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Frequently asked questions and answers on Fungi. In this article we will discuss about:- 1. Definition of Fungi 2. Origin of Fungi 3. Metabolism 4. Characteristics 5. Structures 6. Reproduction 7. Classification 8. Importance 9. Spore Forms 10. Laboratory Diagnosis.
Contents:
- Questions and Answers # Definition of Fungi
- Questions and Answers # Origin of Fungi
- Questions and Answers # Metabolism of Fungi
- Questions and Answers # Characteristics of Fungi
- Questions and Answers # Structures of Fungi
- Questions and Answers # Reproduction of Fungi
- Questions and Answers # Classification of Fungi
- Questions and Answers # Importance of Fungi
- Questions and Answers # Spore Forms in Fungi
- Questions and Answers # Laboratory Diagnosis of Fungal Infection
Questions and Answers # 1. Definition of Fungi:
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The fungi are more evolutionarily advanced forms of microorganisms, as compared to ” the prokaryotes (prions, viruses, bacteria). They are classified as eukaryotes. i.e., they have a diploid number of chromosomes and a nuclear membrane and have sterols in their plasma membrane. Genetic complexity allows morphologic complexity and thus these organisms have complex structural features that are used in speciation.
Fungi can be divided into two basic morphological forms, yeasts and hyphae. Yeasts are unicellular fungi which reproduce asexually by blastoconidia formation (budding) or fission. Hyphae are multi-cellular fungi which reproduce asexually and/or sexually. Dimorphism is the condition where by a fungus can exhibit either the yeast form or the hyphal form, depending on growth conditions.
Very few fungi exhibit dimorphism. Most fungi occur in the hyphae form as branching, threadlike tubular filaments. These filamentous structures either lack cross walls (coenocytic) or have cross walls (septate) depending on the species. In some cases septate hyphae develop clamp connections at the septa which connect the hyphal elements.
A mass of hyphal elements is termed the mycelium (synonymous with mold). Aerial hyphae often produce asexual reproduction propagates termed conidia (synonymous with spores). Relatively large and complex conidia are termed macroconidia while the smaller and simpler conidia are termed microconidias.
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When the conidia are enclosed in a sac (the sporangium), they are called endospores. The presence/absence of conidia and their size, shape and location are major features used in the laboratory to identify the species of fungus in clinical specimens.
Asexual reproduction, via conidia formation, does not involve genetic recombination between two sexual types whereas sexual reproduction does involve genetic recombination between two sexual types.
Questions and Answers # 2. Origin of Fungi:
Ireland of the 1840s was an economically depressed country of eight million people. Most were tenant farmers paying rent to landlords who were responsible, in turn, to the English owners of the property. The sole crop of Irish farmers was potatoes, grown season after season on small tracts of land. What little corn was available was usually fed to the cows and pigs. Early in the decade, heavy rains and dampness portended calamity.
Then, on August 23, 1845, The Gardener’s Chronicle and Agricultural Gazette reported:
“A fatal malady has broken out amongst the potato crop. On all sides we hear of the destruction. In Belgium, the fields are said to have been completely desolated.”
The potatoes had suffered before. There had been scab, drought, “curl,” and too much rain, but nothing was quite so destructive as this new disease. It struck down the plants like frost in the summer. Beginning as black spots, it decayed the leaves and stems, and left the potatoes a rotten, mushy mass with a peculiar and offensive odor. Even the harvested potatoes rotted.
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The winter of 1845-1846 was a disaster for Ireland. Farmers left the rotten potatoes in the fields, and the disease spread. First the farmers ate the animal feed and then the animals. They also devoured the seed potatoes, leaving nothing for spring planting.
As starvation spread, the English government attempted to help by importing corn and establishing relief centers. In England, the potato disease had few repercussions because English agriculture included various grains, but in Ireland famine spread quickly.
After two years, the potato rot seemed to slacken, but in 1847 it was back with a vengeance. Despite relief efforts by the English, over two million Irish suffered death from starvation. Eventually, about 900,000 survivors set off for Canada and the United States. Those who stayed had to deal with economic and political upheaval as well as misery and death.
The potato blight faded in 1848, but did not vanish. Instead, it emerged again during wet seasons and blossomed anew. In the end, hundreds of thousands of Irish left the land and moved to cities or foreign countries. During the 1860s, great waves of Irish immigrants came to the United States. Many Americans are descended from those starving, demoralized farmers.
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Such are the historic, political, economic, and sociological effects of one species of fungus. Other fungal diseases of fruits, grains, and vegetables can be equally devastating. In addition, we shall take note of several widespread human and animal diseases that are due to fungi and we shall encounter many beneficial fungi such as those used to make antibiotics, bread, and foods or used as insecticides. Our study will begin with a focus on the structures, growth patterns, and life cycles of fungi.
Questions and Answers # 3. Metabolism
of Fungi:
All fungi are free living, i.e., they are not obligate intracellular parasites. They do not contain chlorophyll and cannot synthesize macromolecules from carbon dioxide and energy derived from light rays. Therefore all fungi are heterotrophs, living on preformed organic matter.
For medical purposes the important aspects of fungal metabolism are:
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1. The synthesis of chitin, a polymer of N-acetyl glucosamine, and other compounds, for use in forming the cell wall. These induce immune hypersensitivity.
2. The synthesis of ergosterol for incorporation into the plasma membrane. This makes the plasma membrane sensitive to those antimicrobial agents which either block the synthesis of ergosterol or prevent its incorporation into the membrane or bind to it, e.g. amphotericin B.
3. The synthesis of toxins such as:
(a) Ergot alkaloids – these are produced by Claviceps purpurea and cause an alpha adrenergic blockade
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(b) Psychotropic agents – these include psilocybin, psilocin and lysergic acid diethylamide (LSD)
(c) Aflatoxins – these are carcinogens produced by Aspergillus flavus when growing on grain. When these grains are eaten by humans or when they are fed to dairy cattle and they get into the milk supply, they affect humans.
4. The synthesis of proteins on ribosomes that are different from those found in bacteria. This makes the fungi immune to those antimicrobial agents that are directed against the bacterial ribosome, e.g., chloramphenicol.
5. The ability of certain metabolites to alter morphology of yeast and/or be assimilated by yeast with concomitant clinical identification affects.
Questions and Answers # 4. Characteristics
of Fungi:
1. Eukaryotic
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2. Do not contain chlorophyll
3. Nonphotosynthetic
4. Absorptive heterotrophs – digest food first and then absorb it into their bodies.
5. Release digestive enzymes to break down organic material or their host.
6. Store food energy as glycogen
7. Most are saprobes – live on other dead organisms
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8. Important decomposers and recyclers of nutrients in the environment
9. Most are multicellular, but some unicellular like yeast
10. Some are internal or external parasites; a few are predators that capture prey
11. Nonmotile
12. Lack true roots, stems, and leaves
13. Cell walls are made of chitin (a complex polysaccharide)
14. Grow as microscopic tubes or filaments called hyphae that contain cytoplasm and nuclei
15. Hyphal networks are called mycelium
16. Some are edible
17. Reproduce by sexual and asexual spores
18. Antibiotic penicillin comes from Penicillium mold
19. Classified by their sexual reproductive structures
20. Grow best in warm, moist environments preferring shade
21. Mycology – study of fungi
22. Fungicide – chemicals used to kill fungi
23. Includes yeasts, molds, mushrooms, ringworm, puffballs, rusts, smuts, etc.
24. Fungi may have evolved from prokaryotes by endosymbiosis.
Questions and Answers # 5. Structures of Fungi
:
1. Body of a fungus made of tiny filaments or tubes called hyphae.
2. Hyphae contain cytoplasm and nuclei and have a cell wall of chitin.
3. Each hyphae is one continuous cell.
4. Hyphae continually grow and branch.
5. Septum (septa-plural) is cross walls with pores to allow the movement of cytoplasm in hyphae.
6. Hyphae with septa are called septate hyphae.
7. Hyphae without septa are called coenocytic hyphae.
8. Tangled mats of hyphae are known as mycelium
9. All hyphae within a mycelium share the same cytoplasm so materials move quickly.
10. Hyphae grow rapidly from the tips by cell division.
11. Stolon is horizontal hyphae that connect groups of hyphae to each other.
12. Rhizoids are root like parts of hyphae that anchor the fungus.
1. Most fungi reproduce asexually and sexually.
2. Asexual reproduction produces genetically identical organisms and is the most common method used.
3. Sexual reproduction in fungi occurs when nutrients or water are scarce.
4. Fruiting bodies are modified hyphae that make asexual spores.
5. Fruiting bodies consist of an upright stalk or sporangiophore with a sac containing spores called the sporangium.
6. Types of fruiting bodies include basidia, sporangia, and ascus.
7. Spores – haploid cells with dehydrated cytoplasm and a protective coat capable of developing into new individuals.
8. Wind, animals, water, and insects spread spores.
9. When spore lands on moist surface, new hyphae form.
Questions and Answers # 6. Reproduction of Fungi:
Asexual Reproduction in Fungi:
1. Fungi reproduce asexually when environmental conditions are favorable.
2. Some unicellular fungi reproduce by mitosis.
3. Yeast cells reproduce by budding where a part of the cell pinches off to produce more yeast cells.
4. Athlete’s foot fungus reproduces by fragmentation from a small piece of mycelium.
5. Most fungi reproduce asexually by spores.
6. Penicillium mold produces spores called conidia without a protective sac on the top of a stalk called the conidiophore.
1. Fungi reproduce sexually when environmental conditions are unfavorable.
2. No male or female fungi.
3. Two mating types – plus (+) and minus (-)
4. Fertilization occurs when (+) hyphae fuse with (-) hyphae to form a 2N or diploid zygote.
5. Some fungi show dimorphism (ability to change their form in response to their environmental conditions)
Questions and Answers # 7. Classification of Fungi:
Fungi are classified by their reproductive structures. The 4 phyla of fungi are Basidiomycota, Zygomycota, Ascomycota, and Deuteromycota.
1. Called sporangium fungi or common molds.
2. Includes molds and blings such as blights such as Rhizopus stolonifer (bread mold).
3. No septa in hyphae (coenocytic).
4. Asexual reproductive structure called sporangium and produces sporangiospores.
5. Rhizoids anchor the mold, release digestive enzymes, and absorb food.
6. Asexual reproductive structure called sporangium and produces sporangiospores.
7. Sexual spore produced by conjugation when (+) hyphae and (-) fuse is called zygospore.
8. Zygospores can endure harsh environments until conditions improve and new sporangium.
1. Called club fungi
2. Includes mushrooms, toadstools, puffballs, bracket fungi, shelf fungi, stinkhorns, rusts, and smuts
3. Some are used as food (mushroom) and others cause crop damage (rusts and smuts)
4. Seldom reproduce asexually
5. Basdiocarp made up of stalk called the stipe and a flattened cap.
6. Stipe may have a skirt like ring below cap called the annulus.
7. Gills are found on the underside of the cap and are lined with basidia.
8. Basidium – sexual reproductive structure that make basidiospores.
9. Basidiospores are released from the gills and germinate to form new hyphae and mycelia.
10. Vegetative structures found below ground and include rhizoids (anchor and absorb nutrients), hyphae, and mycelia.
1. Called sac fungi
2. Includes yeast, cup fungi, truffles, powdery mildew, and morels
IV. Mycelia:
1. Some are parasites causing Dutch elm disease and chestnut blight.
2. Sac Fungi can reproduce both sexually and asexually.
3. Yeast reproduces asexually by budding (form small, bud-like cells that break off and make more yeast)
4. Asexual spores called conidia form on the tips of specialized hyphae called condiophores.
5. Ascocarp – specialized hyphae formed by parent fungi during sexual reproduction.
6. Ascus – sacs within the ascocarp that form spores called ascospores.
1. Symbiotic association between a sac fungus and a photosynthetic green algae or cyanobacteria.
2. Both organisms benefit (algae makes food and fungus supplies moisture, shelter, and anchorage)
3. Grow on rocks, trees, buildings, etc. and help form soil.
4. Crustose lichens grow on rocks and trees; fructose lichens grow shrub-like; foliose lichens grow mat-like on the soil.
VI. Mycorrhizae:
1. Symbiotic association of a fungus living on plant roots.
2. Most plants have mycorrhizae on their roots.
3. Fungus absorbs sugars made by plant.
4. Plants absorb more water and minerals with aid of the fungus.
Questions and Answers # 8. Importance of Fungi
:
1. Fungal spores cause allergies
2. Molds, mildew, rust, and smuts damage crops
3. Yeasts are used to make beer and bread
4. Antibiotic penicillin
5. Decomposers and recyclers of nutrients
6. Mushrooms eaten as food
7. Help form blue cheeses
8. Aspergillus is used to make soy sauce
9. Cause athlete’s foot and ringworm
10. Amanita is poisonous mushroom
11. Can cause yeast infections
Disease caused by fungi.
Candida:
Species are important human pathogens that are best known for causing opportunist infections in immuno compromised hosts (e.g. transplant patients, AIDS sufferers, cancer patients). Infections are difficult to treat and can be very serious – 30-40% of systemic infections result in death.
The sequencing of the genome of C. albicans and those of several other medically relevant Candida species has provided a major impetus for Candida comparative and functional genomic analyses. These studies are aiding the development of sensitive diagnostic strategies and novel antifungal therapies.
Aerosolized:
Aspergillus spores are found nearly everywhere so we are routinely and almost constantly exposed to them. Such exposure is a normal part of the human condition and generally poses no adverse health effects. Nevertheless, Aspergillus can and does cause disease in three major ways: through the production of mycotoxins; through induction of allergenic responses; and through localized or systemic infections. With the latter two categories, the immune status of the host is pivotal.
Allergies and asthma are thought to be caused by an active host immune response against the presence of fungal spores or hyphae. In contrast, with invasive aspergillosis, the immune system has collapsed and little or no defence can be mounted.
The most common pathogenic species are Aspergillus fumigatus and Aspergillus flavus. Aspergillus flavus produces aflatoxin which is both a toxin and a carcinogen and which can potentially contaminate foods such as nuts. Aspergillus fumigatus and Aspergillus clavatus can cause allergic disease.
Some Aspergillus species cause disease on grain crops, especially maize, and synthesize mycotoxins including aflatoxin. Aspergillosis is the group of diseases caused by Aspergillus. The symptoms include fever, cough, chest pain or breathlessness. Usually, only patients with weakened immune systems or with other lung conditions are susceptible.
Cryptococcus:
Cryptococcus neoformans can cause a severe form of meningitis and meningoencephalitis in patients with HIV infection and AIDS. The majority of Cryptococcus species lives in the soil and do not cause disease in humans. Cryptococcus neoformans is the major human and animal pathogen.
Cryptococcus laurentii and Cryptococcus albidus have been known to occasionally cause moderate-to-severe disease in human patients with compromised immunity. Cryptococcus gattii is endemic to tropical parts of the continent of Africa and Australia and can cause disease in non-immunocompromised people
Histoplasma capsulatum can cause histoplasmosis in humans, dogs and cats. The fungus is most prevalent in the Americas, India and southeastern Asia. It is endemic in certain areas of the United States. Infection is usually due to inhaling contaminated air.
Pneumocystis jirovecii (or Pneumocystis carinii) can cause a form of pneumonia in people with weakened immune systems, such as premature children, the elderly, and AIDS patients.
Stachybotrys chartarum or “black mold” can cause respiratory damage and severe headaches. It frequently occurs in houses in regions that are chronically damp.
Questions and Answers # 9. Spore Forms in Fungi:
Fungi reproduce both asexually and sexually by different types of spores. Some spores are the unit of dispersal, but others function as resting spores to overcome the unfavourable period, and some others have the efficiency to serve both the above functions.
The spores are mainly divided into two groups:
A. Asexual spores, and
B. Sexual spores.
A. Asexual Spores:
The spores formed during asexual reproduction are the asexual spores. Basically the asexual spores are of two types: sporangiospores, and conidia.
The sporangiospores are developed inside a sac-like structure, the sporangium.
Sporangiospores are of two types, zoospores and aplanospores:
1. Zoospores:
Zoospores (Fig. 4.4) are the characteristic spore of the subdivision Mastigomycotina. Three types of zoospores are produced in this group.
These are:
i. Anteriorly uniflagellate, these are pyriform in shape having one whiplash (Synchytrium) or tinsel (Rhizidiomyces) type of flagellum anteriorly.
ii. Anteriorly biflagellate. These are pyriform in shape having two flagella (one whiplash and one tinsel type), placed anteriorly., e.g., Saprolegnia etc.
iii. Laterally biflagellate. These are kidney-shaped having two flagella (one whiplash and one tinsel type), placed laterally, e.g., Phytophthora, Pythium etc.
2. Aplanospores:
The sporangiospores of this type are devoid of flagella and are called aplanospores (Fig. 4.8L). It is the characteristic spore of Mucorales under Zygomycetes, e.g., Mucor, Rhizopus etc.
The conidia (Fig. 4.8A, B, C) differ from aplanospores because they are not enclosed by a separate sporangial wall. They are usually developed at the apex of hyphae.
Conidia show variability in both their form and development. They are mainly divided on the basis of their mode of development.
There are two basic modes of development of conidia: bias- tic and thallic. In blastic type, marked enlargement of conidium initial takes place before it is delimited by the septum. In thallic type, enlargement of the conidium initial takes place only after the initial has been delimited by the septum.
Blastic Types:
These are divided into two types:
1. Blastoconidia. They can develop from a cell of an undifferentiated hypha or conidiophore at o ;e or more points through budding, either apically or laterally. They may develop either singly or in chains, e.g., Aureobasidium, Cladosporium etc.
2. Phialoconidia. They develop from a bottle-shaped cell, the phialide. Single conidium reaches full size before being cut off by septation and immediately another one develops underneath. Thus generally a chain of conidia are developed, where younger one always remains near the mother, e.g., Tricho- derma, Fusarium.
Thallic Types:
1. Thalloconidia. In this type, the conidia are developed on hypha either singly or in short or long, branched or unbranched chains and enlargement of the conidium initial takes place only after the initial has been delimited by septum, e.g., Erysiphe, Penicillium, Geotrichum, Oidium etc.
B. Sexual Spores:
The spores formed after sexual reproduction are the sexual spores. There are four types of sexual spores in fungi.
These are oospores, zygospores, ascospores and basidiospores:
1. Oospores:
These are diploid (2n) spores formed by the union between egg and male nucleus. Coming in contact with the oogonium, the antheridium passes its nucleus inside the oogonium through fertilisation tube and forms oospore. It is a resting spore, e.g., members of Oomycetes under Mastigomycotina.
2. Zygospores:
Like Oospore, it is also a resting spore formed by the union of two gametangia into a single cell. The cell develops into a thick-walled, black and warty structure, the Zygospore, e.g., members of Zygomycetes. In majority of the members of Mastigomycotina and Zygomycotina, the sexually produced spores are developed freely and are not surrounded by sterile hyphae. But in Ascomycotina and Basidiomycotina, the spores are commonly surrounded by sterile hyphae and are called ascocarps and basidiocarps respectively.
3. Ascospores:
Ascospores are developed by the union of two gametangia. The ascospores are always developed in (i.e., endogenously) a sac-like structure, the ascus, and thus the fungi containing asci are commonly called sac fungi.
The ascospores are uninucleate and unicellular; generally oval, round or elongated structure. They are generally 8 in an ascus, but may be 4 (Saccharomyces cerevisiae, Saccharomycodes ludwigii) or numerous.
4. Basidiospores:
The basidiospores are developed on (i.e., exogenously) basidia. The basidia are developed from the dikaryotic cell. During this process a long gap is generally maintained between plasmogamy and karyogamy. Basidia are usually of two types: Holobasidium i.e., aseptate basidium and Phragmobasidium.
The basidiospores are unicellular, uninucleate, thin walled; generally round to oval structure, developed on basidium. They are generally four in number per basidium but may be two or many (e.g., Ustigo nuda tritici).
Questions and Answers # 10. Laboratory Diagnosis of Fungal Infection:
Alkali Wet Prep:
The KOH test for fungus is conducted on an outpatient basis and patients do not need to prepare in advance. Results are usually available while the patient waits or the next day if sent to a clinical laboratory. The KOH test procedure may be performed by a physician, physician assistant, medical assistant, nurse, or medical laboratory technician. If fungal cultures are required, the test is performed by a technologist who specializes in microbiology.
1. Collection – Skin, nail, or hair samples are collected from the infected area on the patient. For skin samples, a scalpel or edge of a glass slide is used to gently scrape skin scales from the infected area. For hair samples, a forceps is used to remove hair shafts and follicles from the infected site. If the test is being sent to a laboratory, the scrapings are placed in a sterile covered container.
2. The scrapings are placed directly onto a microscope slide and are covered with 10% or 20% potassium hydroxide.
3. The slide is left to stand until clear, normally between five and fifteen minutes, in order to dissolve skin cells, hair, and debris.
4. To enhance clearing dimethyl sulfoxide can be added to the slide. To make the fungi easier to see lactophenol cotton blue stain can be added.
5. The slide is gently heated to speed up the action of the KOH.
6. Adding calcofluor-white stain to the slide will cause the fungi to become fluorescent, making them easier to identify under a fluorescent microscope.
7. Place the slide under a microscope to read.
Dermatophytes are easily recognized under the microscope by their long branch-like tubular structures called hyphae. Fungi causing ringworm infections produce septate (segmented) hyphae. Some show the presence of spores formed directly from the hyphae (arthroconidia).
Under the microscope Tinea versicolor is recognized by curved hyphae and round yeast forms that give it a spaghetti-and-meatball appearance. Yeast cells appear round or oval and budding forms may be seen. The KOH prep cannot identify the specific organism; the specimen can be submitted for fungal culture to identify the organism.
8. A normal, or negative, KOH test shows no fungi (no dermatophytes or yeast). Dermatophytes or yeast seen on a KOH test indicate the person has a fungal infection. Follow-up tests are usually unnecessary.
9. The skin may be sore after the test because of the tissue being scraped off the top of the surface of the skin.
Specimen Collection:
Specimens should be collected in sterile containers or with sterile swabs and transported immediately to the laboratory. This product is used in conjunction with other biochemical and serological tests to identify cultures of isolated organisms.
Method of Use:
Mix the specimen with a small drop of India on a clean glass slide. Place a cover slip over the smear and press gently. The preparation should be brownish, not black. Using reduced examine the smear microscopically (100X) for the presence of encapsulated cells as indicated by clear zones surrounding the cells.
Note – The India is ready to use. Further dilution with water is not recommended.
Note – Production of capsular material may be increased by cultivation in a 1% peptone solution (Peptone Broth).
Fungal Culture:
Fungal cultures are a test to try and ascertain the type of fungal organism that is responsible for an infection or even the presence of the organism in the first place. Fungi are one of the five classes of pathogens that cause disease; the other being viruses, bacteria, prions, and helminthes.
It is very important that the correct organism is identified during an infection because the drug treatments differ according to the type of organism and one treatment would not work for the other.
A fungus culture is usually acquired from wound exudates and swabs of areas that are within the body like the mouth and vagina. Very rarely can fungi cause infections within the body because the human immune system is more than adapt at eliminating fungi.
However, sometimes, a fungal blood culture might need to be done because of a condition called fungal sepsis. Sepsis is the presence of bacteria or an infection in the blood. This is a very rare condition and a medical emergency as well. The fungal culture test that is performed in this condition will usually turn up the Candida fungus as the causative organism, though the list is quite endless.
Reasons to Conduct Fungal Culture Test:
A fungal culture is taken from a part of the body in secretions or by taking a blood sample. Most infections with fungi are limited to the skin, oral mucosa, or the genitals. Very rarely does fungal sepsis actually occur unless a patient is severely immune-compromised. This is usually the case in patients with AIDS or with diabetes. Both of these diseases will cause the immune system to stop working altogether giving rise to all sorts of opportunistic infections.
A fungal growth is acquired by analyzing the secretions and exudates from a wound or by swabbing a surface that is infected. A blood sample might also be required for checking for the presence of fungi in the blood. The sample is then cultured in a fungus-friendly environment for up to a week or more before the fungal colonies become visible.
This stain is prepared over two days.
1. On the first day, dissolve the Cotton Blue in the distilled water. Leave overnight to eliminate insoluble dye.
2. On the second day, wearing gloves add the phenol crystals to the lactic acid in a glass beaker, place on magnetic stirrer until the phenol is dissolved.
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3. Add the glycerol.
4. Filter the Cotton Blue and distilled water solution into the phenol/glycerol/lactic acid solution. Mix and store at room temperature.
Other Microscopy Techniques and Stains:
1. Calcofluor White with 10% KOH
2. Potassium Hydroxide (KOH) with Chlorazol Black
3. KOH-DMSO Preparation
4. India Ink Mounts
5. Cello tape Flag Preparations
6. Slide Culture Preparations
7. Southgate’s Mucicarmine stain
8. Periodic acid-Schiff (PAS) and PAS Digest stain
9. Grocott’s Methenamine Silver (GMS) stain
Cryptococcus neoformans, because of its large polysaccharide capsule, can be visualized by the India stain. Organisms that possess a polysaccharide capsule exhibit a halo around the cell against the black background created by the India.
