigate the presence of pathogenic or opportunistic fungi, either from clinical or non-clinical samples. Likewise, it is also ideal for the growth of filamentous bacteria such as Streptomyces and Nocardias. Its use is very wide, as it can be used in human, animal, plant and industrial mycology.
This medium was created in 1896 by the prominent dermatologist Raimond Sabouraud, who became a world-renowned specialist in disorders of the scalp, mainly caused by dermatophytes..
Its creation was so important that it has been used since then and remains today, although with some modifications.
Although it is special for fungi, bacteria can grow in this medium, therefore, for samples with mixed flora, the inclusion of antibiotics in their preparation is required and thus inhibit the growth of the bacterial flora that may be present..
The choice of the antibiotic must be made carefully and taking into account the type of fungus to be recovered, since some are inhibited in the presence of certain substances.
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Sabouraud dextrose agar is a medium that in its original formulation is weakly selective, due to its acidic pH of 5.6 ± 0.2, however bacteria can still develop, mainly in prolonged incubations.
The medium contains casein peptone and pancreatic digest of animal tissue, which provide the source of carbon and nitrogen for the growth of microorganisms..
It also contains a high concentration of glucose, which acts as an energy source, promoting the growth of fungi over bacteria. All mixed with agar-agar, a component that gives it the right consistency..
On the other hand, Sabouraud dextrose agar can be selective if antibiotics are added..
With antibiotics it is especially useful in samples of wounds, open ulcers or any sample in which great bacterial contamination is suspected.
-Sabouraud agar with chloramphenicol: ideal for recovering yeast and filamentous fungi.
-Sabouraud agar with gentamicin and chloramphenicol: almost all filamentous fungi and yeasts grow on this medium and inhibits a large number of bacteria, including Enterobacteriaceae, Pseudomonas and Staphylococcus.
-Sabouraud agar with cycloheximide: it is especially useful for samples from the skin or the respiratory tract, as long as the suspicion is dimorphic fungi.
Cycloheximide should be used with caution; Although it is used to inhibit the growth of non-pathogenic or environmental fungi and yeasts that may be present as contaminants in a sample, it also inhibits the growth of some fungi such as Cryptococcus neoformans, Aspergillus fumigatus, Allescheria boydii, Penicillium sp and other opportunistic fungi.
-Sabouraud agar with chloramphenicol plus cycloheximide: used primarily to isolate dermatophytes and dimorphic fungi. It has the disadvantage that it inhibits some species of opportunistic fungi such as Candida no albicans, Aspergillus, Zygomycetes or C. neoformans.
-Sabouraud agar with chloramphenicol, streptomycin, penicillin G and cycloheximide: it is ideal for samples extremely contaminated with bacteria and saprophytic fungi, but it has the disadvantage that it inhibits the growth of Actinomyces and Nocardias, in addition to the opportunistic fungi mentioned above.
If you have the ingredients separately, it can be prepared as follows:
Weigh:
- 40 gr dextrose
- 10 g of peptone
- 15 gr of agar-agar
- Measure 1000 ml of distilled water
All ingredients are mixed, the pH is adjusted to 5.6. The solutes are dissolved by boiling, 20 ml of the medium is distributed in tubes of 25 x 150 mm, without rim and preferably with a cotton cap..
Other tube sizes can also be used, depending on availability.
They are autoclaved for 10 minutes at an atmosphere of pressure (121 ° C). The autoclaving time should not be exceeded. When leaving the autoclave, the tubes are inclined with the help of a support until they solidify in a flute peak..
Another way is to dissolve the ingredients by heating until it boils. In the same flask, autoclave for 10 minutes and then distribute 20 ml in Petri dishes..
If you have a Sabouraud dextrose agar medium that already contains all the ingredients, proceed to weigh the amount specified by the company for one liter of water. The rest of the steps are the same as those described above.
Weigh:
- 20 gr dextrose
- 10 g of peptone
- 17 gr of agar-agar
- Measure 1000 ml of distilled water
All ingredients are mixed, the pH is adjusted to 6.9. Proceed in the same way as the previous case.
There are commercial houses that offer the medium with all the ingredients. In this case, weigh and prepare as described on the insert..
- Weigh 500 mg of chloramphenicol base
- Measure 100 ml of 95% ethanol
- Mix
Sabouraud dextrose agar medium (Emmons) is prepared as previously described and additionally for each liter of medium add 10 ml of chloramphenicol stock solution before autoclaving..
- Weigh 5 g of cycloheximide
- Measure 100 ml of acetone
- Mix
Sabouraud dextrose agar medium (Emmons) is prepared as previously described and additionally for each liter of medium add 10 ml of cycloheximide stock solution before autoclaving..
Sabouraud dextrose agar medium (Emmons) is prepared as previously described and additionally for each liter of medium add 10 ml of chloramphenicol stock solution and 10 ml of cycloheximide stock solution before autoclaving..
20,000 to 60,000 units of penicillin per liter of medium.
30 mg of streptomycin per liter of medium.
Both must be incorporated after the medium has been autoclaved, slightly cooled (50-55 ° C).
0.04 g of neomycin per liter of medium.
0.04 g of gentamicin per liter of medium.
For safety, it is preferred to sow Sabouraud dextrose agar in wedge-shaped tubes (inclined in flute beak) rather than in Petri dishes, to avoid dispersion and inhalation of spores.
It is important that Sabouraud agar tubes are covered with cotton and not with a screw cap, as semi-anaerobic conditions have been shown to inhibit spore formation in some strains, for example Coccidioides immitis. Also, most fungi are aerobic..
In case of using screw cap, do not close hermetically.
The prepared media should undergo quality control to verify its proper functioning. For this, certain control strains are sown.
For Sabouraud dextrose agar with chloramphenicol, ATCC strains of Candida albicans, which must have excellent growth. Another plate is inoculated with strains Escherichia coli, must be completely inhibited.
An uninoculated plate is also incubated in which no microorganisms should grow..
For Sabouraud dextrose agar with chloramphenicol and cycloheximide, strains of Trichophyton mentagrophytes, should develop well. Another plate is inoculated with a strain of Aspergillus flavus, in which there must be little or no growth. Additionally, an uninoculated plate is incubated to demonstrate its sterility..
For Sabouraud dextrose agar with cycloheximide, strains of Candida albicans, Trichophyton rubrum or Microsporum canis, which must show good growth.
Likewise, a strain of Aspergillus flavus, showing little or no growth. Finally, incubate an uninoculated plate for sterility control..
Classic Sabouraud dextrose agar contains 4 grams of dextrose and is excellent as a primary isolation medium, as it shows the characteristic morphology of each fungus..
It is also excellent for demonstrating pigment production. However, it is not the most suitable means of observing sporulation..
It is also not recommended for growing Blastomyces dermatitidis, which is inhibited by the high concentration of glucose present.
On the other hand, for cultivation certain considerations must be taken into account.
Some fungi grow best at room temperature, like molds, others grow successfully at 37 ° C, like some yeasts, and others can grow at both temperatures (dimorphic fungi).
For this reason, it is sometimes necessary to use several Sabouraud agar plates for the same sample, since duplicate seeding is often performed to incubate one plate at room temperature and another at 37 ° C..
For example, Sporothrix schenckii it is sown in two plates; one is incubated at room temperature to obtain the mold phase and the other is incubated at 37 ° C to obtain the yeast phase, but in the latter it is necessary to add 5% of blood to the medium.
In other cases, such as mycetoma samples, two Sabouraud agar plates are seeded, one with chloramphenicol and the other with cycloheximide. The first will allow the growth of mycetoma causative agents of fungal origin (Eumycetoma) and the second causative agents of mycetoma of bacterial origin, such as actinomycetomas.
Sabouraud dextrose agar modified by Emmons contains 2 grams of dextrose and is not only used for isolation, but also for sporulation and preservation of fungi.
In this medium, if strains of Blastomyces dermatitidis.
To preserve mushroom cultures, they can be stored in a refrigerator (2-8 ° C). The conservation time can vary between 2 to 8 weeks. After this time they must be subcultured to repeat the process.
Some mushrooms keep best at room temperature, such as Epidermophyton foccosum, Trichophyton schoenleinnii, T. violaceum Y Microsporum audounii.
The maintenance of the strain can be lengthened to avoid pleomorphism if the dextrose is completely eliminated from the agar and if the amount of agar in the medium is reduced to avoid dryness.
For the identification of some filamentous fungi, it is necessary to carry out microcultures using Sabouraud agar or other special means to observe the structures of sexual and asexual reproduction..
It is used mainly for the diagnosis of fungal diseases, especially those that affect the skin and its attachments (hair and nails).
The samples can be secretions, exudates, skin, hair, nails, sputum, CSF or urine. Commonly isolated pathogens are dermatophytes, fungi that cause subcutaneous and systemic mycoses..
Animals are frequently affected by fungal infections, therefore Sabouraud agar is as useful in animal mycology as it is in humans..
For example, dermatophytes can often affect animals. Such is the case of Microsporum canis var distortum, It frequently infects dogs, cats, horses, pigs, and monkeys. Likewise, Microsporum gypseum infects dogs, cats, and livestock.
Birds such as chickens, roosters and chickens are affected by Microsporum gallinae.
Other fungi, such as Zymonema farciminosum, They are also a cause of disease in animals, mainly in horses, mules and donkeys, producing significant inflammation in the lymphatic vessels.
Sporothrix schenkii and Histoplasma capsulatum affect domestic animals and humans.
Many pathogenic or opportunistic fungi can concentrate at any given time in a given environment, especially in operating rooms and Intensive Care Units (ICUs) of clinics and hospitals. Therefore it is necessary to carry out a control of them.
Other vulnerable spaces are libraries and old buildings, which can be affected by the concentration of environmental fungi.
In environmental studies, Sabouraud dextrose agar is used for the isolation of fungi..
Sabouraud dextrose agar cannot be absent for the study of contaminating fungi in the production of cosmetics, food, beverages, leather, textiles, among others..
Plants also suffer from diseases caused by fungi, affecting different parts of the plant, which can even end the harvest, causing great losses in agriculture..
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