Cultural Technique: Culture Media and Incubation Condition

Culture media

Selection and Inoculation of culture media

Laboratory culture is preferred when it is not possible to diagnosis a serious fungal infection microscopically or species identification needs to be established or confirmed. The commonly used media for the cultures of fungi are:

1. Sabouraud dextrose agar
2. Sabouraud dextrose agar with antibiotics
3. Chlamydospore agar or corn meal agar
4. Brain heart infusion agar with or without antibiotics
5. Potato dextrose agar (PDA)
6. Potato flake agar (PFA)
7. Inhibitory mold agar (IMA)
8. Sabouraud dextrose agar with heart infusion (SABHI) agar

Generally used nonselective culture media such as brain heart infusion agar allows the growth of virtually all clinically relevant fungi. Whereas, the use of Sabouraud dextrose agar as primary recovery medium is discouraged because it is not sufficiently rich to obtain certain fastidious pathogenic species, particularly dimorphic fungi. Rather, the use of Potato flake agar, inhibitory mold agar, or a combination of Sabouraud dextrose agar with heart infusion agar is recommended for laboratory culture.

Sabouraud agar is sufficient for the recovery of yeasts from vaginal culture also dermatophytes from cutaneous samples. For more fastidious dimorphic fungi such as Blastomyces dermatidis and soistoplasma capsulatum, a enrich agar like IMA or SABHI is preferred and in particular for Histoplasma capsulatum media with the addition of 5-10% sheep blood is recommended. Cryptococcus neoformans, Aspergillus fumigatus may be partially or entirely inhibited by cycloheximide, therefore a nonselective media must always be used in parallel. After the culture plates incubation, is shows growth of probable fungi, the identification of the colonies, characteristic morphology of colony is observed. After the culture plates reveal the growth of probable fungi then the identification of the colonies is done by the characteristic colony morphology. Colonies with smooth, creamy, viscous or pasty appearance, yeast must be considered. Dematiaccous mold produces colonies that are dark. For molds which grow within 3-5 days have a distinct border, with white patel on the surface and for the molds that grow in 7-14 days or that have asymmetrical aerial mycelium, the dimorphic species should be considered.

Sabouraud dextrose agar-It is commonly used for opportunistic and pathogenic fungi.

Sabouraud dextrose agar with antibiotics-It is preferred for specimen where bacterial contamination is suspected.

Chlamydospore agar/corn meal agar-It is preferred for chlamydospore formation for Candida.

Brain heart infusion agar without antibiotics-For fastidious organism

Brain heart infusion agar with antibiotics-Used for yeast phase, the growth of dimorphic fungi.

Potato Dextrose Agar-In PDA, usually two culture with the specimens are done and the plates are incubated one at 25C and other at 37C kept for more than one week. Identification can be done by studying the cultural characteristics like morphology of the culture color and structure of the growth. Similarly, the morphology of conidia and spores produced as well as hyphae quickly the fungus grows and at what temperature the fungus grow.Example Trichophyton rubrum produces wine red diffusible pigment.

Culture ofCandida albicans

C. albicans usually grows in most bacterial culture media as well as fungal culture medium on blood agar under 24-48 hrs incubation, the colonies are white, opaque and convex. On the fungal culture medium like Sabouraud’s agar colonies are smooth, creamy white with yeast like smell.

Culture ofCryptococcus neoformans

C. neoformans is easily cultured on routine fungal culture media. It may appear on bacterial cultural media like blood agar. On BA or Sabouraud’s agar, C. neoformans produces moist creamy white colored mucoid colonies usually after 2-3 days of incubation. When examined microscopically, the yeasty cell appears capsulated but the capsules are often smaller than seen in specimens and occasional capsule may be absent.

Culture ofHistoplasma capsulatum

capsulatum is a dimorphic fungus so it can grow in artificial media at 25-30C as well as at 37C. Specimens when inoculated on Sabouraud’s dextrose agar or brain heart infusion agar in the presence of chloromphenicol and cycloheximide which is then incubated at 25C for 2-6 days, the samples yield white cottony mycelium. Small thin walled, smooth, round microconidia and characteristic large, round, spiny or tubercle macroconidia are also produced. At 37C yeast phase of this dimorphic fungus induces.

Culture ofAspergillus fumigatus

A.fumigatus is a rapidly growing mold (2-6 days) that produces fluffy to granular, white to a blue-green colony on Sabourauds’s dextrose agar. Matured sporulating colonies most often show the blue-green powdery appearance. Microscopically, A. fumigatus is characterized by the presence of septate hyphae with conidiophores on it. The tip of the conidiophores expands into a large dome-shaped that has bottle shaped phialides covering the upper half or two-thirds of its surface. Long chains of rough-walled green conidia, small, spherical form a columnar mass. The culture of A. fumigatus is thermo-tolerant and is able to withstand temperature up to 45C.

A.fumigatus produces yellow-green colonies on Sabouraud’s dextrose agar. However, A.fumigatus produces darkly pigmented rough spores. Growth begins initially as a yellow colony that soon develops a black dotted surface as conidia are produced.

Culture of Dermatophytes

Here, the culture is needed to identify the species of ringworm fungus. A typical; microbiological medium such as malt agar or Sabouraud’s agar is used for the cultivation of dermatophytes. Incubation temperature is maintained at 28-30C which is the optimum temperature for all except one of the dermatophytes, Trichophyton verrucosum, which grows best at 37C. The incubation period is usually for 2-3 weeks. Selective antibiotics for inhibition of contaminating bacterial and antifungal agents such a cycloheximide for reduction of contaminating fungi may be used in the culture media. The dermatophytes are distinguished on the basis of hyphae structure and structure of macroconidia.

If no growth from positive material

Failure to isolate a fungus from a specimen of skin or hair in which hyphae were seen in the microscopy sample can usually be attributed to:

• Insufficient or incorrectly collected material for culture.
• Incorrect storage such as refrigeration or holding material in a container that retains moisture.
• Prior treatment of the lesions with an agent possessing antifungal activity.

Incubation of fungal culture

The fungal sample is cultured in two set of culture media and is incubated at two different temperatures at 30C (Room temperature) and at 35C. All fungal cultures are incubated for a minimum of 30 days before discarding as negative. During culture of the specimen, either use of culture plate or tube is optional. In the case of a tube, the media is poured in a thick slant to prevent dehydration during a long time incubation period. After the medium is inoculated, the screw cap is not closed tightly because fungi require breathing.

Culture media in tube have an advantage during transport but has limitation to prepare stained mounts for microscopic examination. Similarly, Petri dishes have the advantages of providing a larger surface for growth resulting in better colony separation and making the cultures easier to examine and subculture. Also, transparency tape preparations are effectively made from plate cultures. The disadvantage of using the plates results dehydrated during prolonged incubation to prevent drying the plates may be placed into sealed, or the edges are sealed by oxygen permeable tape.

REFERENCE

Cheesbrough, M. Medical Laboratory Manual for Tropical Countries. Vol. Vol 2. ELBS London, 2007.

Tille, P. Diagnostic Microbiology. 13th. Elsevier, 2014.

D, Grenwood, Slack RCB and Peutherer J. Medical Microbiology. Dunclude Livingstone: ELBS, 2001.