FUNGI: MOLDS AND YEAST

Today we will be working with molds that were grown from air exposure on plates of Sabaroud’s Dextrose Agar. Pick out one colony that is dark (not white and fuzzy) and has spores on it. Mark the colony with an arrow and your initials so you remember which colony you chose.

SDA was originally designed to isolate ringworm. It is selective for molds and yeast because its pH is 5.6 (slightly acidic), which inhibits bacteria and favors the growth of molds and yeast.

The study of fungi is called mycology. Chapter 12 in your textbook covers fungi, protozoa, and helminthes. Fungi are ubiquitous (they are everywhere).

CHARACTERISTICS OF FUNGI

Eukaryotic

Microscopic groups:

  1. Yeast: unicellular
  2. Molds: multicellular.

MOLDS

The cells are filamentous structures called hyphae (singular = hypha). Hypae are little tubes. Some have individual cells separated by crosswalls called septum. These are known as septate hyphae. Those without septums are called aseptate or non-septate hyphae. You can only see this under a microscope. The cell walls are made of polysaccharide (chitin or cellulose). When a mass of hyphae are visible, it is called mycelium.

There are two kinds of hyphae in mycelium:

  1. Vegetative hyphae: these procure nutrients by secreting enzymes into the substrate (food source) which catabolize (break down) the nutrients, which are then sucked up through the hyphae. The vegetative hyphae also serve as an anchor, so look for them on the bottom side of your plate (On Penicillium, they are green and the aerial hyphae are white). These hyphae are heterotrophs because they get energy by breaking down organic matter. Molds are the decomposers of the food chain, and that is also their ecological niche. Ecology is the study of the interrelationships of living things with each other and their environment. An environment contains both living and non-living things. These relationships should be balanced. Every living thing has a place. We can disturb the balance with our waste products. Fungi return organic matter to the earth by breaking it down with their vegetative hyphae.
  2. Aerial hyphae: living things also need to reproduce; that is the role of the aerial hyphae. They reproduce by spore production. How are these spores different than bacterial spores? One hyphae produces thousands of spores = reproduction. One bacterium produces one spore = survival. Very few soil bacteria also make reproductive spores; and example is actinomyces, which is used for antibiotics.

SPORE PRODUCTION

This occurs on specialized structures called fruiting bodies that grow into the air like miniature trees. We identify molds by their fruiting bodies. First look at your colony; the surface growth is the aerial mycelium, and the bottom growth is the vegetative mycelium. That is why the color of the top and the bottom of the colony may differ. Take note of the colors and texture (Velvety? Peppered?) of your colony.

To examine a fruiting body, we could just touch a piece of Scotch tape to the aerial mycelium and tape it to a slide, but that damages the morphology. The best way to examine fruiting bodies is to grow them on a slide under a cover slip. The next lab period, you lift up the cover slip to examine them.

Today, we will prepare slide cultures that will allow aerial mycelium to grow under the coverslip. This exercise is not in your lab manual. Remember, when you open your plates, the air currents can take the spores into your eyes, nose, and lungs. Some people are allergic and may sneeze. If your allergies are severe, have your lab partner do this part for you. Do this with aseptic technique: goggles, gloves, lab coat. If these spores get into your eyes, they can cause keratitis (redness and swelling in the eyes).

The goal for this procedure is to identify the mold you have chosen by its morphology of the colony and fruiting bodies. The plan is to grow it under a cover slip. The problem is that some molds grow faster than others, so you will have to share your mold with the rest of the class if yours grows faster.

PROCEDURE

Dip a scalpel in a small beaker of alcohol, and then touch the scalpel to the Bunsen burner flame, being careful to keep the alcohol beaker away from the flame. If the alcohol beaker catches fire, put the lid of a Petri dish over it to smother it. Also be careful to keep the flaming scalpel tilted downward so the alcohol does not drip down your hand and catch your clothes on fire! Once the scalpel has stopped flaming, use it to cut ½ inch squares out of a medium called Potato Dextrose Agar, which is enriched, not selective. It is made from the broth of boiled potatoes (starch) and dextrose (sugar). Make sure you do not set the lid of the Petri dish on the table; hold it over the plate like a shield. One dish of squares is enough for two lab tables. Lift up one square of agar with the scalpel and transfer it to the sterile slide in a sterile glass Petri dish. The slide should sit upon a bent glass tubing to keep the slide dry after we add water to the bottom of the dish.

Flame a loop and the opening of a sterile tube of water. Get one loop of water, then open the mold culture dish and rub the top of the colony. Transfer the loop to the agar square and streak it completely across the top, making sure the edges are inoculated, since molds are aerobic and most of the air will be in the sides once we apply the coverslip. Dip the forceps in alcohol and flame it, then use them to pick up the sterile cover slip in the glass Petri dish and place the slip on top of the agar. Leave the spare coverslip in the dish; it’s there in case you break one. Take the rest of the tube of sterile water and dump in into the bottom of the Petri dish. We have now created a humid chamber. Label your dishes and set them out on the side lab countertops until next lab.

MOLDSFig 12.2

Colonies start as vegetative hyphae, which are white. When they get colored dots on them, those are the fruiting bodies. Fig 12.3

Yeasts look like bacteria; they are smooth with no mycelium. They divide asexually by either binary fission (symmetrical) like bacteria, or by budding (asymmetrical). One yeast that produces alcohol is saccharomyces, and is used in fermentation of beer and other alcoholic beverages.

PATHOLOGICAL FUNGI

These cause disease even in healthy people, and they are characterized by thermodimprphism. Dimorphic means they come in two forms. Thermo means those forms are influenced by temperature. At 37° C, they are yeast-like. At 25 ° C they are mold-like.

FUNGI LIFE CYCLE Fig 12.7

Some fungi life cycles are complex because they have both sexual and asexual life cycles (example Rhizopus). Sexual reproduction means that it takes two forms. In the animal kingdom, they are classified as male and female. In the fungi kingdom, they are called + and -.

There morphology is identical. Some fungi seem to have only an asexual cycle.

Asexual cycle = anomorph stage

Sexual cycle = teleomorph stage

Taxonomy is the grouping of living organisms according to their characteristics. They phylum of fungi are named according to the type of sexual spores they produce.

Ascospores are found in the phylum ascomyota. Their class is ascomycetes. Within this class are Aspergillus, Penicillium, lichens (algae and fungi that grow together on a tree or rock), Morel (brain shaped mushrooms that are good to eat or deadly), and Staghorns. There are more on the chart on the back of the room.

In sexual reproduction, the hyphae of + and the hyphae of – come into contact with each other, their crosswall disappears. The nucleus of the + goes into the – hyphae (called plasmogramy). This is followed by the two nuclei fusing (called Kayrogramy). Now we have two sets of chromosomes in one cell (called diploid, or 2n). All of the cells in human bodies are diploid except for the gametes (sex cells; ova and sperm), which are haploid (n).

To get from diploid to haploid, cell division occurs, which reduces the chromosome number by half in a process called meiosis. This is followed by mitosis, which is a simple nuclear division with no change in the number of chromosomes. The cells that result from this process are called ascospores. They are located inside a structure called an ascus. Only eukaryotes go through this process.

The ascus then ruptures and the haploid spores germinate into haploid hyphae. Some are + and some are -. If they don’t come into contact with each other, they go through an asexual cycle by mitosis only, and all their cells remain haploid.

CHARACTERISTICS OF ASTROMYCOTA

Hyphae are septate, and the fruiting bodies are aerial hyphae. Two main types are Penicillium and Aspergillus. You can tell the difference between them by their fruiting bodies. Both are brush-like, but Aspergillus looks like a toilet brush and Penicillium looks like a paint brush.

Spores form on the fruiting bodies and reproduce by mitosis. The spores of Astromycota are called conidia (naked spores), and they are formed during the asexual cycle of astromycota. The spores form in chains and are carried by wind or insects to other locations, where they germinate into septate hyphae. You need to be able to identify Aspergillus and Penicillium.

DR. VOLK’S WEBSITE: FUNGUS OF THE MONTH

He is a mycologist with the best website. Go there and click on Intro to Fungus, check out the Images, and the Fungus of the Month links.

TRUFFLES

One clinically significant fungus that is not pathogenic is the mushroom called a truffle. These are rare and delicious and sell for about $5 each! In the past, hunting dogs and pigs were used to sniff out where truffles were growing in the woods.

ASPERGILLUS

Aspergillus gets its name from “aspergillum”, the name of the item a Priest uses to bless with Holy water. Aspergillus is a very common fungi, pervasive (found everywhere). There are more than 600 species. Most are saprobic (non-pathogenic) and live off dead matter. In a young fruiting body, the “handle” of the conidia is called the conidiophore. The bulb at the top is called the vesicle. The philiae radiate out from the vesicle, and the spores form chains in the philiae. The older fruiting bodies look like a toilet brush because they are covered with spores at all angles.

Some Aspergillus fungi can ferment. That’s where we get citric acid for soft drinks and also soy sauce. One pathogenic species is called Aspergillus flavus. It produces a toxin called aflatoxin (a mycotoxin) which is carcinogenic, especially in the liver (liver cancer). This species is fond of grains (corn, wheat), and peanuts, and jelly. Therefore, a peanut butter and jelly sandwich is a triple whammy for aflatoxin. It dissolves in the jelly, so scooping it off the top surface of the jar will not do any good. To prevent aflatoxin, only buy peanut butter and jelly in the size jars you use up in a week, keep them refrigerated (the bread, too), and use a clean knife each time. Don’t leave the lid off for long, because that’s when it gets in. Another disease Aspergillus causes is aspirgilosis, a lung disease that will be discussed in lecture this week.

PENICILLIUM (Latin for penicillus = “paint brush”)

The branches off its conidophores are called metula. This mold is famous for its use by Alexander Fleming in making penicillin, but it is also used to make aged cheeses like Brie, Blue cheese, and Roquefort. The white top layer of Brie cheese is where the vegetative hyphae are. The cheese is made with lactic acid bacteria (the curd is the cheese) and it is inoculated with Penicillium for flavor. The colony of Penicillium is green with a white ring.

ASSIGNMENT FOR THIS WEEK’S LECTURE

Google these 11 fungal diseases and print out the information you get. These diseases are clinically important because you will see lots of immunosuppressed people: elderly (people live longer now), catheterized patients, AIDS, diabetes, neonates, cancer patients, etc. Opportunistic pathogens are part of our normal microbiota (normally found on us or nearby in grasses, etc). They only cause disease in immunocompromised people. Truly pathogenic organisms cause disease even in healthy people.

A good website to look for information on these diseases is CDC, but they don’t list the treatment. For treatment of these diseases, go to Merck.

Also become very familiar with Appendix F in the text book. This is a taxonomic guide to infectious diseases.

Pathogenic Fungi and Their Diseases:

OPPORTUNISTIC MYCOSES

Candida albicans (Candidiasis: yeast infection in women and thrush in children)

Aspergillus (aspergillosis)

Cryptococcus neoformins (Cryptococcus)

Pneumocytis (pneumocystic pneumonia; most common pneumonia)

SUPERFICIAL MYCOSES

Disease: White and Black Piedra (“Rocks” on hair shafts; a tropical disease; don’t need

to know this one)

CUTANEOUS MYCOSES (most common; fungus of the hair, skin, nails, esp. ringworm. Caused by dermatophytes. Most common is athlete’s foot).

Microsporum

Epidermophyton

Trichophytum

SUBCUTANEOUS MYCOSES

Sporotrichum schenchii (sporotrichosis, AKA “Rose thorn disease”)

SYSTEMIC MYCOSES (most serious; most often occurs in lungs)

Coccidiodes immitus (Valley Fever)

Histoplasma capsulatum (histoplasmosis)

Blastomyces dermatiditis (blastomycosis)

LAB 21

OBSERVATION OF FUNGI

FUNGI DISEASES

OBSERVATION OF PREPARED SLIDES (use photos from chapter 11.1 to help you identify the structures you see and the names of the fungi). Look at the fruiting bodies, and know the life cycles of the following phylums:

  1. Ascomycota (observe under 400x, not oil!)
  2. Penicillium: conidia and conidiospores (amorph/asexual)
  3. Aspergillus: conidia and conidiospores (amorph/asexual)
  4. Peziza: asci, ascospores (teleomorph/sexual)
  5. Saccharomyces (beer yeast): parent cells and buds; use 1000x for this one.
  6. Zygomycota
  7. Rhizopus: anamorph and teleomorph: sporangia, sporangiospores, zygospores
  8. Basidiomycota: teleomorph; observe the mushroom coprinus

Draw each of these slides on data sheet 11.1; on the last page are some blank circles where you can draw coprinus and peziza.

OBSERVATION OF LIVE CULTURES

Place one drop of lactophenol cotton blue on a clean slide. The phenol in this dye penetrates the chitinous cell wall of the fungi to stain the fruiting body; it will also penetrate your skin, and it is toxic! Use gloves and goggles.

Sterilize a probe needle and stick it into your agar block to hold it down. Use sterile forceps to lift the cover slip off. Take the cover slip, MOLD FACING DOWN, and touch the side of the slide to the dye for a few seconds, and then let it fall down onto the slide. Wick off the excess dye and throw the paper towel into the biohazard bag. Observe under 400x (NOT OIL), and draw your pictures. Discard entire slide and coverslip into the broken glass box. Do not clean and reuse these slides.

CLINICALLY SIGNIFICANT DISEASES:

OPPORTUNISTIC MYCOSES

  1. Aspergillosis: can occur anywhere in the body, but is most common in the lungs. Some people just have allergies to this mold. X-ray of aspergillosis shows aspergilloma (“fungus balls”) which are mycelium and white blood cells. Although opportunistic diseases are usually seen in immunocompromised people, healthy people who are overexposed can also get sick. Since this mold loves grains, farmers are especially at risk. Some species make a mycotoxin called aflatoxin, which is a potent liver carcinogen.
  2. Ringworm: these skin lesions are circular, so they look like a worm. It is caused by a group of fungi called dermatophytes, which live on keratin (found in skin, hair, and nails). Dermatophyte infections are the most common fungal infections in humans. They are known as “tinnea”. Fungi like moisture, and feet are in a dark, moist environment, so infection is common there. Athlete’s foot is known as tinnea pedis. You get it by just walking barefoot, exposing the skin to the air where spores are. If there is a slight scratch in the skin, the fungi can get in. They form itchy vesicles. When scratched, the fungus spreads. Treatment is topical creams. When dermatophytes infect the nails it is called onychomycosis. The nails get thick, chalky, discolored. Topical creams don’t penetrate the nails. Oral medicines like griseofulvin kill the fungus but are also toxic to the liver. Onychomycosis is common with age.

Ringworm can also occur in the scalp, usually in children. The hyphae are fluorescent, so you can use a Wood’s Lamp, which shines with ultraviolet light, and see the hyphae glow.