BASICS OF MICROBIOLOGY - E. Yu. Tyumentseva - 2015

TOPIC 3. STUDY OF MORPHOLOGICAL AND CULTURAL CHARACTERISTICS OF MICROSCOPIC FUNGI AND YEASTS. PREPARATION OF WET MOUNT SLIDES

When categorizing microorganisms, researchers distinguish features that either unite or separate them (taxonomic features). These taxonomic features must meet basic requirements: they must be evolutionarily stable and define phylogenetic relationships. The main taxonomic features used in systematics are morphological ones—shape, size, mutual arrangement, and Structure.

Cultural (or macromorphological) properties refer to the characteristic growth patterns of microorganisms on Solid and liquid nutrient media. On the surface of solid media, depending on the inoculation method, microorganisms may grow as colonies, streaks, or a confluent lawn.

A colony is an isolated accumulation of Cells of the same species grown from a single Cell (a cell clone). Depending on where the microorganism grows (on The surface of a solid nutrient medium or within its depth), surface, subsurface, and bottom colonies are distinguished.

When describing colonies, the following characteristics are taken into account:

a) colony shape — circular, amoeboid, rhizoid, irregular, etc.;

б) size (diameter) of the colony — very small (punctiform) (0.1-0.5 mm), small (0.5-3 mm), medium-sized (3-5 mm), and large (more than 5 mm in diameter);

в) colony surface — smooth, rough, folded, wrinkled, concentrically ringed, or radially striated;

г) colony profile — flat, convex, conic, crateriform, etc.;

д) opacity — dull, matte, shiny, transparent, powdery;

е) colony color (pigment) — colorless or pigmented (white, yellow, golden, red, black); special attention is paid to pigment excretion into the medium with its subsequent coloration;

ж) colony margin — entire, wavy, dentate, fimbriate, etc.;

з) colony structure — uniform, finely or coarsely granular, striate; the margin and STRUCTURE OF THE colony are determined using a magnifying Glass or at low Microscope magnification by placing the Petri dish with the culture upside down on the microscope stage;

е) colony consistency — determined by touching the surface with an inoculation loop: the colony may be dense, soft, growing into the Agar, mucoid (drawing out into a thread behind the loop), or brittle (easily breaking upon contact with the loop).

3.1. Morphology and cultural characteristics of microscopic Fungi

Microscopic fungi belong to the superkingdom Eukaryota, kingdom Fungi, division Eumycota, and are represented by three out of four classes: Phycomycetes, Ascomycetes, and Deuteromycetes. Members of the fungi kingdom are aerobic microorganisms and, regarding their type of Nutrition, are chemoorganoheterotrophs. Most fungi are Saprophytes, though some cause diseases and act as parasites.

The vegetative body of fungi is called the mycelium. The mycelium consists of numerous intertwining tubular filaments called hyphae. The diameter of hyphae ranges from 5 to 50 µm. Depending on The structure of the mycelium, fungi are divided into lower and higher fungi. In higher fungi, the hyphae are divided by cross-walls (septa), in the center of which There is a large pore. The Class Phycomycetes unites lower fungi, while Representatives of the classes Ascomycetes and Deuteromycetes are higher fungi.

Fungi are coenocytic microorganisms. This means they grow while nuclear division occurs, but Cell Division does not follow. Thus, the vegetative body of a fungus represents a single large multinucleated cell.

All microscopic fungi can reproduce vegetatively via a piece of mycelium.

During asexual reproduction, Phycomycetes form sporangiophores, whereas Ascomycetes form conidiophores. Deuteromycetes can reproduce via multicellular conidia.

Phycomycetes and Ascomycetes are perfect fungi. This means that representatives of these classes can reproduce sexually. Deuteromycetes belong to imperfect fungi.

Cultural characteristics of microscopic fungi

Colonies of microscopic fungi exceed the size of single-celled Organism colonies (Bacteria, Yeasts) by many times and frequently spread across the entire surface of the nutrient medium in Petri dishes. The consistency of fungal colonies varies. Felt-like and leathery colonies are formed most often, while crumbly ones are rarer. The colony surface can be cotton-like, velvety, powdery, web-like, filamentous, leathery, or smooth. When grown on solid and liquid media, part of the hyphae grows into the nutrient medium, forming the substrate mycelium, while another part forms the aerial mycelium as a fluffy coating visible to the naked eye. The mycelium can also be colorless (white, grayish) or pigmented (black, brown, green, yellow, etc.). Only the fruiting mycelium is pigmented.

Characteristics of microscopic fungi of various classes

The morphological features of fungi of various classes are shown in Figure 3.

The genus Mucor belongs to the class Phycomycetes. These fungi have a non-septate mycelium. They can reproduce both asexually and sexually, forming sporangiophores (Fig. 3a). Externally, the sporangium is covered with fine spines of calcium oxalate crystals. Upon maturation, the sporangium ruptures, releasing sporangiospores that are dispersed by air currents. After the spores are released from the sporangium, a columella remains on the sporangiophore, with a collar at its base. The mycelium of Mucor fungi is initially white, later turning grayish-olive, with a felt-like appearance.

Mucor fungi grow on the surface of damp grain, malt, ROOT crops, food products, and on the walls of damp rooms as a grayish, fluffy coating. Mucor nigricans is the CAUSATIVE AGENT OF clamp rot in sugar beets. Many Mucor fungi are used industrially to produce various organic acids and alcohol (species such as Mucor javanicus and Mucor racemosus), enzyme preparations, carotenoids, and Steroids.

Fig. 3. Morphological features of fungi of various classes: a — Mucor; b — Penicillium; c — Aspergillus; d — Alternaria

Members of the genera Aspergillus and Penicillium belong to the class Ascomycetes, which comprises the higher microscopic perfect fungi. During asexual reproduction by spores, these fungi form conidiophores (Figs. 3b, 3c). Aspergilli and penicilli are classified as ascomycetous (sac) fungi. This means that during sexual reproduction, they form asci (sacs) containing 8 ascospores on specialized fruiting bodies.

About half of all Molds belong to the genus Penicillium. They are widely distributed in soil and the air of poorly ventilated rooms, causing the spoilage of various products and Materials. This fungus has a branching, septate mycelium (hyphae diameter of 2-3 µm) and septate conidiophores (resembling small brushes) that branch at the tip into structures called sterigmata. From these arise conidia, which consist of chains of spores. Depending on the species, conidia can be of various colors (white, green, etc.). Many penicilli are used industrially to obtain various valuable products. Among the isolated strains of this genus, 25% exhibit antibiotic activity, and species such as Penicillium notatum and Penicillium chrysogenum are used as producers of penicillin. Certain penicillium species are used to produce Enzymes and Lipids. In The production of soft cheeses like Roquefort and Camembert, the noble molds Penicillium roqueforti and Penicillium camemberti are used.

Fungi of the genus Aspergillus comprise more than 200 species. These fungi have a well-developed, branching mycelium with numerous septa. The conidiophores are non-septate, and their upper ends are pear-shaped or spherically dilated into a small vesicle (HEAD). The vesicle bears bottle-shaped sterigmata with chains of conidia, which resemble streams of Water pouring from a watering can. This gave rise to the name "watering-can mold" (aspergere in Latin means to sprinkle or spray). Upon maturation, Aspergillus conidia acquire various colors, which, along with other characteristics, determines their species identity.

Just like penicilli, members of the genus Aspergillus are widely distributed in nature and play an important role in the Mineralization of organic matter. They cause the molding of Many food products. These fungi produce many valuable substances and are widely used in industry. For instance, Aspergillus niger is used industrially for citric acid production; Aspergillus terreus for itaconic acid; Aspergillus flavus and Aspergillus terricola form the most active complex of Proteolytic Enzymes; and Aspergillus oryzae and Aspergillus awamori are the best producers of amylolytic enzymes.

Fungi of the genus Alternaria belong to the class of imperfect fungi, the Deuteromycetes. These are higher fungi. They have a septate mycelium and short, non-septate conidiophores bearing multicellular, pear-shaped or lemon-shaped conidia (see Fig. 3d). The fungus is the causative agent of black rot, a disease affecting root crops and fruits, as well as a cause of food spoilage.

3.2. Yeast Morphology and Characteristics

Yeasts are higher unicellular fungi. Most yeasts belong to two classes of fungi: Ascomycetes and Deuteromycetes.

With respect to oxygen, yeasts are divided into facultative anaerobes and aerobes (under aerobic conditions, they respire and actively accumulate biomass, while under anaerobic conditions, they cause Alcoholic Fermentation).

Morphologically, yeasts are diverse. They differ from one another in Cell size and shape. Depending on the species, yeast cell sizes vary within the following ranges: from 2.5 to 10 µm in diameter and from 4 to 20 µm in length. The Morphological diversity of yeast shapes is shown in Figure 4.

The shape and size of yeast cells depend on the species, age, nutrient medium, and cultivation method.

They reproduce both vegetatively and sexually. Vegetative Reproduction Methods include budding and fission; sexual reproduction involves spore formation. Budding yeasts include representatives of "cultivated" yeasts of the genus Saccharomyces (saccharomycetes). Fission yeasts include species of the genus Schizosaccharomyces (schizosaccharomycetes). During the sexual process, the fusion of vegetative cells leads to The formation of asci with spores, or spores may form first and subsequently copulate with each other. Each ascus contains from 2 to 8, sometimes 12 spores. Among yeasts, there are asporogenous, false yeasts that are incapable of sexual reproduction and spore formation. They belong to the class of imperfect fungi.

Fig. 4. Shapes of yeast cells: a - oval/ovoid; b - cylindrical; c - apiculate/lemon-shaped; d - arrow-shaped; e - triangular; f - sickle-shaped; g - flask-shaped; h - mycelial

Among the yeasts belonging to the class Ascomycetes, the saccharomycetes of the genus Saccharomyces (saccharomycetes) are of great importance and are widely used in the food industry. The main biochemical characteristic of these yeasts is their ability to ferment sugars, producing ethyl alcohol and carbon dioxide. Yeasts used in industry are referred to as cultivated yeasts. For example, top-fermenting yeasts of the species Saccharomyces cerevisiae are used in baking and alcohol production. The species Saccharomyces minor is used in the production of rye bread and kvass. Bottom-fermenting yeasts, Saccharomyces carlsbergensis, are used in brewing. Saccharomycetes are oval-shaped, reproduce vegetatively by budding, and, under unfavorable conditions, reproduce sexually via ascospores.

Some sporogenous yeasts are considered wild yeasts. Like cultivated yeasts, they are capable of alcoholic fermentation, but In addition to alcohol, they produce many by-products (such as aldehydes, higher alcohols, esters, etc.), thereby deteriorating the organoleptic Properties of the product. These yeasts are contaminants in the production of various beverages (beer, wine, non-alcoholic drinks) and are also responsible for the spoilage of many food products.

Deuteromycete yeasts can only reproduce vegetatively. Some of these yeasts (for example, yeasts of the genus Candida) are used industrially to produce Single-Cell Protein (feed protein), organic acids, Vitamins, and other products of microbial synthesis. The yeast species Torulopsis kefir is part of the symbiotic starter culture—the kefir grain. Other representatives of imperfect (asporogenous) yeasts are wild yeasts and cause the spoilage of many food products. Contaminant yeasts in production include species of the genera Pichia, Hansenula, Candida, Rhodotorula, Torula, Torulopsis, Mycoderma, Trichosporon, etc. Among asporogenous yeasts, there are false yeasts that form a pseudomycelium and grow on liquid substrates as films.

Laboratory Procedure

Objective: to study the Morphological Characteristics of fungi and yeasts encountered in food production, their cultural properties, and representatives of individual classes; to master the technique of microscopic examination of fungi and yeasts in "wet mount" (crushed drop) preparations, as well as the technique of Microscopy of living, unstained objects.

Materials, Reagents, and Equipment: microscope; dissecting needles and bacteriological loops; microscope slides and coverslips; pipette; filter paper; alcohol burner; slide staining rack; immersion oil; fungal cultures of the genera Mucor, Penicillium, Aspergillus, Alternaria; pure culture of the yeast Saccharomyces cerevisiae; baker's and wine yeasts; 96% ethyl alcohol; distilled water; 0.5% alcoholic solution of Sudan III dye; Lugol's solution.

Experiment No. 1. Examination of Fungi

Preparation of specimens by the squash mount method:

1. Place a large drop of water or ethanol onto a microscope slide using a tube or pipette.

2. Light the alcohol burner, flame-sterilize the dissecting needle, and aseptically sample a small amount of mycelium from the test tube or Petri dish.

3. Carefully place the mycelium into the drop on the slide and tease it apart in the water using two needles.

4. Cover the specimen with a coverslip and press down gently. Remove excess water with filter paper. If there is insufficient liquid, add more by pipetting it under the edge of the coverslip.

5. Examine the squash mount preparation under a microscope, starting with the 8x objective and then switching to 40 in a darkened field of view (with the condenser lowered and the iris Diaphragm partially closed).

When sampling and microscopically examining fungal specimens, consider the following recommendations:

a) mold of the genus Mucor. Sample the blackish-grey fluffy aerial mycelium. During microscopy, focus on hyphae with spore-filled sporangia and the columellae formed upon sporangial release;

b) mold of the genus Penicillium. When sampling, try to collect young mycelium (at the boundary between the colored and white mycelium) by inserting the needle into the medium. Focus on the septate hyphae with brush-like structures;

c) mold of the genus Aspergillus. Sample a small amount of fluffy mycelium with colored conidia, inserting the needle slightly into the nutrient medium. Focus on the non-septate conidiophores;

d) mold of the genus Alternaria. Sample the mycelium from the black areas, inserting the needles into it. Focus on the septate mycelium, poorly developed conidiophores, and large conidia, which appear as rounded or pointed multicellular structures resembling hand grenades.

Experiment No. 2. STUDY OF YEAST

Baker's yeast can be used for laboratory classes. A few hours before the class, place a small piece of yeast mass into warm sweetened water and keep it in a warm place. A whitish, turbid liquid will form. Place a drop of this liquid on a microscope slide, cover it with a coverslip, apply a drop of immersion oil on top, and examine the specimen using an oil immersion system. The cells are also clearly visible at lower magnifications.

Baker's yeast typically contains two strains: one is represented by round-ellipsoidal cells that quickly separate during budding; the other by elongated-cylindrical cells that form branched clusters (pseudomycelium) during budding. Buds are visible on many cells. In the fine-grained content of living yeast, large transparent vacuoles, sometimes centrally located, are clearly visible. Under microscopy, the Cytoplasm appears as a darker granular mass, lipid droplets are bright and shiny (highly refractive), and Glycogen appears as dense granules.

Microchemical Reactions are used to determine the Chemical Nature of intracellular inclusions.

To detect glycogen, add a drop of Lugol's iodine solution to a drop of yeast (baker's or wine yeast) before covering it with a coverslip: glycogen stains reddish-brown. If there is little or no glycogen in the cells, the yeast is immature or old.

By adding a drop of 0.5% alcoholic solution of Sudan III dye to a drop of yeast (round cells), lipids can be detected (staining reddish-yellow).

Recording and Analysis of Research Results

1. Briefly summarize the theoretical material.

2. Examine molds and yeasts (baker's, wine, and kefir yeasts) under a microscope (using a 40x objective in a dimly lit field of view).

3. Observe and draw the microscopic views of the studied mold and yeast cultures, taking into account the morphological characteristics of each microorganism. Label each drawing with its Latin name and the magnification used.

4. Measure the baker's yeast cells, selecting non-budding cells.

5. Confirm the lack of motility in yeast (taking into account the possible movement of cells with the liquid flow).

6. Describe the cultural CHARACTERISTICS OF THE studied molds.

Upon completing this topic, students will be able to identify the shapes and species of molds and yeasts encountered in food production, as well as their cultural characteristics and morphological features. They will master the technique of microscopic examination of molds and yeasts in squash mount preparations, as well as the MICROSCOPY OF LIVE, unstained specimens, which are essential for analyzing Food Products and other samples.

Review Questions

1. How are fungi classified based on the structure of their mycelium?

2. Which classes of fungi are classified as higher fungi?

3. Name the method of reproduction of microscopic fungi.

4. How are microscopic fungi and yeast specimens prepared?

5. Describe the morphological and cultural characteristics of microscopic fungi.

6. Which fungi are used in industry to produce organic acids, enzymes, Antibiotics, and other valuable products?

7. Describe fungi of the genus Penicillium.

8. Describe the morphological characteristics of yeast.

9. What is cultivated yeast? In which Branches of the food industry is it used?

10. Are yeasts motile?

11. How are yeast cells measured?

12. How can glycogen and fat be detected in yeast cells?