Candida tropicalis on SDA, PDA, CMA, CHROMagar, AFS: Introduction and Fungal Culture Media Details

Candida tropicalis on SDA,PDA, CMA, CHROMagar, AFST

Candida tropicalis on SDA, PDA, CMA, CHROMagar, AFST

Candida tropicalis on SDA, PDA, CMA, CHROMagar, and AFST as shown above picture.  C. tropicais colonies on Sabouraud Dextrose Agar (SDA) are white to cream, with a creamy texture and smooth appearance, and may have slightly wrinkled edges. Thus, it is indistinguishable from other Candida species. C. tropicalis has emerged as one of the most important Candida species. It is the second most virulent Candida species after C. albicans. Besides, this is also a very strong biofilm producer, surpassing C. albicans in most of the studies. It produces a wide range of other virulence factors, including adhesion to buccal epithelial and endothelial cells; the secretion of lytic enzymes, such as proteinases, phospholipases, and hemolysins, bud-to-hyphae transition, and the phenomenon called phenotypic switching. This species is very closely related to C. albicans and has been easily identified with both phenotypic and molecular methods. C. tropicalis is a clinically relevant species and maybe the second or third etiological agent of candidemia, specifically in Asia and  Latin American countries.  Antifungal drugs resistant to this organism are azoles, polyenes, and echinocandins.  C. tropicalis is an osmotolerant microorganism and this ability to survive to high salt concentration may be important for fungal persistence in saline environments. This physiological feature makes this species suitable for use in biotechnology processes.

Fungal Culture Media and AFST in Details

This topic contains  Sabouraud Dextrose Agar (SDA), Potato Dextrose Agar ( PDA), Corn Meal Agar (CMA), HiCrome candida differential agar, Antifungal Sensitivity Testing (AFST) medium.

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 lowered 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 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 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 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 Malasezzia species to 2 to 4 weeks for 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 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 which 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 further modified 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 potato dextrose agar ( PDA) in 1 liter 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), 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) whereas another 35-37ºC 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 seen in 7 days and it may take 3-4 weeks for 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 sporulation.
  4. Various modified PDAs and their applications are as follows-PDA with TA 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 (CMA)

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 (CMA)

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 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 (CMA)

           (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 (CMA)

  1. Suspend 17.0 grams of Corn Meal Agar (CMA) in 1 liter 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 (CMA)

  • 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), 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 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 sporulation of molds. Incubate until the mold is visible and mount 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 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 seen in 7 days and it may take 3-4 weeks for red color on the reverse side of colony to be visible.

Uses of Corn Meal Agar (CMA)

  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. Cornmeal 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 CMA

  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 (CMA)

  • Recommended temperature is 25ºC for the best results of Chlamydospore formation because of 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 result in a loss of their ability to produce chlamydospores.

Use of CHROMagar Candida

CHROMagar Candida or HiCrome candida differential agar recommendation is for rapid isolation and identification of Candida species from mixed cultures in clinical and non-clinical samples.

Composition of CHROMagar Candida

Ingredients       Gm / Litre

Peptone, special      15.000

Yeast extract            4.000

Dipotassium hydrogen phosphate    1.000

Chromogenic mixture         7.220

Chloramphenicol      0.500

Agar       15.000

Final pH (at 25°C) 6.3±0.2

Preparation of CHROMagar Candida medium

  1. Suspend 42.72 grams in 1000 ml distilled water.
  2. Heat to boiling to dissolve the medium completely.
  3. DO NOT AUTOCLAVE.
  4. Cool to 45-50°C.
  5. Mix well and pour into sterile Petri plates.

Principle And Interpretation of CHROMagar Candida

Perry and Miller reported that Candida albicans produces an enzyme b -N-acetyl- galactosaminidase and according to Rousselle et al incorporation of chromogenic or fluorogenic hexosaminidase substrates into the growth medium helps in the identification of C. albicans isolates directly on primary isolation. HiCrome Candida Differential Agar is a selective and differential medium, which facilitates the rapid isolation of yeasts from mixed cultures and allows differentiation of Candida species namely C. albicansC. kruseiC. tropicalis, and C. glabrata on the basis of coloration and colony morphology. On this medium results are obtained within 48 hours and it is useful for the rapid and presumptive identification of common yeasts in Mycology and Clinical Microbiology Laboratory. Peptone special and yeast extract provides nitrogenous, carbonaceous compounds, and other essential growth nutrients. Phosphate buffers the medium-well. Chloramphenicol suppresses the accompanying bacterial flora. C. albicans appear as light green colored smooth colonies, C. tropicalis appear as blue to metallic blue colored raised colonies. C. glabrata colonies appear as a cream to white smooth colonies, while C. krusei appear as purple fuzzy colonies.

Type of specimen

Clinical samples – skin scrapings, urine or stored Candida species or yeast colonies

Procedure

  1. Stored Candida species were inoculated in HiCrome candida differential agar.
  2. It was then incubated at 25-30°C for 48 hours.

Observation of CHROMagar Candida

After two days of incubation above-shown type of figure observed.

  1. Green
  2. Green
  3. purple, fuzzy
  4. blue to purple

Result of CHROMagar Candida

  1. Candida albicans
  2. Candida albicans
  3. Candida krusei
  4. Candida tropicalis

Note: as shown above figure

Cultural characteristics of CHROMagar Candida

After incubation at 25-30°C for 40-48 hours.

Organism                                               Growth                       Color of Colony

Candida albicans ATCC 10231      good-luxuriant          green

Candida glabrata ATCC 15126    good-luxuriant           cream to white

Candida krusei ATCC 24408        good-luxuriant          purple, fuzzy

Candida tropicalis ATCC 750       good-luxuriant           blue to purple

Escherichia coli ATCC 25922        inhibited

Staphylococcus aureus ATCC 25923  inhibited

Limitations of using CHROMagar Candida

  1. Variations in color intensity may be observed for Candida isolates depending on the presence of enzymes.
  2. Other Candida species may produce light mauve-colored colonies which are also produced by other yeast cells. This must be confirmed by further biochemical tests.
  3. Other filamentous fungi also exhibit color on this medium.

Introduction of Antifungal Drugs Susceptibility Testing

Antifungal drugs and their susceptibility testing as shown above picture. There are various testing methods like disc diffusion, broth dilution, E-test, and VITEK. Among them, we tested antifungal susceptibility testing (AFST) by the disc diffusion method.

List of common anti-fungal drugs are as follows-

  • Clotrimazole
  • Econazole
  • Miconazole
  • Terbinafine
  • Fluconazole
  • Ketoconazole
  • Itraconazole
  • Voriconazole
  • Posaconazole,
  • Ravuconazole
  • Amphotericin-B,
  • 5-Fluorocytosine,

The mode of action of some antifungal agents are as follow-

Amphotericin B binds to the plasma membrane creating pores.

Azoles inhibit cytochrome P450 enzymes in the fungal cell 5FC converts to 5FU, incorporated into RNA, abnormal proteins.

Griseofulvin binds microtubule proteins, inhibits cell wall synthesis.

Terbinafine is an ergosterol inhibitor useful for systemic mycosis.

Echinocandins target their action on the fungal cell walls.

Griseofulvin:  Anti-inflammatory properties Inhibits keratolytic action

Antifungal Sensitivity Testing (AFST) can be performed by following methods and they are-

  1. Disc diffusion method
  2. Broth dilution: Synthetic Media: RPMI 1640 buffered with MOPS (Morpholine propane sulfonic acid) for Candida species and Yeast Nitrogen Broth for Cryptococcus neoformans
  3. E-test
  4. VITEK

AFST by Disc diffusion method

  • It is only applicable for Candida species.
  • Other genera are not covered and also have not been used in the studies of the yeast form of the dimorphic fungi.

Requirements for test

  • Mueller-Hinton Agar + 2% Glucose + 0.5 mcg/ml Methylene Blue Dye medium (GMB) having  pH 7.2 to 7.4
  • Antifungal drugs to be tested
  • McFarland Std
  • Incubator
  • Fungal growth of test organism
  • Test tubes
  • Sterile cotton swabs

Test procedure

  1. Preparation of test inoculum
  2. Inoculation of the test organisms into a  plate.
  3. Application of Disks to the inoculated agar plate.
  4. Incubate the plate.

Observation

  •  Examine each plate after 24 to 24 Hr of incubation
  • Measure the zone diameter to the nearest whole millimeter at the point at which there is a prominent reduction in growth.
  • Pinpoint micro-colonies at the zone edge or larger colonies within a zone are encountered frequently and they should be ignored as shown above picture.

Result Interpretation

Susceptible (S): Infection due to the strain may be appropriately treated with the dose of the antimicrobial agent recommended.

 Susceptible Dose-Dependent (S-DD): It includes isolates with antimicrobial agent MICs that approach usually attainable blood and tissue levels but the response rate may be lower than for susceptible isolates (Only applies when multiple approved dosage options exist)

Resistant (R): Isolates are not inhibited by the usually achievable concentration of the agent with normal dosage schedules.

Importance of AFST

  1. Increase of Fungal Infections Several factors have contributed to the increase in fungal infections – most notably, an increasing number of immunosuppressed cases e.g AIDS, cancer, or diabetes, the use of broad-spectrum antibiotics, cytotoxic chemotherapy, and organ transplantation.
  2.  Increase in incidence of immunosuppressive states
  3. Increasing incidence of invasive mycosis and life-threatening infections as a significant public health issue
  4.  Emerging resistance
  5. Correlate with in vivo activity and predict the likely outcome of therapy
  6.  Provide a reliable measure of the relative activities of two or more antifungal agents.
  7. Growing concern about a shortage of effective antifungal agents and an increase in the resistance of fungal pathogens to the existing agents
  8.  Among the invasive mycoses, none is more important or common than candidiasis. Candidiasis, specifical candidemia, has been shown in numerous studies to be the most frequent mycotic infection in hospitalized patients and is associated with significant attributable mortality and excess length of hospital stay
  9.  Fungal Infections have increased Morbidity and Mortality The increasing incidence of opportunistic severe fungal infections has greatly enhanced the interest in novel methods for in vitro antifungal susceptibility testing, the standardized methodology.

Further Readings

  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. 3rd 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 Textbook 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. Simmous, 4th ed, Publisher Churchill Living Stone, New York, Melborne, Sans Franscisco 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://www.ncbi.nlm.nih.gov/pmc/articles/PMC5645804/
  13. https://catalog.hardydiagnostics.com/cp_prod/Content/hugo/PotatoDextroseAgar.htm
  14. http://himedialabs.com/TD/M096.pdf
  15. https://en.wikipedia.org/wiki/Potato_dextrose_agar
  16. http://himedialabs.com/TD/M146.pdf
  17. http://www.oxoid.com/UK/blue/prod_detail/prod_detail.asp
  18. https://catalog.hardydiagnostics.com/cp_prod/Content/hugo/CornMealAgarTween.htm
  19. https://www.sigmaaldrich.com/catalog/product/sial/42347
  20. https://cmr.asm.org/content/33/3/e00069-19
  21. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC88997/
  22. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4711191/
  23. https://academic.oup.com/mmy/article/45/7/569/956049
  24. https://www.cdc.gov/fungal/candida-auris/c-auris-antifungal.html
  25. https://techlib.biomerieux.com/wcm/techlib/techlib/documents/docLink/
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