Introduction

The Fungi Kingdom includes spore-forming eukaryotes that lack undulipodia (amastigote) at all stages of their life cycle, but some taxonomists include chytrids and oomycetes, which are undulipodia, in the Kingdom.

Almost all fungi are aerobic, and all fungi are heterotrophs, which means that they absorb their food without ingesting it. Fungi secrete powerful digestive enzymes that break down the food material, usually decaying plant or animal matter, into compounds that can be absorbed through the fungal membrane.

Fungi are abundant in most terrestrial habitats. There are about 100,000 known species, a few of which are marine. Because of their abundance and diversity, there are likely many more species to be discovered and described.

Reproduction in fungi is generally asexual and occurs through vegetative spores (conidia). These spores are distributed by wind and water, and are highly resistant to adverse environmental conditions. Under favorable conditions they will germinate and initiate the growth of a new organism. The germinating spore produces a thin, tubular structure called a hypha. Cross walls called septa divide the hypha into “cells”, though the septa rarely completely enclose an entire portion of the hypha. Some hyphae lack septa entirely. Hyphae grow in large masses called mycelia, which constitutes the vegetative body of the fungus. The cell walls of most fungi are composed of chitin, similar to that found in insects, which is very impervious to desiccation, thus enabling the fungi to survive under harsh conditions (fig. 1).

Sexual reproduction can also occur in fungi. This occurs when hyphae of opposite mating types grow together and fuse. The haploid hyphal nuclei grow and undergo subsequent division, but they remain in pairs, one nucleus from each of the two parental hyphae. Such hyphae are called dikaryotic. Nuclear fusion occurs in time, forming diploid zygotes. The zygote then immediately undergoes meiosis to form haploid spores that are distributed in the same manner as the vegetative spores.

Sexual reproduction modes and structures form the basis for classifying the major groups of fungi. There are four phyla of fungi.

1.Zygomycota. These are the “algae-like” fungi. There are no septa in the hyphae, though the reproductive structures are separated from the rest of the mycelium. Genetic material is exchanged in thick-walled zygospores, which are formed by the conjugation of opposite mating types, and then the haploid spores are released from the zygospores. Asexual reproduction is by conidia, resistant spores that develop within the sporangium. The common bread molds are examples. Some examples are common bread molds and parasites of protists, nematodes, insects, and small animals (fig. 2).

2. Ascomycota. The mycelium of Ascomycetes form a cottony mass of multi- branched hyphae. This phylum is characterized by sac-like reproductive structures called asci, which result from the conjugation of two compatible mating strains. Each ascus typically contains eight ascospores, which form when the short-lived diploid nuclei undergo meiosis. Many serious plant pathogens are in this phylum, including Dutch Elm Disease and apple scab. The group also includes beneficial yeasts (vital to baking and brewing industries) and highly prized edible fungi, including the morels and truffles (fig. 3).

3. Basidiomycota. The phylum is distinguished by its reproductive structure, the club-like basidium, which contains the products of sexual reproduction called basidiospores. Germinating basidiospores produce a mycelium that develops septa as it grows. Hyphae of compatible mating types conjugate to form a secondary mycelium. A tertiary mycelium develops to form the familiar reproductive structures of mushrooms, puffballs, and bracket fungi. Some basidiomycetes, the rusts and smuts, are serious crop pests, while others can be used for food or medical products. Some members of this phyla can also be deadly because of their poison. (fig. 4)

4. Deuteromycota. These fungi are often termed “fungi imperfecti” because they lack structures for sexual reproduction. Only asexual reproduction is known to occur naturally, although some genetic recombination has occurred under laboratory conditions. Germinating spores produce mycelia with septate hyphae. The phylum includes economically important species like the pathogenic Candida, as well as Penicillium, source of the powerful antibiotic Penicillin. It also includes an interesting genus of predaceous fungi, Arthrobotrys, which actively captures and feeds upon nematodes. (fig. 5)

Biotechnology has enabled us to use the unique biochemical makeup of these organisms to our advantage. Fungi are important in the processing of many foods, including tea, coffee, cheese, vinegar, beverages, and breads. They are also influential in the manufacture of industrial chemicals such as acetone, alcohols, and citric acid, as well as in the production of antibiotics and vitamins. Last, but by no means least, is the recycling of organic matter. Fungi are vitally important in initiating the breakdown of matter through decay, helping to return vital nutrients to the soil.

Why WARD’S is Better

• We maintain all cultures in our own laboratory
• Cultures are maintained under optimum conditions by subculturing regularly
• You are assured of getting fresh material in the maximum growth phase
• Cultures are available in a number of formats to meet specific teaching requirements
  • Tubes
  • Plates (two week lead time may be required)
  • Freeze-dried
  • Jars
• Fully labeled cultures indicate species, media, and incubation temperature
• Instructional literature is supplied with every order

Care of cultures

Unpack your shipment carefully. Read the labels on the culture containers for information on storage temperatures and conditions. Most fungi cultures can be kept at room temperature, where they will last for several weeks if kept in subdued light. Avoid excessively high temperatures and keep out of direct sunlight. Loosen the caps on tube cultures – fungi are aerobic. Lyophilized (freeze-dried) cultures can be stored at room temperature if you intend to use them within a week. Refrigeration or freezing is recommended for long-term storage.

Handling Cultures

Precautions: Sterile technique is essential in handling all cultures of fungi. This will eliminate any risk of contamination in the culture, and, more importantly, will protect you and your students from accidental exposures to the organisms. Even though most fungi are non-pathogenic, they should be treated as potentially pathogenic to eliminate all possible risks of exposure.

1. All media and glassware should be sterilized prior to use. An autoclave or pressure cooker at 15 psi for 15 minutes at 100°C or dry heat sterilization of glassware at 160°C for two hours will suffice. WARD’S prepared media has already been sterilized for your convenience.
2. Make sure that the work area is free of clutter. Wash the work surface with a strong disinfectant, such as 5% Lysol or 70% alcohol, both before and after use.
3. Wear a lab coat, smock, or apron to protect your clothes and reduce further the chance of contaminating the cultures.
4. Avoid any hand-to-mouth operations such as eating, drinking, smoking, or licking adhesive labels while in the lab.
5. Wash hands thoroughly with soap and water both before and after working with cultures.

Culture Transfers

Materials needed for slants: Inoculating loop or needle, Bunsen burner or similar flame source (CAUTION – do not use alcohol near an open flame), culture, second container with media for culture transfer.

Slants: Follow the sequence of drawings Fig. 6a-e for a successful transfer

1. Hold both tubes in the left hand (fig. 6a)
2. Hold the inoculating loop or needle in your right hand. Pass the entire length of the wire through the flame until it all has been red hot (fig. 6b)
3. While still holding the needle, quickly remove the caps (or plugs) from the tubes, holding them between the fingers of the right hand (fig. 6c).
4. Flame the mouths of both culture tubes by passing them two or three times through the flame (fig. 6d). Hold the
   tubes almost parallel to the work surface, to reduce the possibility of air-borne contaminants.
5. Touch the needle or loop to the medium in the culture tube, to be sure it is cooled, then to the culture mass, making sure that a portion of the fungal culture adheres to the loop or needle (you do not need a large amount) (fig. 6e).
6. Quickly withdraw the needle or loop and insert it into the second culture tube, gently wiping it across the surface of the agar.
7. Flame the mouths of each tube as before and replace the caps or plugs.
8. Flame the inoculating needle or loop until it is red hot. You have now completed the tube transfer.

Materials needed for plates: Sterile Petri plates, bottled media, large beaker, hot plate, thermometer, inoculating needle or loop, Bunsen or similar flame source

Plates: Use sterile technique as outlined above.

To make plates using bottled solid media:

1. Place the bottle of media in a large beaker. Add water to the beaker until the water level is just above the level of the medium in the bottle.
2. Loosen the cap on the bottle.
3. Put a thermometer in the beaker; set it on a hot plate and heat the water to boiling.
4. Meanwhile, set the Petri plates (each bottle of media will supply 5 – 7 plates, depending on the thickness poured) on the work surface
5. Boil the water gently for several minutes until the medium is completely liquefied. Swirl the bottle gently to be
   sure all is melted.
6. Turn off the heat. Allow the water temperature to cool to between 45°C and 50°C.
7. Using an insulated glove, pick up the bottle, remove the cap, and flame the bottle mouth.
8. Lift the lid of a Petri plate just enough to admit the neck of the bottle, and pour the first plate, using just enough medium to slightly more than cover the bottom of the plate. Replace the lid.
9. Swirl the plate gently to distribute the medium.
10. Flame the mouth of the bottle and proceed to pour the rest of the plates

To transfer cultures to plates:

1. Follow aseptic technique as described for slants
2. Lift the lid of the Petri dish just enough to introduce the inoculating loop. Gently wipe the needle or loop across the culture (you only need a small amount ).
3. Replace the lid, and then lift the lid of the sterile petri dish containing fresh medium just enough to introduce the needle or loop. Gently wipe the needle or loop across the surface of the media, then replace the lid and sterilize the needle or loop as before. You have now transferred the fungal culture to the plate.

Lyophilized (Freeze-dried) Cultures: Specimens to be lyophilized are grown under optimal conditions to achieve the maximum growth rate. 0.5 ml cell suspensions are then taken and pipetted into sterile vials and freeze-dried. Instructions for rehydrating freeze-dried cultures are included with each culture that we ship. The great advantage of lyophilized cultures is their longevity. Unopened cultures can be stored under refrigeration for ten years or more. Cultures stored at room temperature can be kept for up to two years. Most media supplied with lyophilized cultures can be stored for up to six months. Lyophilized cultures can be conveniently stored, so that you will have them on hand whenever you need them. There is no need for specific ship dates to meet your schedule. Cultures are easily rehydrated with fungi outgrowth occuring 7 – 14 days.

Macrofungi Cultures: Corprinus and Schizophyllum cultures are shipped in jars. They can be subcultured, using sterile technique, by excising a bit of the media upon which they are growing, and transferring it to fresh medium in another jar. The jar should be large enough to allow the reproductive structures to develop. Other larger fungi may be kept in jars to display the morphology of the reproductive structures and in some cases part of the mycelium. Larger fungi may also be maintained in a terrarium. They should be collected with a large portion of the substrate to be sure that the mycelium is included. Mushrooms, bracket fungi, coral fungi, and others are easily collected and can demonstrate diversity within the fungi kingdom. They can also be used to illustrate the structural differences between the phyla.

Culture Disposal/Handling Spills: Safety concerns should always be kept in mind when conducting any microbiology work. You should have on hand at all times:

• Autoclavable Bio-Hazard bags
• Paper towels
• Latex or vinyl gloves
• Beaker tongs or bottle forceps
• 70% alcohol or 10% Lysol in squeeze bottles

To dispose of cultures:

1. All cultures must be autoclaved at 121°C at 15 psi for 15 minutes
2. Contents of containers can then be discarded. If autoclave is unavailable, soak in bleach or incinerate.
3. Glassware should then be washed in hot water and rinsed well for re-use.
4. Note: Do not pour melted agar down the drain, as it will solidify and plug the drain.

To handle a spill:

1. Put on protective gloves. Pour disinfectant (70% alcohol or 10% Lysol) on all broken glass and contaminated surfaces. Extend coverage at least 3” beyond contaminated area. Make sure that all ignition sources are eliminated if using alcohol.
2. Cover the spill area with paper towels. Add more disinfectant solution to saturate toweling. Allow to stand for 30
   minutes.
3. Using gloves and tongs or forceps, pick up all broken glass, residue, and saturated paper towels and place in the Bio-Hazard bag.
4. Disinfect the area once more as in steps 1– 3. Dispose of your gloves in the Bio-Hazard Bag.
5. Seal the Bio-Hazard bag and autoclave contents for disposal.
6. Wash hands thoroughly.

Special Techniques

General: The techniques previously outlined apply to most fungal cultures. Some special techniques apply to specific fungi:

Saprolegnia ferax, (water mold). This species is best grown on cornmeal agar. It can be grown in liquid medium by adding sterile rice grains to distilled water and inoculating it with Saprolegnia.

Demonstration of sexual reproduction in fungi can easily be done using Rhizopus stolonifer, Phycomyces blakesleeanus, or Mucor hiemalis.

Inoculate plus and minus strains on opposite sides of a Petri plate containing sabouraud dextrose agar or potato dextrose agar. Be careful not to mix the strains when plating them.

When hyphae of the opposite strains grow to meet in the center of the Petri plate, a line of mature zygospores will develop where the strains meet. Have students observe these under a dissecting microscope and sketch what they see.

Making wet mount preparations:

Observe the mold colony under a dissecting microscope. Following sterile technique, use an inoculating needle or loop to remove a small amount of mycelium bearing conidia or sporangia. Place this in a drop of water on a clean microscope slide. Tease it apart with the needle, if necessary. If staining is desired, add one or two drops of methylene blue. Cover with a coverslip and observe under low (40X) then high (400X) power of a compound scope. Draw what you see.

Constructing a moist chamber:

Use WARD’S Silicone Culture Gum to construct a moist chamber, so that you can grow a fungus as a slide culture and observe the entire life cycle. This simple yet beautiful preparation will let you see all phases in the growth of the fungus. The fungus is grown on a block of agar under a coverslip on a glass slide.

1. Roll a marble-sized ball of culture gum and flatten it to form a disc about 1⁄4” thick.
2. Press the disc to a clean microscope slide. Use a cork borer to cut and remove the center of the disc.
3. Immerse the slide into 70% alcohol to sterilize and let dry. Cover with a sterile coverslip.
4. Use a sterile scalpel to cut a small block of agar. Remove the coverslip from the slide.
5. Place the agar block in the center of the slide. Using a sterile needle, inoculate the center of each of the four sides of the agar block with mycelia or spores. Replace the coverslip. You can observe the growth of the fungus over a number of days. If necessary, remove the coverslip and add a drop of distilled water to keep the chamber moist.

This guide is also available in PDF format on wardsci.com.

Related Products

WARD’S offers a wide variety of fungi cultures, kits, and lab activities.

• Predatory Fungi Kit - For a dramatic demonstration of predatory behavior, add a nematode to the culture of fungus you grow. The fungus, commonly found in soil, has adhesive loops at the ends of its hyphal branches. As the nematode passes through a loop, it becomes trapped, and the fungus digests it.
• Introduction to Genetics: A Monohybrid Cross in Yeast Lab Activity - Students will investigate the concepts of inheritance, dominant and recessive alleles, and phenotype versus genotype, as well as learn how to predict phenotypic ratios. Using yeast allows students to obtain results much more quickly than with other organisms, and the results are observable with the naked eye.
• Introduction to Genetics: A Dihybrid Cross in Yeast Lab Activity - Dihybrid crosses can be complex and abstract in nature, but this activity allows students to perform a cross that clearly demonstrates the properties of dominant and recessive alleles, genotypic and phenotypic ratios, independent assortment, and F₁ and F₂ inheritance patterns on just two Petri plates.
• Introduction to the Yeast Life Cycle Lab Activity - Using select strains of Saccharomyces cerevisiae (common Baker’s yeast), students can observe the entire yeast life cycle, including sexual and asexual reproduction, diploid and haploid life stages, and sporulation.

Share This