Introduction of Fungal Culture Media

Fungal Culture Media are very important for the isolation of fungal etiological agents from clinical specimens in the Clinical Mycology Laboratory and their detailed study. The common fungal culture media are Sabouraud Dextrose Agar (SDA), Potato Dextrose Agar (PDA), Corn Meal Agar (CMA), Dermatophyte Test Medium (DTM), Czapek Dox Agar, Ascospore Agar, and Bird Seed Agar. The introduction, Principle, Preparation, Test Procedure, Colony Morphology, Result-Interpretation, and Keynotes of those fungal culture media are described below detailed.

Introduction of SDA

SDA stands for Sabouraud Dextrose Agar and  Sabouraud is the surname of creator Raymond Jacques Adrien Sabouraud a French physician born in Nantes. He formulated SDA in 1892 for culturing dermatophytes. SDA  is the most common fungal medium to recover the growth of fungi in Mycology. It is different from bacterial medium due to having main two properties, comparatively low pH (5.6) and high concentration of sugar. It is useful for the isolation, cultivation, and maintenance of non-pathogenic and pathogenic species of fungi ( yeasts and molds). The pH is adjusted to approximately 5.6 in order to enhance the growth of fungi, especially dermatophytes, and to slightly inhibit bacterial growth in clinical specimens.

Principle of SDA 

Carlier’s modification of SDA contains ingredients like dextrose, mycological, peptone, agar, and pH 5.6. Mycological peptone provides nitrogen and vitamin source required for organisms in this medium. Dextrose provides an energy source while agar acts as a solidifying agent. High dextrose concentration and low pH favor fungal growth and inhibit contaminating bacteria from test specimens. This modification is useful for the cultivation of fungi (yeasts, molds), particularly useful for the fungi associated with skin infections. This medium is also employed to determine microbial contamination in food, cosmetics, and clinical samples.

Composition of SDA

Sabouraud  original formulation

• Peptone:10 g
• Glucose: 40 g
• Agar: 15 g
• Distilled Water (D/W): 1000 ml

Note:  The acidic pH of traditional Sabouraud agar inhibits bacterial growth.

Emmon’s modification

• Neo-peptone: 10 g
• Glucose: 20 g
• Agar: 20 g
• Distilled Water (D/W): 1000 ml

Note: Shifting pH towards neutral and lowering the concentration of sugar of the Emmon’s modification enhances to support the growth of other microorganisms and some pathogenic fungi also, such as dermatophytes.

Carlier’s modification

Ingredients                         Gms / Litre
Dextrose (Glucose):       40.000
Mycological, peptone:  10.000
Agar:                                       15.000
Final pH ( at 25°C:          5.6±0.2

Note: High sugar (dextrose) concentration and low pH(5.6) favor fungal growth and inhibit contaminating bacteria from test specimens.

Modified SDA with the incorporation of antimicrobial agents

(Gentamicin, chloramphenicol, tetracycline, cycloheximide)

1. SDA with chloramphenicol contains 50.0 mg of chloramphenicol and the final pH of the medium should be 5.6 +/- 0.3 at 25ºC. Chloramphenicol inhibits a wide range of gram-positive negative bacteria.
2. SDA with chloramphenicol and gentamicin contains 50.0 mg of chloramphenicol and 5.0 mg of gentamicin. The final pH of the medium should be  5.6 +/- 0.3 at 25ºC. Chloramphenicol inhibits a wide range of gram-positive negative bacteria whereas gentamicin inhibits gram-negative bacteria.
3. SDA with chloramphenicol and tetracycline contains 50.0 mg of chloramphenicol and 10.0 mg of tetracycline. The final pH of the medium should be 5.6 +/- 0.3 at 25ºC. Chloramphenicol inhibits a wide range of both gram-positive and negative bacteria while tetracycline inhibits a wide range of microorganisms including gram-positive and gram-negative bacteria, chlamydiae, mycoplasmas, rickettsiae, and protozoan parasites.
4. SDA with cycloheximide: Cycloheximide inhibits primarily saprophytic fungi but not dermatophytes or yeasts. It is incorporated as an amount of 0.5 g/liter.

Note: Antimicrobial agents should only be added after media has been autoclaved and then cooled to 45 to 50°C. Keep all plates at 4°C until they are used, regardless of whether they contain antibiotics.

Preparation of SDA various modifications

SDA original (Sabouraud formulation)

1. Add all ingredients in nearly 900 ml of distilled water or deionized water.
2. Adjust pH to 5.6 with hydrochloric acid.
3. Then adjust the final volume to 1 liter.
4. Autoclave 20 minutes at 121°C, 15 lbs.
5.  Cool to 45 to 50°C and pour into Petri plates or tubes for slants.

Note: The addition of an antimicrobial agent, chloramphenicol is optional and its use depends on the user’s preference. If desired add 5 mg to the above recipe.

Emmons modification SDA

• Add all ingredients in nearly 900 ml of distilled water.
• Adjust pH to 6.8 to 7.0 with hydrochloric acid (HCl).
• Then adjust the final volume to 1 liter.
• Sterilize by autoclaving 20 minutes at 121°C, 15 lbs.
•  Cool to 45 to 50°C and pour into Petri plates or tubes for slants.

Carlier’s modification of SDA

1. Suspend 65.0 grams in 1000 ml distilled water.
2. Heat to boiling to dissolve the medium completely.
3. Autoclave at 15 lbs pressure (121°C) for 15 minutes.
4. Cool to 45-50°C.
5. Mix well and pour into sterile Petri plates or test tubes.
6. Store prepared SDA plates or tubes at 2-8°C until any defects appear on them.

Requirements for testing SDA

• SDA
• Suspected fungal specimen
• Quality control strains ( as positive controls: Positive controls:
  Candida albicans ATCC® 10231 and Aspergillus brasiliensis ATCC® 16404 while as negative control uninoculated medium)
• Incubator/s
• Biological safety cabinet (BSC)
• Inoculating loop  or wire
• Bunsen burner
• Personal protective equipment (PPE) as required

Test procedure

1. Inoculate the specimens on SDA.
2. Incubate the medium ( according to the required temperature and time depending on the nature of fungi to be recovered e.g. as you know, dimorphic fungi occur in two forms-yeast forms ( parasitic phase) and mold ( spores and filamentous form). Typically, molds are incubated at room temperature (22 to 25°C) and yeasts are incubated at 37°C if suspected of being dimorphic fungi. Incubation times will vary, from approximately 2 days for the growth of yeast colonies such as Malassezia species to 2 to 4 weeks for the growth of dermatophytes ( Trichophyton, Microsporum, and Epidermophyton) or dimorphic fungi such as Histoplasma capsulatum. Indeed, the incubation time required to acquire fungal growth is one of the  diagnostic tools used to identify or confirm fungal species.)
3. Observe for fungal growth.

Result Interpretation of fungal growth on Sabouraud Dextrose Agar

• Yeasts: Creamy to white colonies
• Molds: Filamentous colonies of various color
• Positive controls: Candida albicans: Luxuriant
  (white colonies)
• Aspergillus brasiliensis : White mycelium; black spores
• Negative control:  No change in Uninoculated medium( SDA)

Uses of SDA

1. SDA is recommended for the cultivation of yeasts, molds, and aciduric bacteria from clinical samples.
2. The medium with the addition of antimicrobial agents (antibiotics) makes it more selective for the isolation of pathogenic fungi from material containing large numbers of microbial load i.e. other fungi or bacteria.
3. This medium is also applicable to determine microbial contamination in food and cosmetics specimens.

Limitations of SDA

1. For heavily contaminated specimens, the plate must be supplemented with inhibitory agents for inhibiting bacterial growth with lower pH.
2. Avoid overheating the SDA medium during preparation with an acidic pH, this may result in a soft medium
3. SDA is not recommended as a primary isolation medium because it is insufficiently rich to recover certain fastidious pathogenic species, particularly most of the dimorphic fungi like e.g. Blastomyces dermatitidis, coccidioides immitis, Histoplasma capsulatum.
4.  Some pathogenic fungi may produce infective spores which are easily dispersed in the air, so examination should be carried out in the safety cabinet.
5. It does not promote the conidiation of filamentous fungi.
6. Further biochemical or serological tests should be performed for confirmation
7. For identification, organisms must be in pure culture.
8. Antibiotics added into a medium to inhibit bacteria may also inhibit certain pathogenic fungi.

Keynotes of Sabouraud Dextrose Agar

• Colony characteristics,  microscopic structures, rate of growth, media which support the growth of the organism, and source of the specimen are very helpful for the isolation of fungi.
• A variety of biochemical tests are available for the identification of yeasts.
• Sabouraud Dextrose Broth (SDB) is the same formulation as SDA, without agar.

Colony Characteristics of various fungi ( yeasts and molds) are given below-

Sporothrix schenckii growth on SDA

Acremonium on SDA and LPCB preparation

Aspergillus fumigatus Colony on SDA, LPCB tease mount under microscopy

Introduction of Potato Dextrose Agar (PDA)

Potato Dextrose Agar ( PDA) is a fungal medium and its ingredients are clear from its name it contains potato ( vegetable), dextrose ( sugar), and agar ( solidifying agent). PDA  is recommended for the isolation and enumeration of fungi (yeasts and molds) from water, dairy, other food products, and even clinical specimens ( skin scrappings). The nutritionally rich base, potato infusion encourages mold sporulation and pigment production in some dermatophytes (e.g.Trichophyton rubrum).

Principle of Potato Dextrose Agar (PDA)

Potato dextrose agar (PDA) contains dehydrated Potato infusions and Dextrose. Potato infusion provides a nutrient base for the luxuriant growth of most fungi whereas dextrose serves as a growth stimulant. Agar in the medium acts as the solidifying agent. PDA has been further modified by incorporating agents like tartaric acid, chloramphenicol, and chlortetracycline. The incorporation of tartaric acid (TA) in the medium lowers the pH to 3.5 which inhibits bacterial growth. Chloramphenicol acts as a selective agent to inhibit the bacterial overgrowth of competing microorganisms from mixed specimens while permitting the selective isolation of fungi. The application of chlortetracycline in PDA is for the evaluation of yeast and mold from cosmetic products.

Composition of Potato Dextrose Agar (PDA)

Potato dextrose agar ( PDA) ingredients and their amounts are as follows-

Ingredients                        Gms / Litre

• Potatoes, infusion from:  200.0
• Dextrose: 20.0
• Agar: 15.0
• Distilled water(D/W): 1000 ml
  Final pH ( at 25°C) 5.6±0.2

Preparation of Potato Dextrose Agar ( PDA) 

1. Suspend 39.0 grams of potato dextrose agar ( PDA) in 1 liter of purified/distilled or deionized water.
2. Heat to boiling to dissolve the medium completely.
3. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
4. After autoclaving,  leave for cooling to 45-50°C.
5. Note: In specific work, when pH 3.5 is required, acidify the medium with sterile 10% tartaric acid. The amount of acid required for 100 ml of sterile, cooled medium is approximately 1 ml, and do not heat the medium again after the addition of the acid.
6. Mix well before dispensing.
7. Pour Potato Dextrose Agar into each plate and leave plates on the sterile surface until the agar has solidified.
8. Store the plates in a refrigerator at 2-8°C.

Storage and Shelf life of Potato Dextrose Agar ( PDA) 

• Store at 2-8ºC  and away from direct light.
• Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), or contamination.
• The product is light and temperature-sensitive; protects from light, excessive heat, moisture, and freezing.

Test Requirements

• Test specimens ( samples or fungal growth)
• Inoculating loop
• Bunsen burner
• Incubator
• Control strains (Candida albicans ATCC ® 10231 and Trichophyton rubrum
  ATCC ® 28188)

Test procedure (specimen/organism inoculation)

1. Allow the plates to warm at 37°C or to room temperature, and the agar surface to dry before inoculating.
2. Take two plates for one specimen.
3. Inoculate and streak the specimen as soon as possible after collection.
4. If the specimen to be cultured is on a swab, roll the swab over a small area of the agar surface.
5. Streak for isolation with a sterile loop.
6. Incubate one plate aerobically at room  temperature ( 20-25ºC) and another 35-37ºC for up to 4 weeks ( depending on the type of organisms suspected)
7. Examine colony characteristics.

Result Interpretation  of PDA

• Control strains i.e. Candida albicans ATCC ® 10231 ( rapid grower)  and Trichophyton rubrum ( slow grower) ATCC ® 28188): Presence of growth
• Presence of fungi in specimen: Presence of  growth on PDA

Colony Characteristics of various organisms in PDA

Candida albicans: Growth; smooth white colonies after 24-48 hours

Trichophyton rubrum: Growth is seen in 7 days and it may take 3-4 weeks for the red color on the reverse side of the colony to be visible.

Modification of PDA

1. Potato Dextrose Agar with TA (Tartaric Acid): It uses for the microbial examination of food and dairy products.
2. Potato Dextrose Agar with Chlortetracycline: It uses is for the microbial enumeration of yeast and mold from cosmetics.
3. Potato Dextrose Agar with Chloramphenicol: It uses for the selective cultivation of fungi from mixed samples.

Uses of Potato Dextrose Agar ( PDA) 

1. PDA uses for the detection of fungi from dairy products, prepared foods as well as water samples.
2. It also uses for the cultivation of fungi (yeasts and molds) from clinical specimens.
3. This medium is also applicable for pigment expression as well as sporulation.
4. Various modified PDAs and their applications are as follows-PDA TA is used for the microbial examination of food and dairy products while PDA with chlortetracycline uses are for the microbial enumeration of yeast and mold from cosmetics. PDA with chloramphenicol uses for the selective cultivation of fungi from mixed samples.

Keynotes on PDA

1. Heating the medium after acidification (incorporation of tartaric acid) should be avoided as it may hydrolyze the agar which can render the agar unable to solidify.
2. 4.0gm of potato extract is equivalent to 200 gm of potato infusion.
3. The original potato dextrose agar not only supports the growth of fungi but also some acidic bacteria and thus its modifications are preferred.
4. The amount of chlortetracycline 40.0 mg, chloramphenicol 25.0 mg, and tartaric acid 1.4 gm can use in 1000 ml of PDA in modified forms additionally.

Introduction of Corn Meal Agar 

Corn Meal Agar (CMA) is a well-established fungal medium that is a suitable substrate for chlamydospore production by Candida albicans and the maintenance of fungal stock cultures. This is a very simple formulation containing only cornmeal infusion and agar. The addition of glucose (0.2 g% w/v) to CMA will enhance the chromogenesis of some species of Trichophyton e.g. Trichophyton rubrum. Cornmeal agar is an enrichment medium developed by Hazen and Reed for use in the cultivation of fungi. Walker and Huppert, in 1960, found that the addition of Tween 80 to CMA resulted in rapid and abundant chlamydospore formation.

Principle of Corn Meal Agar

The medium consists of only cornmeal infusion and agar which is a very simple formulation. However, this infusion has enough nutrients to enhance the growth of fungi. Agar acts as a solidifying agent that provides a solid physical form. Cornmeal agar with tween 80, tween 80, a mixture of oleic esters, stimulates the production of chlamydospores by C. albicans, C. stellatoidea, and occasionally C. tropicalis and is also useful for the maintenance of fungal stock cultures.

Composition of Corn Meal Agar 

           (Hardy Diagnostics)

Corn Meal Agar (CMA) ingredients and their amounts are as follows-

Ingredients                        Gms / Litre

• Corn Meal Infusion: 2.0gm
• Tween  80: 7.0ml
• Agar: 15.0
• Distilled water(D/W): 1000 ml
  Final pH 6.2 +/- 0.3 at 25ºC.

Preparation of Corn Meal Agar 

1. Suspend 17.0 grams of Corn Meal Agar (CMA) in 1 liter of purified/distilled or deionized water.
2. Heat to boiling to dissolve the medium completely.
3. Add 7 ml Tween 80.
4. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
5. After autoclaving,  leave for cooling to 45-50°C.
6. Mix well before dispensing.
7. Pour Corn Meal Agar into each plate and leave plates on the sterile surface until the agar has solidified.
8. Store the plates in a refrigerator at 2-8°C.

Storage and Shelf life of Corn Meal Agar 

• Store at 2-8ºC  and away from direct light.
• Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), or contamination.
• The product is light and temperature-sensitive; protects from light, excessive heat, moisture, and freezing.

Test Requirements

• Test specimens ( samples or fungal growth)
• Inoculating loop
• Bunsen burner
• Incubator
• Control strains (Candida albicans ATCC® 10231 and Candida krusei ATCC 6258)
• Biological Safety Cabinet (BSC)

Test procedure

1. Allow the plates to warm at 37°C or to room temperature, and the agar surface to dry before inoculating.
2. Take plates.
3. Using an inoculating needle, obtain the visible paste of the organism. Draw the needle through the agar making two perpendicular lines in the shape of an “x”.
4. Flame a coverslip and allow it to cool. Once the coverslip has cooled, place it over the central area of the “x” in order to reduce oxygen tension. Reduced oxygen tension stimulates chlamydospore production. Leave some growth uncovered.
5. Seal the plate with tape or MycoSeals and incubate aerobically at room temperature (25-30ºC) for up to 72 hours in the dark. Examine daily for typical colonial growth and morphology.
6. Invert the plate and examine microscopically using a low-power objective (10 X). View along the edge of the coverslip for detection of chlamydospore formation.
7.  Follow steps one through six of the above procedure for the examination of the sporulation of molds. Incubate until the mold is visible and mount the coverslip in glycine.
8. Examine microscopically for characteristic structures.

Result Interpretation  of CMA

Using low-power magnification, examine for the presence of budding cells, hyphae, blastospores, and chlamydospores. Most strains of C. albicans and C. stellatoidea form typical chlamydospores after 24-48 hours of incubation. Examine daily for up to four days. C. dubliniensis will also form chlamydospores, but in clusters, rather than singles as with the C. albicans. Control strains, Candida albicans: Good growth; white colonies and chlamydospores while Candida krusei: Good growth; white / cream colonies, no chlamydospores.

Colony Characteristics of various organisms in CMA

Candida albicans: Growth; smooth white colonies after 24-48 hours

Trichophyton rubrum: Growth is seen in 7 days and it may take 3-4 weeks for the red color on the reverse side of the colony to be visible.

Uses of Corn Meal Agar

1. Corn Meal Agar (CMA) is a well-established fungal medium for chlamydospore production by Candida species.
2. It is also useful for the cultivation of fungi.
3. Corn meal agar is a nutritionally impoverished medium and thus it is useful for the maintenance of stock cultures of fungi, especially the black-pigmented varieties.
4. The addition of glucose (0.2 g% w/v) to CMA is applicable for the chromogenesis of some species of Trichophyton e.g. Trichophyton rubrum.

Keynotes on Corn Meal Agar

1. The addition of `Tween 80’ (e.g. 1%) to Corn Meal Agar greatly enhances the development of chlamydospores on the medium
2. Mackenzie found that all 163 isolates of Candida albicans obtained from laboratories in the United Kingdom produced chlamydospores on Oxoid Corn Meal Agar but Dawson using only 27 isolates of Candida albicans, found that Oxoid Czapek Dox Agar and rice infusion agar were slightly superior for chlamydospore production
3. A single Petri dish containing Corn Meal Agar may be used to identify four or five different colonies of Candida grown on Sabouraud Dextrose Agar. Using a straight wire, pick a colony off the surface of the latter medium and make a deep cut in the Corn Meal Agar (i.e. a horizontal furrow). Repeat for each colony. Place a flamed sterile coverslip over the line of inoculum. After incubation for 24 to 48 hours at 22°C, the streaks are examined microscopically, through the coverslip, using a low-power objective. Along with such streaks, Candida albicans produces mycelium-bearing ball-like clusters of budding cells and the characteristic thick-walled round chlamydospores.
4. Glucose-supplemented Corn Meal Agar should not be used for chlamydospore production.
5. Corn Meal Agar with `Tween 80’ (or other wetting agents) will allow Candida stellatoides and Candida tropicalis to produce chlamydospores.
6. Some Candida strains lose their ability to produce chlamydospores after repeated subculturing.

Limitations of Corn Meal Agar

• Recommended temperature is 25ºC for the best results of Chlamydospore formation because of the inhibition of Chlamydospore At 30-37ºC.
• Further tests need colonies from pure culture for complete identification like biochemical, immunological, molecular, or mass spectrometry
• A non-selective and selective medium should be inoculated for the isolation of fungi from potentially contaminated specimens.
• Repeated subculturing of some Candida strains results in a loss of their ability to produce chlamydospores.

Introduction of Dermatophyte Test Medium (DTM)

Dermatophyte Test Medium (DTM) is a selective and differential medium recommended for the cultivation and isolation of pathogenic dermatophytic fungi. The Dermatophytes are a distinct group of fungi that infect the hair, skin, and nails in humans producing a variety of cutaneous infections known as ringworm. Dermatophytes are a group of three genera like Trichophyton, Microsporum and Epidermatophyton are responsible for most of cutaneous fungal infections.

Principle of Dermatophyte Test Medium (DTM)

Soya peptone provides nitrogenous and carbonaceous substances essential for growth. Dextrose( glucose) serves as the energy source for metabolism. The pH indicator, phenol red, is used to detect amine production. Cycloheximide inhibits most of the saprophytic fungi. Chloramphenicol acts as a broad-spectrum antimicrobic that inhibits a wide range of gram-positive and gram-negative bacteria.  The presence of growth on the medium provides presumptive identification of dermatophytes. DTM helps in the isolation and early recognition of members of the Microsporum, Trichophyton by means of the distinct color change from yellow to red. Rapidly growing species may effect a complete color change within 3 days while slow growers will change color proportionately for a longer time. Non-Dermatophytes can be recognized by the absence of color change. A few saprophytes, yeasts, and bacteria change the medium from yellow to red, but can be easily distinguished by colonial morphology.

Composition of Dermatophyte Test Medium (DTM)

           (Hardy Diagnostics)

Dermatophyte Test Medium (DTM) ingredients and their amounts are as follows-

Ingredients                        Gms / Litre

• Papaic Digest of Soybean Meal:  10.0
• Dextrose:  10.0
• Cycloheximide:   0.5
• Phenol Red: 0.2
• Chloramphenicol:  0.05
• Agar:  20.0
• Distilled water (D/W): 1000 ml

Final pH 5.6 +/- 0.2 at 25ºC.

Preparation of Dermatophyte Test Medium (DTM)

1. Suspend 40.75 grams of Dermatophyte Test Medium in 1 liter of purified/distilled or deionized water.
2. Heat to boiling to dissolve the medium completely.
3. Sterilize by autoclaving at 15 lbs pressure (121°C) for 10 minutes. Note:  Avoid overheating at any time.
4. After autoclaving,  leave for cooling to 45-50°C.
5. Mix well before dispensing.
6. Pour Dermatophyte Test Medium into each plate or tube and leave plates or tubes on the sterile surface until the agar has solidified.
7. Store the plates in a refrigerator at 2-8°C.

Storage and Shelf life of Dermatophyte Test Medium (DTM)

• Store at 2-8ºC  and away from direct light.
• Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), or contamination.
• The product is light and temperature-sensitive; protects from light, excessive heat, moisture, and freezing.

Test Requirements

• Test specimens ( samples or fungal growth about Specimen Collection: Submit Infectious material directly to the laboratory without delay and protected from excessive heat and cold. If there is to be a delay in processing, the specimen should be inoculated onto an appropriate transport medium and refrigerated until inoculation.)
• Inoculating loop
• Bunsen burner
• Incubator
• Control strains (For positive control: Trichophyton mentagrophytes
  ATCC 9533, Candida albicans ATCC 10231 and for negative control: Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923)
• Biological Safety Cabinet (BSC)

Test procedure

1. Allow the plates to warm at room temperature, and the agar surface to dry before inoculating.
2. Take plates or tubes.
3. Inoculate the specimen directly onto the medium by pressing the specimen lightly into the surface of the agar.
4. Alternatively, place a small amount of fungus on the agar surface if sub-culturing from another culture medium.
5. A control medium was inoculated in Sabouraud Dextrose Agar parallelly.
6. Incubate media at room temperature (15-30ºC.), aerobically, for up to 14 days.
7. Examine media daily and observe for the development of a red color change in the medium.
8. Most pathogenic dermatophytes will produce a color change in three to six days.
9. Examined media daily for up to 14 days.

Result Interpretation  of DTM

• Positive: The appearance of white aerial hyphae and red color around the fungal growth is positive for the presence of dermatophytes fungi.
• Negative: Growth, without a color change to red, indicates that the organism is probably not a dermatophyte. Further biochemical and/or serological testing needs for complete identification.
• If growth appears on the control medium (Sabouraud Dextrose Agar) and no growth appears on DTM, the organism is not a dermatophyte. Colonies with green or black hyphae are not typical of dermatophytes even though the media may turn red.
• Control strains: Positive control- Shows growth of Trichophyton mentagrophytes ( color change in the medium) and Candida albicans ( no color change in the medium) while Negative control- shows no growth of Escherichia coli  and Staphylococcus aureus ( growth inhibited)

Colony Characteristics of various organisms in Dermatophyte Test Medium (DTM)

Trichophyton mentagrophytes: Growth; white colonies and a red color change develops in the medium surrounding the colonies.
Candida albicans: Growth; small white colonies and no color change in the medium

Microsporum audouinii: pink-red

Aspergillus brasiliensis: No growth
Escherichia coli: No growth
Staphylococcus aureus: No growth

Pseudomonas aeruginosa: No growth or poor growth

Uses of Dermatophyte Test Medium (DTM)

1. Dermatophyte Test Medium (DTM)  is used for the primary isolation and identification of dermatophytes fungi like Epidermophyton, Microsporum, and Trichophyton species from hair, nails, or skin scrapings and scaling scalp lesions.
2. It is also applicable for the selective isolation of dermatophytes in veterinary specimens.

Keynotes on DTM

1. The infection caused by these organisms( dermatophytes) is commonly referred to as a ringworm.
2. The pH indicator is useful in distinguishing dermatophytes, which utilize nitrogenous material for preferred metabolism, producing alkaline by-products, imparting a red color change to the medium while typical saprotrophic fungi utilize carbohydrates in the medium producing acidic by-products and no red color change.
3. The lack of availability of chlortetracycline in late 1992 resulted in the substitution of chloramphenicol for chlortetracycline.
4. Some manufacturers provide antimicrobial agents ( i.e. cycloheximide,
   chlortetracycline, chloramphenicol, and gentamicin) from outside to add on Dermatophyte Test Agar Base like Oxoid (Cycloheximide and
   Chloramphenicol on Dermasel agar base), Himedia (cycloheximide,
   chlortetracycline and gentamicin on DTM agar base) whereas Hardy Diagnostics, Remel, and BBL  incorporate directly on Dermatophyte Test Medium.
5. Gentamicin inhibits gram-negative bacteria including Pseudomonas species while chlortetracycline inhibits a wide range of gram-positive and gram-negative bacteria.
6. Sterilize DTM by having antimicrobial agents by autoclaving at 121°C for only  10 minutes and also avoid overheating at any time.
7. Himedia suggests aseptically addition of Dermato Supplement ( antimicrobial agents rehydrated vial)  in the cooling step (45-50°C) before pouring into sterile Petri plates whereas Oxoid ( Dermasel selective supplement -vials ) prior to autoclaving.

Limitations of Dermatophyte Test Medium (DTM)

1. Colonies from pure culture for complete identification need other tests like biochemical, immunological, molecular, or mass spectrometry.
2. This medium is more useful as a general screening test, as opposed to an identification medium.
3. Don does not do result interpretations beyond 6 days of incubation otherwise false-positive reactions may result. An alkaline reaction will eventually be produced by most non-dermatophytic fungi that are capable of growing on this medium.
4. Don’t culture the dormant area of an infection otherwise false-negative reactions may arise.
5. Keep loose the caps or lids of inoculated media to assure optimal recovery of dermatophytes.
6. Certain strains of yeast may produce a color change in the medium. These organisms will produce a characteristic white, creamy, bacteria-like colony will and thus allow differentiation from dermatophytic fungi.
7. In heavily contaminated specimens, saprophytic fungi may result in a color change on the medium.  Recognize some of these organisms by their dark green to black hyphae; white aerial hyphae are unique by dermatophytes.

Introduction of Czapek Dox Agar

Czapek medium is also called Czapek’s agar (CZA) from the surname of inventor Czech botanist Friedrich Johann Franz Czapek (1902-1903). Later CZA was modified by another American inventor, chemist Arthur Wayland Dox  (1910) and thus its name became’ Czapek-Dox medium’ and formula prepared according to Thom and Church. It is recommended for use in qualitative procedures for the cultivation of saprophytic fungi and soil bacteria. The medium contains sucrose as the sole source of carbon and nitrate as the only inorganic source of nitrogen. Czapek Dox Agar (Modified) is an Oxoid modification called Oxoid Czapek Dox Agar which contains magnesium glycerophosphate and potassium sulfate to replace the magnesium sulfate and potassium phosphate of the original. This modification prevents the precipitation of magnesium phosphate. The medium is also a highly satisfactory substrate for chlamydospore production by Candida albicans.

Czapek’s  agar

↓

Czapek-Dox Agar

↓

Czapek Dox Agar (Modified)

Principle of Czapek Dox Agar

Czapek Dox Agar is a semisynthetic medium used for the cultivation of fungi, containing sodium nitrate as the sole source of nitrogen. Sucrose serves as the sole source of carbon while sodium nitrate serves as the sole source of nitrogen. Dipotassium phosphate buffers the medium. Magnesium sulfate, potassium chloride, ferrous sulfate serves as sources of essential ions. Agar acts as a solidifying agent. In modified  Czapek Dox Agar,  magnesium glycerophosphate and potassium sulfate replace the magnesium sulfate and potassium phosphate of the original which prevents the precipitation of magnesium phosphate and it is also a highly satisfactory substrate for chlamydospore production by Candida albicans.

Composition of Czapek  Agar ( original)

• Cane sugar: 30 gm
• Monopotassium phosphate: 1 gm
• magnesium sulfate: 0.5 gm
• Potassium chloride: 0.5 gm
• Iron sulfate: 0.01 gm
• Distilled water (D/W): 1000 ml

Composition of Czapek Dox Agar

• Sucrose 30.0 gm
• Sodium Nitrate 2.0 gm
• Dipotassium Phosphate 1.0 gm
• Magnesium Sulfate 0.5 gm
• Potassium Chloride 0.5 gm
• Ferrous Sulfate 0.01 gm
• Agar 15.0 gm
• Distilled Water(D/W): 1000 ml

Final pH 7.3 +/- 0.3 at 25ºC.

Composition of Czapek Dox Agar ( modified)

                   (Oxoid Czapek Dox Agar )

• Sodium nitrate: 2.0 gm
• Potassium chloride: 0.5 gm
• Magnesium glycerophosphate: 0.5 gm
• Ferrous sulfate: 0.01 gm
• Potassium sulfate: 0.35 gm
• Sucrose: 30.0 gm
• Agar: 12.0 gm
• Distilled Water (D/W): 1000 ml

pH 6.8 ± 0.2 @ 25°C

Preparation of Czapek Dox Agar

1. Suspend 49.01 grams of Czapek Dox Agar in 1 liter of purified/distilled or deionized water.
2. Heat to boiling to dissolve the medium completely.
3. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
4. After autoclaving,  leave for cooling to 45-50°C.
5. Mix well before dispensing.
6. Pour Czapek Dox Agar into each plate and leave plates on the sterile surface until the agar has solidified.
7. Store the plates in a refrigerator at 2-8°C.

Storage and Shelf life of Czapek Dox Agar

• Store at 2-8ºC  and away from direct light.
• Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), or contamination.
• The product is light and temperature-sensitive; protects from light, excessive heat, moisture, and freezing.

Test Requirements

• Test specimens ( samples or fungal growth)
• Inoculating loop
• Bunsen burner
• Incubator
• Control strains (For positive control: Candida albicans ATCC 10231 and Aspergillus niger ATCC 9642, For negative control: Uninoculated medium)
• Biological Safety Cabinet (BSC)

Test procedure

1. Allow the plates to warm at 37°C or to room temperature, and the agar surface to dry before inoculating.
2. Take plates.
3. Inoculate by spreading the sample material thinly on the surface of the culture medium.
4. Incubate for up to 1 week at 28ºC. Optimal incubation temperatures are as follows: Penicillium species at 20-25ºC, Aspergillus species at 30ºC, and Candida species at 28ºC.

Result Interpretation  of Czapek Dox Agar

Presence of yeast or mold or both: Fungal growth

Control strains-

Candida albicans: Luxuriant growth with cream-colored colonies

Aspergillus niger: Luxuriant growth with White/yellow mycelium, black spores

Uninoculated medium: No growth

Colony Characteristics of various organisms in Czapek Dox Agar

Aspergillus brasiliensis: Good growth

Candida albicans:  Good growth; cream-colored colonies

Saccharomyces cerevisiae: Good growth

Aspergillus niger: White/yellow mycelium, black spores

Aspergillus flavus: Macroscopic features: Colonies on Czapek Dox agar are granular, flat, often with radial grooves and yellow whereas microscopic features in LPCB preparation are globose to subglobose conidia ( 3-6 µm in size),  septate hyphae, stipe, conidiophore, vesicle and phialides as shown above picture.

Uses of Czapek Dox Agar

The application of Czapek Dox Agar is as follows-

1. Czapek Dox Agar is  APHA( American Public Health Association)  recommended fungal medium for the isolation of Aspergillus, Penicillium, Paecilomyces, and some other fungi with similar physiological requirements.
2. It is also recommended for the general cultivation of fungi from water samples.
3. The medium is widely used for taxonomic studies of Aspergillus, Penicillium, and actinomycetes.
4. Czapek-Dox Agar produces luxuriant growth of almost all saprophytic Aspergilli causing the organisms to produce characteristic mycelia and conidia.
5. The acidity of the medium may be increased for the cultivation of acidophilic organisms such as yeasts.
6. The medium is useful in a variety of microbiological procedures, including fungi and mildew resistance tests and soil microbiology testing.
7. Oxoid Czapek Dox is the most satisfactory medium compared to rice infusion agar and corn meal agar for chlamydospore production by Candida albicans.

Keynotes on Czapek Dox Agar

• Czapek-Dox Agar is recommended for use in cultivating fungi and bacteria capable of using inorganic nitrogen.
• Czapek and Dox did not add agar but many recipes add 15 g to make a solid medium.
• Most Penicillium species have an optimum growth temperature between 20° and 25°C, while many Aspergillus species grow best at about 30°C. However, different fungi grow over a wide range of temperatures; Aspergillus fumigatus grows well at 50°C and Cladosporium herbarium will grow on meat at -6°C.

Limitations of Czapek Dox Agar

• This medium is a general-purpose medium and may not support the growth of fastidious organisms.
• Further tests need colonies from pure culture for complete identification like biochemical, immunological, molecular, or mass spectrometry.
• Modification is preferred for two reasons, 1. due to prevention of precipitation of magnesium phosphate and 2.  chlamydospore production by Candida albicans.

Introduction of Ascospore Agar

Ascospore Agar is recommended medium for the expression of ascospores and therefore it is useful for the enrichment and detection of ascosporogenous yeasts. It is the modification of the McClary medium with the addition of potassium acetate in place of sodium acetate. Saccharomyces cerevisiae is the most common yeast used to show expression ascopores.

Principle of Ascospore Agar

Yeast extract and dextrose supply the nutrients required for growth and also stimulate ascospore formation in yeasts while acetate salt of potassium enhances sporulation in Saccharomyces. The slightly acidic pH of the ascospore agar favors the growth of S. cerevisiae. Agar is the solidifying agent and water is the source of hydrogen and oxygen.

Composition of Ascospore Agar

Ingredients  Gms / Litre
Yeast extract: 2.5
Dextrose:  1.0
Potassium acetate:  10.0
Agar: 30.0
Final pH ( at 25°C) 6.4±0.2

Preparation of Ascospore Agar

1. Suspend 43.5 grams of Ascospore Agar in 1 liter of purified/distilled or deionized water.
2. Heat to boiling to dissolve the medium completely.
3. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
4. After autoclaving,  leave for cooling to 45-50°C.
5. Mix well before dispensing.
6. Pour Ascospore Agar into each plate and leave plates on the sterile surface until the agar has solidified.
7. Store the plates in a refrigerator at 2-8°C.

Storage and Shelf life 

• Store at 2-8ºC  and away from direct light.
• Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), or contamination.
• The product is light and temperature-sensitive; protects from light, excessive heat, moisture, and freezing.

Test Requirements

• Test specimens ( samples or fungal growth)
• Inoculating loop
• Bunsen burner
• Incubator
• Control strains (Candida albicans ATCC  10231 and Saccharomyces cerevisiae ATCC 9763)

Procedure of Ascospore Agar,

1. Allow the plates to warm at 37°C or to room temperature, and the agar surface to dry before inoculating.
2. Inoculate and streak the specimen as soon as possible after collection.
3. If the specimen to be cultured is on a swab, roll the swab over a small area of the agar surface.
4. Streak for isolation with a sterile loop.
5. Incubate one plate aerobically at room temperature ( 25-30°C) for up to 3-6 days.
6. Examine the growth and perform LPCB tease mount Microscopy.

Result Interpretation 

In general, ascospores microscopy have no attachment points and are sometimes enclosed in gelatinous sheaths or within a sac.

Presence of ascospores: Positive

Absence of ascospores: Negative

Candida albicans: Luxuriant growth but no ascospores

Saccharomyces cerevisiae: Luxuriant growth with ascospores

Control strains

Candida albicans ATCC  10231: Negative

and Saccharomyces cerevisiae ATCC 9763: Positive

Use of Ascospore Agar

It is the medium used for the formation of ascopores of ascosporogenous yeasts.

Keynotes on Ascospore Agar

• Ascospore Agar is the McClary modification medium in which sodium acetate has been replaced by potassium acetate to better enhance the rate of ascospores formation.
• In general, ascospores have no attachment points and are sometimes enclosed in gelatinous sheaths or within a sac.

Introduction of Bird Seed Agar

Bird Seed Agar sounds seed of a bird but actually, it is a niger seed, the botanical name Guizotia abyssinica. Niger seed contains caffeic acid that serves as a substrate for the detection of phenoloxidase, an enzyme produced by Cryptococcus neoformans. The action of phenoloxidase on caffeic acid results in the production of melanin which is occupied by the yeast cell wall forming a tan to reddish-brown pigmentation. Bird seed agar in brief is also called BSA.  Stab’s formulation of BSA  is a selective and differential medium for both C. neoformans and C. gattii while modification of Staib’s formulation ignores C. gattii.

Principle of Bird Seed Agar

Niger seeds, creatinine, and glucose provide nutrients for the growth of Cryptococcus neoformans. Chloramphenicol inhibits the bacteria as well as rapidly growing molds that often overgrow the slow-growing dimorphic fungi. The addition of diphenyl also assists in preventing the growth of molds and fungus and is therefore used as a selective agent in the medium.

Composition of Bird Seed Agar

Ingredients         Gms / Litre

• Guizotia abyssinica (niger) seeds: 70.000
• Creatinine:  0.78
• Dextrose (Glucose): 10.0
• Chloramphenicol: 0.05
• Agar: 20.0
• Distilled water: 1000 mL
• Final pH ( at 25°C):  6.7±0.2

Preparation of Bird Seed Agar

1. Suspend 10.8 grams of Bird Seed Agar ( BSA) in 99 mL of purified/distilled or deionized water.
2. Heat to boiling to dissolve the medium completely.
3. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
4. After autoclaving,  leave for cooling to 45-50°C and add 100 µg diphenyl per ml of medium (1 ml of sterile 1% w/v aqueous solution of diphenyl).
5. Mix well before dispensing.
6. Pour BSA into each plate and leave plates on the sterile surface until the agar has solidified.
7. Store the plates in a refrigerator at 2-8°C.

Storage and Shelf life of Bird Seed Agar

• Store at 2-8ºC  and away from direct light.
• Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), or contamination.
• The product is light and temperature-sensitive; protects from light, excessive heat, moisture, and freezing.

Test Requirements

• Test specimens ( Clinical samples-Cerebrospinal Fluid (CSF), tissue, exudates, sputum, blood, and urine or fungal growth)
• Inoculating loop
• Bunsen burner
• Incubator
• Control strains ( Cryptococcus neoformans ATCC 32045
  and Candida albicans ATCC 10231)

Test Procedure of Bird Seed Agar (specimen/organism inoculation)

1. Allow the plates to warm at 37°C or to room temperature, and the agar surface to dry before inoculating.
2. Take a plate for one specimen.
3. Inoculate and streak the specimen as soon as possible after collection.
4. If the specimen to be cultured is on a swab, roll the swab over a small area of the agar surface.
5. Streak for isolation with a sterile loop.
6. Incubate the plate aerobically at 30°C for 7 days.
7. Examine colony characteristics.

Colony Characteristics of Bird Seed Agar

Development of brown to black pigmented smooth colonies-Cryptococcus neoformans

Non-pigmented colonies-Cryptococcus laurentii, Candida, Saccharomyces cerevisiae

Uses of Bird Seed Agar

BSA is the recommended medium for selective isolation and differentiation of Cryptococcus neoformans from other Cryptococcus and other yeasts.

Keynotes on Bird Seed Agar

• Staib’s Medium (Bird Seed Agar) is formulated for selective isolation and differentiation of C.neoformans and  C. gattii.  from other Cryptococcus species and other yeasts.
• The above medium is formulated by Staib and Shields and Ajello which is selective isolation and differentiation of only C. neoformans from other Cryptococcus species and other yeasts.
• Compositiuon of Staib Formunation

Ingredients             Gms/Litre

• Guizotia abyssinica (niger seed): 50.0
• Dextrose (glucose): 1.0
• KH2PO4 (potassium dihydrogen orthophosphate): 1.0
• Creatinin: 1.0
• Agar: 15.0
• Final pH:  6.5 +/- 0.3 at 25ºC.

Additives: to each 500 ml bottle

• Penicillin G (20 units/ml) 1 mL
• Gentamicin (40 mg/ml) 1 mL
• Other species of Cryptococcus are-

1. Candida  albidus ( nitrate positive)
2. C. laurentii ( melibiose fermentation)
3. C. gattii ( Trehalose positive)
4. Cryptococcus neoformans: All the above tests are negative.
5. C. neoformans var. grubii ( serotype A)- worldwide distribution
6. C. neoformans var. neoformans  (serotype D)-Restricted distribution and prevalent in France, Italy, and Denmark.

• Diphenyl prevents the growth of molds and fungus and is therefore used as a selective agent in the medium.
• Bird seed agar is also known as Caffeic acid agar or niger seed agar or Staib agar.

Further Readings on Fungal Culture Media

1. Medical Mycology. Editors:  Emmons and Binford, 2nd ed 1970, Publisher Lea and Febiger, Philadelphia.
2. Rippon’s JW: Medical Microbiology. The pathogenic fungi and the Pathogenic Actinomycetes. 3^rd ed 1988 Publisher WB Saunder co, Philadelphia.
3. Clinical Microbiology Procedure Handbook, Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
4. A Text-Book of Medical Mycology. Editor: Jagdish Chander.  Publication Mehata, India.
5.  Practical Laboratory Mycology. Editors: Koneman E.W. and G.D. Roberts, 3rd ed 1985, Publisher Williams and Wilkins, Baltimore.
6. Textbook of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.
7. Mackie and Mc Cartney Practical Medical Microbiology. Editors: J.G. Colle, A.G. Fraser, B.P. Marmion, A. Simmons, 4th ed, Publisher Churchill Living Stone, New York, Melborne, Sans Francisco 1996.
8. Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
9. https://www.thomassci.com/Laboratory-Supplies/Microbiological-Media/_/Sabouraud-Dextrose-Agar
10. http://himedialabs.com/TD/M063.pdf
11. https://en.wikipedia.org/wiki/Sabouraud_agarhttps://drfungus.org/knowledge-base/sda/
12. https://catalog.hardydiagnostics.com/cp_prod/Content/hugo/PotatoDextroseAgar.
13. htmhttp://himedialabs.com/TD/M096.pdf
14. https://en.wikipedia.org/wiki/Potato_dextrose_agar
15. http://himedialabs.com/TD/M146.pdf
16. http://www.oxoid.com/UK/blue/prod_detail/prod_detail.asp
17. https://catalog.hardydiagnostics.com/cp_prod/Content/hugo/CornMealAgarTween.htm
18. https://www.sigmaaldrich.com/catalog/product/sial/42347
19. https://assets.fishersci.com/TFS-Assets/LSG/manuals/IFU1365.pd
20. https://catalog.hardydiagnostics.com/cp_prod/content/hugo/DTM.htm
21. https://legacy.bd.com/ds/technicalCenter/inserts/8814091(03).pdf
22. https://himedialabs.com/TD/M188.pdf
23. http://www.oxoid.com/UK/blue/prod_detail/prod_detail.asp
24. https://catalog.hardydiagnostics.com/cp_prod/Content/hugo/Czapek-DoxAgar.html
25. http://www.oxoid.com/uk/blue/prod_detail/prod_detail.asp?pr=CM0097
26. https://en.wikipedia.org/wiki/Czapek_medium
27. http://www.himedialabs.com/TD/M075.pdf
28. https://assets.fishersci.com/TFSAssets/LSG/manuals/IFU9046.pdf
29. https://www.cdhfinechemical.com/images/product/specs/DM%201804.pdf
30. https://himedialabs.com/TD/M804.pdf
31. https://www.himedialabs.com/TD/M675.pdf
32. http://www.essentialpaservices.com/listing/birdseed-agar-thermo-fisher-scientific-425b-ce549f
33. https://catalog.hardydiagnostics.com/cp_prod/product/g213-caffeic-acid-agar-bird-seed-agar-for-cryptococcus-15x100mm-plate-order-by-the-package-of-10-by-hardy-diagnosticsbr-bminimum-order-10-packsb-media-prepared
34. https://en.wikipedia.org/wiki/Biphenyl
35. Casadevall A, Perfect J. R., 1998, Cryptococcus neoformans, ASM Press, Washington, D.C.