GENERAL AND FOOD MICROBIOLOGY PART I - L. V. Krasnikova - 2016

6. STUDY OF YEAST MORPHOLOGY

Objective: to examine the Morphology of cultivated Yeasts used in brewing and baking, as well as wild yeasts responsible for food spoilage.

Yeasts are eukaryotic, unicellular microscopic Fungi. Depending on their sexual reproduction cycle or its absence, yeasts are classified into three Major Groups of higher fungi: Ascomycetes, Basidiomycetes, and Deuteromycetes.

The Class Ascomycetes comprises three Yeast families: Saccharomycetaceae, Schizosaccharomycetaceae, and Saccharomycodaceae. Characteristic Features of these fungi include sexual sporulation and The formation of two, four, or eight ascospores within an ascus.

In the food and Fermentation industries, yeasts of the genus Saccharomyces (family Saccharomycetaceae) are widely utilized. Saccharomyces species are essential in The production of ethanol, wine, beer, kvass, bread, and baker's yeast. Brewing typically employs various strains of Saccharomyces cerevisiae, whereas baking utilizes strains of both Saccharomyces cerevisiae and Candida milleri (formerly Saccharomyces minor).

Certain members of the family Saccharomycodaceae (genera Nadsonia and Saenko) participate in fermentation during the production of sherry and sparkling wines. All other yeast species are considered contaminants ("wild" yeasts) that disrupt the biotechnological process at various stages.

Imperfect yeasts of the class Deuteromycetes are either anamorphs of known ascomycetous or basidiomycetous fungi, or species in which the sexual stage has not been identified or is entirely absent. This class includes the family Candidaceae, which exhibits ascomycetous affinities, along with two families displaying basidiomycetous affinities: Cryptococcaceae (which do not form ballistospores) and Sporobolomycetaceae (which form ballistospores). Yeasts belonging to the genera Candida and Trichosporon are capable of forming pseudomycelia.

6.1. Characteristics of Selected Cultivated and Wild Yeast Species

Yeasts are unicellular eukaryotic microorganisms that reproduce via budding, spore formation, or binary fission. The Cell dimensions of yeasts used in the food industry typically range from 3 to 6 µm in width and 8 to 12 µm in length.

To study yeast morphology, they are cultured in sterile malt wort with a dry matter concentration of 8 to 10% or in a glucose-peptone medium at 25-28 °C for 2-3 days.

The vegetative cell shape varies considerably across different yeast species. Both cell shape and size depend on the species, age, growth medium, and cultivation method. Yeast Cells may appear round, oval, egg-shaped, or—less commonly—cylindrical or lemon-shaped (apiculate). Imperfect yeasts often exhibit more distinctive morphologies, such as arrow-shaped (genus Brettanomyces), triangular (genus Trigonopsis), sickle-shaped (genus Selenotila), or flask-shaped (genus Schizoblastosporion). In addition to round and oval cells, cultures of Candida and Trichosporon frequently contain elongated pseudomycelial cells.

Cultivated Yeasts

Saccharomyces cerevisiae (Fig. 6.1).

Fig. 6.1. Saccharomyces cerevisiae cells: a - young cells; b - mature cells; c - old cells

Morphological features. Cells of this species are oval, round, egg-shaped, or slightly elongated, measuring (5-6) × (9-14) µm. Cytological characteristics of yeast cells can vary significantly with age. A new, young cell develops either from the germination of a spore or vegetatively via budding.

Young, active yeast cells (from a 12-18-hour culture) observed under a Microscope feature a thin, transparent Cell wall, homogenous Cytoplasm lacking visible inclusions, and a small vacuole. Occasionally, one or two polyphosphate granules may be present in the cytoplasm. Young yeasts multiply vigorously, with the proportion of budding cells reaching 70-80%.

Mature yeasts (24-48-hour culture) exhibit a granular, heterogeneous cytoplasm. The number of vacuolated cells increases, and individuals with two or more vacuoles are sometimes observed. The rate of reproduction slows down, with budding cells averaging 10-15%. Fat inclusions begin to appear inside the cells and are readily visible without special staining using a semi-closed Diaphragm. Mature cultures contain up to 2-4% dead cells, which can be identified via vital staining with a weak methylene blue solution. Methylene blue penetrates the cytoplasm exclusively through the cell walls of dead cells, staining them blue, whereas live cells remain colorless. The proportion of storage reserves—such as Lipids and Glycogen—increases as the cell develops. A hallmark of mature yeast cultures is a high intracellular accumulation of glycogen. Glycogen is stored within vacuoles as a reserve substance and is consumed as the cell ages. When yeasts are cultivated on carbohydrate-rich media without aeration, the percentage of glycogen-rich cells reaches 65-70%, indicating a well-nourished, mature state. Glycogen is detected by staining live yeast preparations with an iodine solution. Alongside glycogen, mature yeasts accumulate another reserve substance: metachromatin (volutin), which stains reddish-violet when fixed yeast smears are treated with methylene blue.

Old yeasts (cultivated for 48 hours or more) possess thickened cell walls that are clearly discernible microscopically. The cytoplasm is granular and heterogeneous, containing large vacuoles, often multiple per cell. The older population features numerous cells with lipid droplets appearing as round, highly light-refractive inclusions. A defining characteristic of aged cultures is the presence of a high proportion of dead cells.

Significant cytological shifts occur when cultivation conditions change—such as transitioning from aerobic to anaerobic states, altering media composition and limiting nutrient concentrations, or applying physiological stress.

Under aerobic conditions, yeasts proliferate rapidly; the cells are smaller and more uniform, and their Mitochondria actively divide, showing a marked increase in number.

Under anaerobic fermentation, mitochondria fuse together, cells accumulate higher levels of glycogen, and it appears in the form of large granules.

Cultural characteristics. Colonies on wort Agar are white, round, with smooth edges and a convex center, featuring a smooth, dull-glossy surface and a diameter of 0.5 – 1.0 cm. When grown in liquid malt wort, yeasts of this species form a modest yellowish-white sediment.

Candida milleri (formerly Saccharomyces minor). Yeasts of this species are specifically associated with rye dough.

Morphological characteristics. Cells are small and round, 1.5 — 3.0 µm in diameter, with characteristic budding: buds form at one of the cell poles. When yeasts are grown in a liquid medium, buds develop at both ends of the cells, or two buds form at one end.

Cultural characteristics. On wort agar, they form small, grayish-white colonies with smooth edges and a raised center, ranging from 0.4 to 0.8 cm in diameter. The colony surface is smooth and glossy. In malt wort, they form a small grayish-white sediment that is easily resuspended upon agitation.

Food-Spoilage Yeasts

Alongside cultured yeasts, biotechnological processes may encounter so-called "wild" yeasts. These compete with cultured strains for nutrients in the substrate and cause spoilage or a deterioration of the organoleptic Properties of the finished product. The most common wild yeasts belong to the genera Saccharomyces, Kloeckera, Candida, Torulopsis, Pichia, Hansenula, Brettanomyces, Rhodotorula, and several others.

Saccharomyces pastorianus (Fig. 6.2, a) features ellipsoid or sausage-shaped cells, and less frequently, oval cells. They do not sediment with cultured yeasts during primary beer fermentation; as they proliferate, the beer becomes turbid, clears poorly, and develops an off-odor and an astringent-bitter taste.

Saccharomyces turbidans (Fig. 6.2, b) has oval or ellipsoid cells that often join into chains of 4–5 cells. In beer, they cause severe turbidity and flavor spoilage.

Kloeckera apiculata (Fig. 6.2, c) has small, lemon-shaped cells pointed at one or both poles, occurring singly or less often in pairs. As they age, they become elongated and spindle-shaped. Cell dimensions are (5–8) × (2–4) µm. When proliferating in beer, they accumulate volatile acids, esters, and hydrogen sulfide, imparting an unpleasant, specific off-odor to the product.

Saccharomyces bayanus (Fig. 6.2, d) features large, oval cells. They are found in beer, cider, and wine Materials, imparting unacceptable flavors and aromas to the final product due to the formation of significant amounts of esters, diacetyl, acetic acid, and sulfur-containing volatile acids.

Saccharomyces validus (Fig. 6.2, e) has oval or markedly elongated cells that occur in pairs or small clusters. They cause beer turbidity and an unpleasant specific off-odor.

Hansenula anomalia (Fig. 6.2, f) has round, ellipsoid, and sometimes sausage-shaped or club-shaped cells. Yeasts of the genus Hansenula produce large amounts of alcohols and esters, giving beer a pungent odor. They inhibit the growth of cultured yeasts.

Pichia farinosa (Fig. 6.2, g) features rounded to oval cells that occur singly or in pairs. Cell dimensions are (4.0–7.5) × (3–6) µm. Yeasts of this species, as well as P. membranifaciens, form a white film On the surface of beer. They accumulate aldehydes, esters, and volatile acids in the product, imparting a fruity or medicinal off-flavor to the beer. When exposed to air during bottling, they proliferate and cause beer turbidity.

Fig. 6.2 (a–d). Selected wild yeast species (T.P. Slyusarenko, 1984): a — Saccharomyces pastorianus; b — Saccharomyces turbidans; c — Kloeckera apiculata; d — Saccharomyces bayanus

Brettanomyces bruxellensis (Fig. 6.2, h) exhibits polymorphic cells: oval, highly elongated, rod-shaped, and sometimes with pointed, lancet-like ends. B. bruxellensis contaminates wort and beer. The proliferation of these yeasts is accompanied by an off-flavor, fruity aroma, and turbidity in the beer. Other

species (B. anomalus, B. intermedius) cause spoilage in pasteurized beer, imparting a specific, unpleasant odor.

Fig. 6.2 (e–k). Selected wild yeast species (T.P. Slyusarenko, 1984): e — Saccharomyces validus; f — Hansenula anomalia; g — Pichia farinosa; h — Brettanomyces bruxellensis; i — Candida mycoderma; j — Torulopsis colliculosa

Genus Candida. The principal representatives of this genus — C. mycoderma (Fig. 6.2, i), C. utilis, C. krusei, C. guillermondii — possess oval or elongated-cylindrical cells. Cell dimensions are (4–16) × (2–5) µm. Yeasts of this genus do not form spores and are asporogenous. Many species are pest organisms in the production of wine, beer, and baker's yeast. They frequently form a dense white or grayish dry pellicle on The surface of wort and beer, causing turbidity and imparting an unpleasant taste and odor.

During biomass accumulation in baker's yeast production, Candida yeasts compete with cultured strains for substrate nutrients and reduce the maltase activity of the final yeast product.

Genus Torulopsis. Cells of the genus Torulopsis are small, round, or oval, measuring (5–8) × (2–5) µm. Yeasts of this genus are frequently isolated from kefir grains (Torulopsis kefiri). The species Torulopsis colliculosa (Fig. 6.2, j) is a contaminant in brewing and yeast manufacturing.

Rhodotorula roseum features round or oval cells, occurring singly or in pairs, and does not form pseudomycelium. Cell dimensions are (5–8) × (2.5–5.0) µm. On nutrient agar, R. roseum forms round, glossy, pink colonies. When proliferating on the surface of food products, they cause pigmentation.

6.2. Determination of Dead and Budding Cell Counts in Yeast Suspensions

The morphological state of yeast is determined by Microscopy. For this purpose, counts of budding and dead cells are performed.

Determination of dead cell count. The proportion of dead cells is a crucial quality indicator for brewing and baker's yeast. High-quality yeast should contain no more than 5% dead cells. An increase in the number of dead cells indicates adverse conditions during yeast cultivation. A significant proportion of dead cells can lead to technological process disruptions, such as sluggish fermentation and the proliferation of contaminant microorganisms.

When determining the number of dead cells in yeasts cultivated on unstained nutrient media, staining with methylene blue is convenient. For this purpose, a dye solution in distilled Water is prepared at a 1:10,000 dilution. The specimen is prepared as follows: a drop of the test culture is placed on a Glass microscope slide, a drop of the methylene blue solution is added, and it is covered with a coverslip. A drop of immersion oil is applied to the coverslip, and examination is performed using 90× magnification. Dead cells stain blue, whereas live cells remain colorless. The number of dead cells (V) is counted across 10 microscope fields of the prepared specimen.

Determining the number of budding cells. Place a drop of the test yeast suspension on a glass slide, add a drop of methylene blue, mix well, and cover with a coverslip. Examine under a 60x or 90x objective. The total count in the field of view should not exceed 30–40. Count the number of budding cells (C) and the total number of cells (A) in each field of view. A cell is considered a bud if its size does not exceed 1/2 of the mother cell. A cell with a bud smaller than 1/3 of the mother cell's size is counted as a single cell. During active yeast proliferation, the number of budding cells reaches 50% or more. A low number of budding forms indicates low yeast reproductive activity. Calculate the total and budding cell counts in at least 10 fields of view.

Record the experimental results in Table 6.1 and calculate the percentage of dead and budding cells in the tested yeast suspension.

Table 6.1. Results of yeast cell counts in 10 fields of view

Content of dead cells in the tested yeast suspension:

X = В/А • 100, %.

Content of budding cells in the tested yeast suspension:

У = С/А • 100, %.

6.3. Staining of Reserve Nutrients in Yeast Cells

Glycogen staining. Young yeast cultures accumulate large amounts of glycogen as a reserve nutrient. In normally well-nourished yeast, 70% to 75% of the cells contain glycogen. A lower proportion of glycogen-containing cells in industrial yeast indicates cell Aging or insufficient Nutrition.

To detect glycogen, place a drop of the yeast suspension on a slide using a pipette and add a small amount of iodine solution. Cover the suspension with a coverslip, add a drop of cedar oil on top, and examine the specimen under an immersion objective. Glycogen stains reddish-brown, while the yeast cells remain colorless.

Lipid staining. Older yeast cultures typically accumulate large amounts of lipids. Lipid synthesis in *Saccharomyces* yeasts occurs under conditions of carbohydrate excess, intensive aeration, and nitrogen deficiency in the nutrient medium. Lipids are also synthesized in the presence of inhibitors in the medium.

Method 1. To stain lipids, place a drop of the yeast suspension on a slide with a pipette, add a drop of alcoholic Sudan III stain, and cover with a coverslip. Apply cedar oil to the slide with a glass rod and examine under a 90x objective. Fat droplets stain yellow, whereas the yeast cells remain colorless.

Method 2. Place a small drop of a 40% formalin solution on a slide. Using an inoculation loop, introduce the yeast culture into the drop. Formalin kills the cells and increases cell wall permeability. After 5 minutes, add a small drop of methylene blue, and after another 10 minutes, add a drop of Sudan III. Cover the specimen with a coverslip, remove excess liquid with filter paper, and examine using an immersion objective. The cytoplasm stains blue, and the lipid droplets stain pinkish-orange.

Neisser's (Loeffler's) polyphosphate staining. Polyphosphates are also frequently referred to as metachromatic granules or volutin granules. To detect polyphosphates, prepare a heat-fixed yeast smear and stain it with methylene blue. Within the vacuoles, polyphosphates stain reddish-purple, while the cytoplasm of the yeast cells stains light blue.

6.4. Sporulation in Yeasts

Ascosporogenous yeasts can form ascospores under unfavorable conditions. Sporulation requires specific conditions:

✵ young culture age (pre-cultivation on nutrient-rich media for 1–2 days);

✵ inoculation of actively growing yeast onto starvation media (Gorodkova's medium);

✵ incubation on the starvation medium for 2–4 weeks at a Temperature slightly below optimal (20–25 °C), with weekly microscopic examination.

From 1 to 8 ascospores are formed within a yeast cell or an ascus.

Staining of yeast spores. To stain spores, use a culture of the yeast species *Saccharomyces cerevisiae* previously grown for 2 weeks on Gorodkova's medium. Prepare a fixed smear, flood it with Ziehl's carbol fuchsin, and heat over a spirit lamp for 2–3 minutes until vapors appear. Then decolorize the specimen by immersing it in a 2% lactic acid solution or 95% ethanol containing 1% concentrated Hydrochloric acid. Rinse with water and counterstain for 3–5 minutes with a 1% methylene blue solution, rinse again with water, and blot dry with filter paper. Apply immersion oil and examine under a 90x objective.

Mature ascospores stain red, while vegetative cells stain blue.

Gorodkova's medium for ascospore formation contains (in g per 1 L of tap water): peptone – 10; sodium chloride – 5.0; glucose – 1.0; agar – 20. Adjust the medium pH to 7.3 and sterilize for 15 minutes at a pressure of 0.1 MPa.

Control Questions

1. What types of yeast are used in the production of beer, wine, and bread?

2. Which species of yeast act as spoilage organisms in the brewing and baking industries?

3. What are the Morphological Characteristics of young, old, and dead yeast cells?

4. What reserve nutrients are contained in the cytoplasm of yeast cells? How can they be detected?

5. Under what conditions do yeasts form spores?