RECOMMENDED MEDIA FOR FUNGI ISOLATION
Clinical specimens are processed promptly and plated to isolation media as a means to recover fungi that may be causing disease. Media and incubation temperatures are selected to allow for the growth of pathogenic and opportunistic yeasts and fungi.
Isolation Media
The following Table is intended as a guideline for media required for the primary isolation of common isolates in ocular infections. Because some of these sites may be sterile sources and others are nonsterile sources, isolates considered pathogenic differ by site.
* Anaerobic cultures are performed on intraocular fluids wherever necessary. Media abbreviated as follows:
BA : Blood agar
CA : Chocolate blood agar
BHI : Brain heart infusion broth
Thio : Thioglycolate broth culture
SDA : Sabouraud’s dextrose agar (Emmons modification)
LJ : Lowenstein jensen agar slant
BHI : Brain heart infusion broth
A variety of media are available for the primary inoculation and recovery of fungi from clinical specimens. No one specific medium or combination of media is adequate for all specimens. Media must be carefully selected based on specimen type and fungal suspected agents. Media is dispensed into bottles or 100 mm Petri dishes. Petri plates offer the advantage of a large surface area for isolation and dilution of inhibitory substances in the specimens, but must be poured thick with at least 25 ml of medium to resist dehydration during incubation. Because plates are vented, they are more likely to become contaminated during incubation. Each Petri plate must be labeled on the bottom, and the lid must be taped at two points to prevent accidental opening of the plate. All inoculated media should be read every day initially following incubation. Plates must be opened only within a biological safety cabinet to prevent contamination of the plate and exposure of personnel to potentially dangerous fungi.
Media in bottles have a smaller surface area but offer maximum safety and resistance to dehydration and contamination. If the specimen is from a contaminated site, it is important to include media that contain inhibitory substances such as chloramphenicol, gentamicin or cycloheximide. Chloramphenicol or gentamicin will inhibit most bacterial contaminants, while cycloheximide inhibits most saprobic moulds. It is important to remember that Cycloheximide may also inhibit opportunistic fungi such as some species of Aspergillus, Fusarium, Scopulariopsis, Pseudallescheria, Zygomycetes, some dematiaceous fungi, and yeasts such as Cryptococcus neoformans and some Candida species.
Antibacterial agents may inhibit the growth of aerobic Actinomycetes like Nocardia sp. It is important to use media with and without inhibitory agents. Specimens from normally sterile sites can be inoculated to media without inhibitory substances.
Potato Glucose Agar (Potato Dextrose Agar)
Composition
Potatoes 200 gm
Glucose 10 gm
Agar 18 gm
Distilled water 1000 ml
Preparation
• Peel the potatoes, cut into cubes and boil in water for one hour
• Filter through cheesecloth, add glucose and agar; bring to a boil to dissolve agar completelyand filter again on Whatman #2 filter paper.
• Adjust the volume to 1 liter
• Sterilize at 121°C for 15 minutes.
Quality Control
Appearance: Colorless or light yellow, solid medium, transparent or translucent
Final pH at 25°C: 5.6 ± 0.2
Sterility Check
One representative sample of the prepared culture plate is incubated at 25 - 30°C for 4 days to check the sterility of the media.
This medium is also available commercially.
Sabouraud’s Dextrose Agar (SDA)
Principle
SDA agar formulation was originally described by Sabouraud for the cultivation of fungi. Emmons' modification contains 2% glucose and is slightly acidic (pH 6.5). It is the standard medium for recovery and maintenance of a wide variety of fungi commonly isolated in the clinical laboratory. The original SDA formulation specifies 4% glucose. Emmons' modification with less glucose is preferred as an isolation medium because some isolates, notably Blastomyces dermatitidis may not be recovered using the original Sabouraud's formulation. Peptone is the source of nitrogenous growth factors while dextrose provides an energy source for the growth of microorganisms.
a. SDA (acidic Ph)
Ingredients grams/liter of distilled water
Dextrose 40
Peptone 10
Agar 20
Final pH: 5.5-5.6
b. SDA (neutral Ph-Emmon's modification)
Ingredients grams/liter of distilled water
Dextrose 20
Peptone 18
Agar 20
Final pH: 6.8-7.0
c. Directions
• Dissolve all the ingredients by boiling.
• Dispense in plates and autoclave at 121°C for 15 minutes.
• Adjust the final pH.
• The tubes are cooled in a slanted position and stored in the refrigerator (4°C).
d . Quality control
Colour and clarity: Light amber colored. Clear to slightly opalescent gel.
Reaction: Reaction of the molten media is pH 6.8-7
Cultural response:
• Cultural characteristics after 48-72 hr at 30°C.
• Candida albicans will produce luxuriant growth
Sterility check: One representative sample of the prepared culture plate is incubated at 25-30°C for 4 days to check the sterility of the media.
Brain-heart Infusion Agar (BHI)
Principle
It is a highly nutritious medium that can support the growth of wide variety of microorganisms. It can be further enriched by the addition of blood or rendered selective by adding different antibiotics. BHI is an enriched medium that enhances the recovery of Cryptococcus neoformans from sterile specimens such as CSF. BHI is also used in yeast-mould conversions for Sporothrix and Paracoccidioides.
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Ingredients
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gms/liter
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Calf brain infusion
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200
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Beef heart infusion
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250
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Peptone
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10
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Dextrose
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2.00
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Sodium chloride
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5.00
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Disodium phosphate
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2.50
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Agar
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15
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Final pH (at 25°C): 7.4 ± 0.2
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Directions
• Dissolve ingredients by boiling
• Dispense in bottles and sterilize by autoclaving at 15lbs pressure for 15 minutes.
Quality Control
Sterility
Cultural response: Cultural characteristics after 48-72 hrs at 30°C.
Organisms Growth
Cryptococcus neoformans Luxuriant
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