Fungi are needed to be cultured in the laboratory for various purposes like identification of the causative agent associated with the pathology, for research purposes as well as for the productions of certain proteins and enzymes that are helpful in the fermentation of food, etc.
The simplest and most widely used artificial medium which is commonly used in the laboratories for the growth of fungi is Sabouraud Dextrose Agar (SDA) medium. These media provide all the basic components that are required by the fungal cell for the rapid growth in the laboratory.
The Sabouraud Dextrose Agar (SDA) medium was created by and is named after a French Physician Dr. Raymond Jacques Adrien Sabouraud in 1892. The pH of the medium was slightly acidic to support the growth of Fungi and inhibits the growth of many bacteria.
Sabouraud Dextrose Agar (SDA) Medium is a selective medium for fungal culture and primarily used for the isolation of Dermatophytes, Yeasts and various other Pathogenic and Non- pathogenic fungi as well as it also supports the growth of Nocardia spp. and other filamentous bacteria. Moreover, the slightly acidic pH of this SDA medium inhibits the growth of most of the bacteria and permits the growth of almost all the fungi with various modifications applicable.
In this article, I’m gonna explain the procedure of preparing Sabouraud Dextrose Agar (SDA) Medium in Laboratory
PRINCIPLE OF SABOURAUD DEXTROSE AGAR MEDIUM
Fungi, in contrast with bacteria, are routinely cultured on a solid medium i.e. Sabouraud Dextrose Agar Medium (SDA) to obtain the discrete colonies of the Fungi present in the specimen or to get the information about cultural characteristics of Fungi on a solid medium, colony morphology and patterns of growth etc. The Solid basal medium i.e. SDA medium, commonly used in Microbiology laboratory for the growth of fungi constitutes the 3 essential components –
Dextrose – The Dextrose present in the Sabouraud Dextrose Agar (SDA) Medium provides a rich source of carbohydrate for the rapid growth of the fungal cell.
Mycological Peptone – Mycological Peptone is a mixture of animal and plant peptones required for obtaining luxuriant growth of fungi- yeasts and moulds. It is light yellow in color and does not mask the appearance of media containing dyes and indicators, provides the rich source of protein to the Fungal cells for the rapid growth.
Agar-Agar – It is often called as Agar, is a complex polysaccharide, a carbohydrate consisting of 3, 6-anhydro-L-galactose and D-galactopyranose, free of nitrogen, produced from various red-purple algae belonging to Gelidium, Gracilaria, Gigastina etc. It liquefies on heating to 96 °C and hardens into a jelly on cooling 40-45 °C. It basically solidifies the medium and commonly used in the microbiology laboratory.
The final pH of the solutions is adjusted to 5.4 – 5.8 preferably the 5.6 at 25 °C. The above-mentioned components can be modified by adding the various substances in a variety of ways as per the requirements of the fungi so that the rapid and satisfactory growth of the fungal cell takes place.
The methodology explained in this article is based on HiMedia Labs Sabouraud Dextrose Agar (SDA) Medium which you can check here
Also, SDA medium is often modified by adding various antibiotics to suppress the growth of bacteria and promote the growth of fungal cells. Some of the modifications are described as follows:
MODIFICATIONS IN SABOURAUD DEXTROSE AGAR (SDA) MEDIA
The Sabouraud Dextrose Agar (SDA) Medium can be modified in no. of ways as per the requirements of the organisms and to prevent the growth of contaminants etc. The values described below are for the preparation of 1000 ml of media. However, the value may vary from lab to lab as per their Standard Operating Procedure (SOPs).
The liquid form of this medium i.e. Sabouraud Dextrose Broth consists of the same contents as described above except for agar. The final pH of the content is same as that of SDA medium i.e. 5.6 ± 0.2 at 25ºC.
Sabouraud Dextrose Agar with Antibiotic Chloramphenicol contains 50.0 mg of Chloramphenicol which inhibits the growth of bacteria. The final pH of the content is 5.6 ± 0.3 at 25ºC.
Sabouraud Dextrose Agar medium with Antibiotics Chloramphenicol and Gentamycin contains 50.0 mg (milligram) of Chloramphenicol and 5.0 mg (milligram) of Gentamycin that inhibits the growth of bacteria. The final pH of the content is 5.6 ± 0.3 at 25ºC.
Sabouraud Dextrose Agar medium with Antibiotics Chloramphenicol and Tetracycline contains 50.0 mg (milligram) of Chloramphenicol and 10.0 mg (milligram) of tetracycline which inhibits the growth of bacteria. The final pH of the content is 5.6 ± 0.3 at 25ºC.
Sabouraud Dextrose Agar medium with Cycloheximide is used whenever there is need to grow only Dermatophytes as Cycloheximide inhibits the growth of Non-Dermatophytes.
The Emmons modified Sabouraud Dextrose Agar medium, modified by Chester W. Emmons, has 20.0 gm of dextrose & 10.0 gram Neopeptone and the final pH of the content is adjusted to 7.0 ± 0.2 at 25ºC. The neutral formula of the agar and low dextrose concentration supports the growth of other microbes.
MATERIALS REQUIRED FOR PREPARATION OF SABOURAUD DEXTROSE AGAR MEDIUM
- Sterile Conical Flask / Erlenmeyer Flask
- Dextrose powder
- Mycological Peptone
- Agar-Agar powder
- Measuring Cylinder
- 1N HCl
- 1N NaOH
- pH Strip
- Weighing Scale
- Distilled Water
- Butter Paper
PROCEDURE FOR THE PREPARATION OF SABOURAUD DEXTROSE AGAR MEDIUM
⇒ Weigh the quantity of Mycological Peptone, Dextrose, and Agar using the weighing scale for 1000 ml of Sabouraud Dextrose Agar (SDA) as follows:
COMPONENTS QUANTITY (in grams)
Dextrose (Glucose) 40
Mycological Peptone 10
⇒ Put the butter paper on the weighing scale and transfer the required quantity of Mycological peptone on to the paper using a spatula. Repeat the step to obtain the required quantity of Dextrose and Agar-Agar powder.
Note: Use the separate piece of butter paper to avoid the errors in measurements. Also, you can use the simple peptone instead of Mycological peptone but the results may vary as Mycological peptone is specific for fungal growth.
Alternatively, You can use the commercially available SDA media powders and simply weigh the mixture of content as prescribed on the carton or box of the manufacturer. As per HiMedia, its 65 grams for 1000 ml medium.
⇒ Take a clean and dry Conical Flask/ Erlenmeyer flask.
Note: The size of the flask should be at least 1.5 times larger than the quantity of media you are preparing, for e.g. use 1500 or 2000 ml flask to prepare 1000ml of broth.
⇒ Pour 500 ml of distilled water to the flask and add the weighed quantity of Mycological Peptone, and Dextrose. Mix well the content by swirling the flask.
⇒ Now add the weighed quantity of Agar-Agar to the above solution.
⇒ Mix well the content and Heat it with continuous agitation to dissolve the constituents.
⇒ Now add more distilled water to the medium and make the volume 1000 ml.
⇒ Check the pH of the solution using pH strip, it should be 5.6 ±2. If required, adjust the pH by adding either 1N HCl (acid) or 1N NaOH (base) as per the case.
⇒ Mix well the content and apply the Non-absorbent cotton plug to the flask.
⇒ Autoclave the content at 121 °C and 15 psi pressure for 15 minutes.
⇒ Allow the content to cool down to 40-45 °C and pour in the empty media plates under a strict aseptic atmosphere (preferably in Laminar Air Flow) and allowed it to cool at room temperature.
⇒ Use the prepared media plates to inoculate the specimen to be cultured and then place in the incubator at optimum temperature.
PRECAUTIONS TO BE TAKEN WHILE PREPARING SABOURAUD DEXTROSE AGAR MEDIUM
⇒ Carefully measure the quantity of Mycological peptone, Dextrose, and Agar-Agar as per the quantity of medium to be prepared.
⇒ Maintain the pH of the medium carefully. A single extra drop can change the pH of the entire solution.
⇒ Store the Medium at low temperature in dust and contamination free environment for later use.
⇒ Acid and Alkali are corrosive to the skin, handle with care.