Culture media and incubation requirements of Fungi
المؤلف:
Patricia M. Tille, PhD, MLS(ASCP)
المصدر:
Bailey & Scotts Diagnostic Microbiology
الجزء والصفحة:
13th Edition , p714-717
2025-11-10
127
A number of fungal culture media are satisfactory for use in the clinical microbiology laboratory (Table 1). Most are adequate for the recovery of fungi, and the selection usually is left up to the laboratory director. For optimal recovery, a battery of media should be used; the following are recommended:
• Media with and without cycloheximide
• Media with and without an antibacterial agent (Media with an antibacterial agent are used for specimens likely to contain contaminating bacteria; they are not necessary for specimens from sterile sites.)
Agar plates or screw-capped agar tubes are satisfactory for the recovery of fungi; however, plates are preferred, because they provide better aeration of cultures, a large surface area for better isolation of colonies, and greater ease of handling by technologists when making microscopic preparations for examination. Agar tends to dehydrate during the extended incubation period required for fungal recovery, but this problem can be minimized by using culture dishes containing at least 40 mL of agar and placing them in a humidified incubator. Dishes should be opened and examined only in a certified biologic safety cabinet (BSC). Many laboratories discourage the use of culture dishes because of safety considerations; however, the advantages outweigh the disadvantages.
Table1. Fungal Culture Media: Indications for Use
Table1. Fungal Culture Media: Indications for Use—cont’d
Compared with agar plates, screw-capped culture tubes are more easily stored, require less space for incubation, and are more easily handled. In addition, they have a lower dehydration rate, and laboratory workers believe cultures are less hazardous to handle when in tubes. However, disadvantages, such as relatively poor isolation of colonies, a reduced surface area for culturing, and a tendency to promote anaerobiosis, discourage routine use in most clinical microbiology laboratories. If culture tubes are used, the tube should be as large as possible to provide an adequate surface area for isolation. After inoculation, tubes should be placed in a horizontal position for at least 1 to 2 hours to allow the specimen to absorb to the agar surface and avoid settling at the bottom of the tube. Cotton-plugged tubes are unsatisfactory for fungal cultures.
Cultures should be incubated at room temperature, or preferably at 30°C, for 21 to 30 days before they are reported as negative. A relative humidity in the range of 40% to 50% can be achieved by placing an open pan of water in the incubator. Cultures should be examined at least three times weekly during incubation.
As previously mentioned, some clinical specimens are contaminated with bacteria or rapidly growing fungi or both, requiring the use of antifungal and antibacterial agents. The addition of 0.5 µg/mL of cycloheximide and 16 µg/mL of chloramphenicol to media traditionally has been advocated to inhibit the growth of contaminating molds and bacteria, respectively. However, better results have been achieved using a combination of 5 µg/mL of gentamicin and 16 µg/mL of chloramphenicol as antibacterial agents. Ciprofloxacin at a concentration of 5 µg/mL may be used.
Cycloheximide may be added to any of the media that contain or lack antibacterial antibiotics. However, if cycloheximide is included in the battery of culture media, a medium lacking this ingredient should also be included. Pathogenic fungi, such as C. neoformans complex, Candida krusei and other Candida spp., Trichosporon spp., P. boydii, and Aspergillus spp., are partially or completely inhibited by cycloheximide.
Although use of antibiotics in fungal culture media is necessary for optimal recovery of organisms, the use of decontamination and concentration methods advocated for the recovery of mycobacteria is not appropriate, because many fungi are killed by sodium hydroxide treatment.
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