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 artificial liquid medium which is commonly used in the laboratories for the growth of fungi especially the Candida species is Sabouraud Dextrose Broth 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 Broth medium which is similar to the well known solid medium for fungal growth that is Sabouraud Dextrose Agar (SDA) Medium except for solidifying agent i.e. Agar-Agar that was created by and 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.
The Sabouraud Dextrose Broth 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 and especially used for the isolation of Candida species. Moreover, the slightly acidic pH of this Sabouraud Dextrose Broth 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 Broth Medium in Laboratory
PRINCIPLE OF SABOURAUD DEXTROSE BROTH MEDIUM
Fungi, in contrast with bacteria, are routinely cultured in a medium that is rich in sugar and protein content and Sabouraud Dextrose Broth medium is such a medium that particularly supports the growth of fungi. The culture of fungi is commonly done to get the information about cultural characteristics of Fungi in a liquid medium, colony morphology and patterns of growth etc.
The Liquid basal medium for the fungus culture i.e. Sabouraud Dextrose Broth medium, commonly used in Microbiology laboratory for the growth of fungi constitutes only 2 essential components –
Dextrose – The Dextrose present in the Sabouraud Dextrose Broth Medium provides a rich source of carbohydrate for the rapid growth of the fungal cells.
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.
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 Broth Medium which you can check here
Also, Sabouraud Dextrose Broth 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 BROTH MEDIA
The Sabouraud Dextrose Broth 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) and the Results required.
The solid form of this medium i.e. Sabouraud Dextrose Agar (SDA) consists of the same contents as described above and a solidifying agent that is Agar-Agar which makes it a solid basal medium for the cultivation of fungi in the laboratory. The final pH of the content is same as that of Sabouraud Dextrose Broth medium i.e. 5.6 ± 0.2 at 25ºC.
Sabouraud Dextrose Broth Medium with Antibiotic Chloramphenicol contains 50.0 mg (milligram) of Chloramphenicol which inhibits the growth of bacteria. The final pH of the content is 5.6 ± 0.3 at 25ºC.
Sabouraud Dextrose Broth 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 Broth 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 Broth medium with Cycloheximide is used whenever there is need to grow only Dermatophytes as Cycloheximide inhibits the growth of Non-Dermatophytes.
The Modified Sabouraud Dextrose Broth medium is the 5% solution of the broth medium and has 40.0 gm of dextrose & 10.0 gram Polypeptone and the final pH of the content is adjusted to 5.6 ± 0.2 at 25ºC. The high dextrose concentration and Polypeptone content of the modified medium enhances the cultivation of fungi and is commonly used for the cultivation of fungi in the cosmetic products.
REQUIREMENTS FOR PREPARING SABOURAUD DEXTROSE BROTH MEDIUM
- Sterile Conical Flask / Erlenmeyer Flask
- Dextrose powder
- Mycological Peptone
- Measuring Cylinder
- 1N HCl
- 1N NaOH
- pH Strip
- Weighing Scale
- Distilled Water
- Butter Paper
PROCEDURE FOR PREPARING SABOURAUD DEXTROSE BROTH MEDIUM
Weigh the quantity of Mycological Peptone, and Dextrose using the weighing scale for 1000 ml of Sabouraud Dextrose Broth 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 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 Sabouraud Dextrose Broth medium powder and simply weigh the mixture of content as prescribed on the carton or box of the manufacturer. As per HiMedia, its 30 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 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.
⇒ Now Pour about 5-10 ml of the broth medium in Test tubes and apply the Non-absorbent cotton plug to all the tubes.
⇒ Autoclave the content at 121 °C and 15 psi pressure for 15 minutes.
⇒ Allow the content to cool down and then use the prepared broth tubes to inoculate the specimen to be cultured under a strict aseptic atmosphere (preferably in Laminar Air Flow) and then place in the incubator at optimum temperature.
PRECAUTIONS TO BE TAKEN WHILE PREPARING SABOURAUD DEXTROSE BROTH MEDIUM
⇒ Carefully measure the quantity of Mycological peptone and Dextrose 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.