The potato dextrose agar It is a solid, non-selective nutritive culture medium. Bacterial and fungal species can grow in it, but its use is especially indicated for the isolation of filamentous fungi and yeasts. It is also known as PDA medium for the English expression Potato Dextrose Agar.
It is particularly useful for the isolation of phytopathogenic fungi, that is, those that affect plants. To sow the samples from infected vegetables, other means such as Sabouraud agar or malta-agar can be used, however, for routine use, potato dextrose agar is preferred due to obtaining greater sporulation..
It is also used for the counting of fungal colonies in samples of cosmetics, pharmaceutical products and some dairy foods. Likewise, it is suitable for sowing samples of skin scrapings in search of dermatophytes, which grow very well in this medium, developing their characteristic pigments..
Potato dextrose medium is a very simple and easy medium to prepare in the laboratory. Contains, as its name implies, infusion of potato, dextrose and agar-agar. Additionally, inhibitory substances can be added to prevent bacterial growth and increase the selectivity for fungal species..
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Potato dextrose agar is a culture medium that provides the nutritional elements necessary for the development of filamentous fungi and yeasts..
The combination of the infusion of potato with glucose provides the perfect energy source for a satisfactory growth of fungi. While the agar is the one that provides the consistency to the medium.
The medium by itself does not inhibit the growth of bacteria, therefore it is a non-selective medium. To make it selective, it needs the addition of inhibitory substances such as tartaric acid or antibiotics..
It is prepared as follows:
In the first place, the potatoes are washed very well, removing the soil they possess. They are cut into thin slices with everything and shell. 200 g of potatoes are weighed and boiled in a liter of distilled water for half an hour.
At the end of the time, filter or strain all the preparation through a cheesecloth.
The liquid obtained is completed with distilled water until reaching one liter. Add 20 g of agar-agar and 20 g of dextrose to the infusion, mix well and autoclave at 121 ° C, at 15 pounds of pressure for 15 minutes..
Allow to cool to 50 ° C and serve in sterile Petri dishes. The prepared plates are stored in a refrigerator..
Potato dextrose agar wedges can also be prepared..
In this case, before sterilizing in the autoclave, 12 to 15 ml of the medium are placed in tubes, later they are autoclaved and when leaving they lie on special supports until it solidifies. Keep in fridge.
The medium remains at a pH of 5.6 ± 0.2, however, some laboratories add 10% tartaric acid to lower the pH to 3.1 ± 0.1 in order to inhibit bacterial growth..
In this same sense, other laboratories prefer to add antibiotics to make it selective for the cultivation of fungi and prevent bacterial development..
Weigh out 39 g of the commercially available dehydrated medium and dissolve in one liter of distilled water. Let stand for 5 minutes.
The mixture is heated with frequent stirring until completely dissolved. Subsequently, it is sterilized in an autoclave at 121 ° C for 15 minutes..
Plates or wedges can be prepared. Proceed as described above.
The pH remains at 5.6 ± 0.2. If a pH of 3.1 is desired, add 14 ml of sterile 20% tartaric acid before serving on the plates..
The raw medium is beige and the prepared medium is light amber with a slightly cloudy or opalescent appearance..
The leaves are cut into pieces.
In a 50 cc glass with 50% alcohol, place the pieces of the leaves (stained and healthy pieces), to disinfect the surface for 20 to 30 seconds. Throw away the alcohol and add 20% sodium hypochlorite for 40 to 50 seconds if they are thin leaves and increase the time to 80 seconds if it is bark and logs.
Discard the sodium hypochlorite and take the disinfected pieces with a sterile forceps and place them on the surface of the medium (a maximum of 10 pieces). Date and incubate at 20-30 ° C.
If the fruit is fleshy, open the fruit affected by the fungus and take pieces with a sterile scalpel, both from the diseased and healthy parts and place them on the surface of the agar.
If the fruit is citrus, such as a lemon or an orange, it must be opened and its seeds sown.
When the surface of the fruit is affected and spores are observed, the ideal is to use the grating method on the plate; This consists of touching the spores with a sterilized and cooled “L” -shaped spatula, and then making a zigzag seeding 2 to 3 times on the agar.
They are disinfected as described in the leaves and later placed on the agar.
A scraping of the bark is made and later pieces of the healthy and diseased part are taken and sown directly on the agar.
The seeded plates are incubated aerobically at 20-30 ° C for 72 hours..
The sample should be taken using a No. 11 scalpel blade, either to cut affected hair, skin scales or nails in search of dermatophytes. Before taking the sample, the area must be disinfected with 70% alcohol..
In scaly lesions, the edge of the lesion should be scraped, as the fungus is more likely to be found there.
In exudative lesions, the sample is taken with a dry or wet swab. Sow immediately on potato dextrose agar or Sabouraud agar. Avoid means of transportation.
Another method of sampling is through the carpet square technique of Mariat and Adan Campos. In this case, the affected area is rubbed 5 times with a piece of sterile wool for later cultivation..
The sample can be placed directly into the culture medium.
Depending on the pathology, the affected part can be cut off or uprooted. Place the sample in the culture medium.
A specific part of the affected nail can be scraped or cut. It will depend on the type of injury.
Cut the sample into 1 mm pieces before sowing in order to increase the probability of contact of the fungus with the culture medium..
The colonies obtained in the plate are isolated in tubes containing potato dextrose agar to carry out the macroscopic study of the colonies (appearance, color, consistency, degree of development.
The microscopic study (observation of structures and their formations) can be done by microcultures or direct observation under the microscope between lamina and lamella..
This medium can also be used to determine the fungal and yeast load present in plant, food, cosmetic or drug samples. For this purpose, potato dextrose agar supplemented with antibiotics is used, such as: (chloramphenicol, chlorotetracycline or both).
Pour 1 ml of the sample - preferably diluted - into a sterile and empty Petri dish, then melt a plug of potato dextrose agar and allow to cool to 45 ° C. Pour onto the Petri dish and rotate until homogenized. Let stand until solid.
Incubate aerobically at 20-25 ° C (molds) or 30-32 ° C (yeasts) for 5 to 7 days or more, depending on the type of fungus sought and the type of sample. Two plates can be used to incubate in both temperature ranges.
Potato Dextrose Agar can be used to maintain viable fungal strains for several years.
To do this, the fungus is grown in wedges of potato dextrose agar and once the fungus has grown, it is covered with mineral oil. The oil should be sterilized in an autoclave for 45 minutes, and have a viscosity of approximately 300 to 330 Saybolt. The oil should be 1 to 2 cm above the tip of the bevel.
From each batch prepared, 1 or 2 plates should be taken and incubated at 25 ° C for 48 hours or at 20 ° C for 96 hours. A good sterility control is one in which colony development is not observed..
Known or certified control strains such as:
Saccharomyces cerevisiae ATCC 9763, Candida albicans ATCC 10231, Aspergillus brasiliensis ATCC 16404, Trichophyton mentagrophytes ATCC 9533. Good growth is expected in all cases.
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