FUNDAMENTALS OF MICROBIOLOGY - V. M. Samygin - 2015

CHAPTER 4. MORPHOLOGY OF MICROORGANISMS

All known unicellular and Multicellular Organisms are morphologically divided into two major groups: PROKARYOTES AND EUKARYOTES. Prokaryotes include Bacteria and cyanobacteria, while eukaryotes encompass Fungi, Algae, Protozoa, green plants, and animals. Prokaryotic Cells lack a true nucleus. In other words, the genetic material (DNA) of prokaryotes is located directly in the Cytoplasm and is not enclosed by a nuclear membrane. Eukaryotes (from the Greek eu, meaning true, and karion, meaning kernel or nucleus) possess a true nucleus, meaning their genetic material is enclosed within a double membrane (nuclear envelope) and forms a well-defined cellular Structure that is easily identifiable.

4.1. Methods FOR STUDYING the Morphology of Microorganisms

Microbial cells are investigated through Cytology, cytochemistry, biochemistry, biophysics, molecular biology, genetics, Cell/15.html">Microscopy, and other scientific disciplines. These fields employ various methods to "peer" inside The Cell, examining its structure and the life processes associated with it.

The primary method for studying microbial cell morphology is microscopy, which involves observing living or dead cells using a microscope. Initially, this was the Light Microscope, followed later by phase-contrast, stereoscopic, ultraviolet, luminescent, polarizing, and, finally, electron microscopes.

Light microscopes are subdivided into student, working, laboratory, and research categories, which differ in design and configuration.

Microbiological research typically begins with the microscopy of native material (biological samples, food products, etc.). In the vast majority of cases, microscopic results alone are insufficient for microorganism identification, but they provide a preliminary estimate of the quantity of microorganisms present and indicate whether they belong to rod-shaped or coccoid microflora. To determine the shape of microorganisms using a light microscope, researchers most frequently use preparations of fixed (killed) cells stained with various Dyes, which are categorized by their ability to reveal specific Structural components of microbial cells. Typically, there are 4 major groups of dyes:

- basic (or nuclear) dyes, which selectively stain The Nucleus and basophilic (from Lat. basis, base) structures of bacterial cells;

- acidic (or cytoplasmic) dyes, which stain predominantly the cytoplasm and, less frequently, cell walls;

- neutral dyes, which selectively stain specific cytoplasmic components; for example, Sudan III or Nile blue stain fat droplets;

- fluorochromes, a group of dyes capable of fluorescence under excitation light of a specific wavelength.

There are specific staining methods for microbial nuclear material (Romanowsky-Giemsa method), spores (Ožهشko [Ozershky] and Peshkov methods), capsules (Burri method), cell walls (Gutstein and Ziehl-Neelsen methods), flagella (Morozov method), and volutin granules (polyphosphate, Raskina method).

As early as the 19th century, scientists established that the resolving power of optical microscopes is limited by the wavelength of light. The smallest bacteria lie near the limit of visibility for the most advanced optical microscopes. The phase-contrast microscope did not push past the lower bound of observable sizes, but it allows for the observation of living microbial cells. In a phase-contrast microscope, altering the optical path length of light waves creates a "phase shift of one-quarter wavelength." As a result, surface relief is enhanced, making it possible to visualize certain minute Structural elements of the cells.

The Electron microscope offers the greatest potential for morphological research. In electron microscopes, beams of electrons play The Role of the light rays used to produce magnified images in other microscopes. Their movement is controlled by electromagnets acting as optical lenses. Modern electron microscopes make it possible to magnify objects several hundred thousand times. A specialized microsurgical device called an ultramicrotome is used to study the Internal Structure of cells. It produces ultrathin cell sections (0.02 µm) for viewing under an electron microscope. However, the electron microscope has one drawback: it can only be used to observe dead cells. This is because air molecules present an insurmountable obstacle to electrons, meaning all observations must be conducted in a vacuum, which leads to the immediate dehydration and death of all living cells.

4.2. Morphological Types of Bacteria

The dimensions of microbial cells are characterized using the following Units of Measurement: micrometer (1 µm = 10^-6 m) and nanometer (1 nm = 10^-9 m). When working with an electron microscope, an even smaller unit is used: the angstrom (Å), or 10^-10 m. Most bacterial cells average 1–3 µm in size, while Yeast cells reach 5–10 µm. Naturally, such tiny entities must also be exceptionally lightweight. There are 5 billion bacteria per milligram of weight, meaning a single cell weighs ~0.2 • 10⁹ mg.

Based on their external appearance, three main bacterial shapes are distinguished: spherical or coccoid (from the Greek coccus, berry), rod-shaped or cylindrical, and tortuous or spiral.

Cocci can be spherical, ellipsoidal, bean-shaped, or lanceolate (double-edged blades). Depending on the relative arrangement of individual cells, cocci are divided into the following groups:

- micrococci — characterized by a single, random arrangement of cells (Fig. 1, a);

- diplococci — paired cocci (pneumococcus, the CAUSATIVE AGENT OF Pneumonia; meningococcus, the causative agent of meningitis) (Fig. 1, b);

- streptococci — arranged in chains (species causing Sepsis and inflammatory processes) (Fig. 1, c);

- staphylococci — arranged in grape-like clusters (causing boils, abscesses, etc.) (Fig. 1, d);

- tetrads — consist of four closely positioned cocci (Fig. 1, e);

- sarcinae — arranged in packet-like clusters of 8, 16, or more cells (Fig. 1, f).

Class="center">Fig. 1. Shape of spherical bacterial cells

Rod-shaped (cylindrical) forms are subdivided into bacteria, bacilli, and clostridia. Bacteria include rod-shaped microorganisms that typically do not form spores (such as Escherichia coli, Salmonella typhi, Shigella, Mycobacterium tuberculosis, and others). Bacilli (from Lat. bacillus – rod) and clostridia (from Lat. closter – spindle) comprise microbes that mostly form spores (hay bacillus, anthrax bacillus, tetanus bacillus, and causative agents of anaerobic infections) (Fig. 2).

Fig. 2. Rod-shaped forms of microorganisms: a – Escherichia coli, b – anthrax bacilli, c – Clostridium botulinum

The total number of rod-shaped bacteria significantly exceeds that of coccoid forms.

Convoluted, or spiral, forms. This group of bacteria includes (Fig. 3):

- vibrios (from Lat. vibrio – to bend) – comma-shaped cells (cholera agent, aquatic vibrios);

- spirilla (from Lat. spira – coil) – convoluted forms of bacteria featuring one or more turns of a spiral;

- spirochetes have a corkscrew-like spiral shape.

Fig. 3. Convoluted (spiral) forms of microorganisms: a – vibrios, b – spirilla, c – spirochetes

Bacteria with triangular, filamentous, star-shaped, and other morphologies have also been discovered.

Microbes exhibit polymorphism – an individual Variability characterized by a diversity of SHAPES AND SIZES that manifests independently of age and developmental stage. They are readily altered by various environmental factors: Temperature, nutrient medium, salt concentration, acidity, metabolic products, disinfectants, medications, etc. Polymorphism is particularly pronounced in bacteria when cultured on artificial media. Such morphological variations are associated with disruptions in either Cell wall synthesis or the Cell Division regulatory mechanism. Depending on the intensity and depth of the impact on the microbial cell, these changes can be hereditary or non-hereditary.

4.3. ULTRASTRUCTURE OF THE Bacterial Cell

The structure of a typical bacterial cell is shown in Fig. 4.

Fig. 4. Schematic representation of a Introduction/4.html">Prokaryotic Cell: 1 – cytoplasm, 2 – cytoplasmic membrane, 3 – cell wall, 4 – capsule, 5 – flagellum, 6 – pili (fimbriae), 7 – nucleoid, 8 – Plasmids, 9 – mesosome, 10 – Ribosomes, 11 – inclusions

The main structures of the microbial cell are the envelope, cytoplasm, nuclear apparatus (nucleoid), and various cytoplasmic inclusions.

The bacterial envelope consists of the cytoplasmic membrane, cell wall, and a capsular layer, which transforms into a true capsule in certain species.

Under METABOLISM/18.html">The Influence of various environmental factors, some microbes have The ability to deposit a thick mucous layer over The cell wall on their surface, known as the capsule. Depending on the density and thickness of this mucous layer, macro-capsules, micro-capsules, mucous sheaths (glycocalyx), and S-layers are distinguished:

- macro-capsule (proper capsule);

- mucous sheath (glycocalyx), like the capsule, is formed by high-molecular-weight mucopolysaccharides, differing from the micro-capsule in that it lacks a permanent, rigid attachment to the cell wall;

- S-layers line the outer surface of the cell wall with regularly packed, uniformly shaped protein structures. They are arranged like parquet tiles that tightly envelop the cell.

The capsular substance of bacteria consists of Polysaccharides, mucopolysaccharides (hyaluronic acid), or Polypeptides. Depending on the Specific characteristics of the MICROORGANISMS AND THE COMPOSITION OF THE nutrient medium, The chemical composition of capsules is highly variable. Due to their high Water content (up to 98%), they stain poorly with dyes. Capsule formation is considered an adaptive function of microbes. Capsules protect the cell against toxic substances, mechanical damage, and desiccation. Pathogenic encapsulated microbes are more resistant to phagocytosis and the host's defense mechanisms. Capsules are not an essential part of the cell. Although capsule formation is a species-specific trait, it can be lost under both natural and artificial conditions.

Flagella. Many bacteria are motile, which is the basis for their division into swimming and gliding forms. The motility of swimming bacteria (such as enterobacteria, pseudomonads, and spirilla) is driven by one or more flagella. Flagella are microtube-like structures composed of identical spherical subunits of the protein flagellin arranged in a spiral to form a hollow cylinder. They typically measure 10–20 µm in length and 12–20 nm in thickness.

Based on flagellar arrangement, motile microbes are divided into four groups: monotrichates, bacteria with a single flagellum at one pole (e.g., Vibrio cholerae, Pseudomonas aeruginosa); lophotrichates, bacteria with a tuft of flagella at one end; amphitrichates, bacteria with two polar flagella or a tuft of flagella at both ends; and peritrichates, bacteria whose entire body surface is covered with flagella (e.g., Escherichia coli, Salmonella typhi) (Fig. 5).

Fig. 5. Arrangement of bacterial flagella: a - monotrichous; b - lophotrichous; c - amphitrichous; d - peritrichous

Cell movement is driven by the Rotation of the flagellum clockwise or counterclockwise (in monotrichs), while the cell itself slowly rotates clockwise. When the flagellum rotates clockwise, the cell is pulled forward by the flagellum (flagellum leading). When it rotates counterclockwise, the cell is pushed forward by the flagellum (flagellum trailing). Some monotrichs rotate their flagella exclusively clockwise, requiring them to stop and reorient themselves to change direction. Peritrichs rotate their flagella (or flagellar bundle) counterclockwise, keeping the flagella trailing behind the cell in the direction of movement. Monotrichs exhibit the highest motility (up to 60 µm/s).

All motile bacteria move in directions determined by external stimuli. Depending on The Nature of the environmental factor triggering the movement, we distinguish chemotaxis (movement in response to specific chemical compounds), aerotaxis (driven by oxygen concentration gradients), magnetotaxis (the ability to align and move along Earth's magnetic field lines), and viscotaxis (bacterial response to changes in solution viscosity).

Another form of bacterial locomotion is gliding, where cells move across a solid surface through wave-like contractions. The Mechanism of this movement type remains insufficiently understood. In some cases, gliding is driven by outer Membrane Proteins, where forward motion along a surface acts as a form of propulsion. Gliding bacteria lack flagella and leave a slime trail as they move. Additionally, some bacteria exhibit a "jumping" motility whose exact nature remains unclear.

Pili. Various microbial species possess thin, straight, Hair-like appendages known as pili (or fimbriae). Shorter and thinner than flagella, they cover the entire cell surface and are found in both motile and non-motile organisms. A single cell may bear anywhere from 50 to 400 pili. A typical fimbria is about 1.5 µm long and 7 nm in diameter. Pili facilitate the attachment of microbial cells to one another or to surfaces. Another type, known as sex pili or F-pili, contain an internal channel through which genetic material is transferred between cells during bacterial conjugation. Pili are composed of the protein pilin and, much like flagella, are not essential structural components of bacterial cells.

The cell wall is a critically important structural element of bacteria. It gives the cell its shape and rigidity, protects its internal contents, and prevents osmotic Swelling and lysis when cells enter a hypotonic environment. It also plays a vital role in regulating bacterial growth and division. Water, other small molecules, and ions easily pass through the tiny pores of the cell wall, whereas large PROTEIN AND NUCLEIC acid molecules cannot. The thickness of the cell wall ranges from 20 to 100 nm, accounting for about 20% of the dry weight of a bacterial cell.

Based on cell wall architecture, bacteria are divided into two main groups. Some retain the Gram stain and are called Gram-positive, while others lose the stain during the decolorization step and are called Gram-negative. The cell walls of both groups contain a rigid lattice composed of murein, or peptidoglycan. The murein molecule forms a regular network of parallel polysaccharide chains cross-linked by short peptide chains. In Gram-positive bacteria, peptidoglycan accounts for 40–60% of the dry mass of the cell wall. These microorganisms incorporate additional structures formed by polysaccharides, proteins, or Glycoproteins directly into their murein network. In Gram-negative bacteria, the cell wall is thinner but structurally more complex. The peptidoglycan layer accounts for only about 10% of the cell wall mass and is externally covered by a smooth lipid layer known as the outer membrane, which consists of Phospholipids, lipopolysaccharides, Lipoproteins, and proteins. This lipid layer renders the cell resistant to certain Enzymes and Antibiotics (such as penicillin). Beneath the outer membrane lies the periplasm, or periplasmic space. The periplasm contains the peptidoglycan layer, periplasm-specific proteins and Oligosaccharides, and Inorganic Compounds. Bacteria that completely lack a cell wall are called protoplasts; they are spherical and capable of division, Respiration, Protein Synthesis, and sporulation. However, protoplasts are highly sensitive to osmotic pressure changes, mechanical stress, and aeration, and they lack active motility. Partial dissolution of the cell wall transforms bacteria into spherical bodies known as spheroplasts.

The cytoplasmic membrane tightly adheres to the inner surface of the cell wall and is 7–8 nm thick. The cytoplasmic membrane (CPM) serves as the primary barrier between the Cell Cytoplasm and the external environment. The Key Components of Biological Membranes—which form bilayer structures—are polar Lipids, predominantly fatty acid-containing phospholipids. The CPM acts as a separating partition, continuously utilizing enzymes to drive The Active Transport of various substances and ions essential for cell survival. In some bacteria, The Plasma Membrane infolds into the cell interior to form mesosomes.

Mesosomes are folded membranous structures composed of tubules, vesicles, and lamellae, On the surface of which reside the enzymes involved in cellular respiration, thereby functioning in energy generation. Alongside respiratory enzymes and permeability-regulation machinery, bacterial mesosomes—together with the CPM—house specific enzyme systems involved in Nitrogen Fixation and Chemosynthesis. Furthermore, mesosomes play a role in cell wall synthesis, cell division, exoenzyme secretion, and sporulation.

The prokaryotic genetic apparatus (the chromosome or nucleoid), which stores the Genetic information for all cellular traits, is represented by a giant double-stranded DNA molecule about 1 mm long and roughly 2 nm thick. The nucleoid is a closed structure that is neither segregated from the cytoplasm by any membrane nor complexed with bacterial Histones. During rapid bacterial growth, Chromosome Replication typically outpaces cell division; consequently, young cells may contain multiple identical copies of a single chromosome. For instance, rapidly growing E. coli cells in a nutrient-rich medium contain up to 11 nucleoids, whereas slowly growing cells harbor 1–2 Chromosomes.

Most bacteria house all their genes on a single chromosome. However, accumulating evidence suggests that essential genes can also reside on multiple chromosomes. Two circular chromosomes have been discovered in certain vibrios, brucellae, and leptospires, while Burkholderia cepacia has between two and four. Moreover, the number of species with more than one linkage group (multiple chromosomes) may be far greater than we currently realize, given that modern molecular biology estimates we know less than 1% of all microorganisms existing on Earth.

In addition to the main chromosome, many bacteria harbor extra-nuclear (extrachromosomal) circular and linear double-stranded DNA molecules that can exist and replicate independently or integrate into the chromosome. These include plasmids, Transposons, and insertion (IS) sequences. Bacterial plasmids are double-stranded DNA molecules ranging from 10⁶ to 10⁸ Da in size, carrying between 40 and 50 genes. They perform regulatory and coding Functions. Plasmid DNA typically accounts for no more than a few percent of chromosomal DNA, with plasmid copy numbers ranging from 1 to 200. Plasmids contain anywhere from 1,500 to 40,000 nucleotide pairs. Like other extrachromosomal elements, plasmids are not strictly vital because they do not encode enzymes required for basic bacterial metabolism. However, they can alter bacterial nutritional requirements and confer traits such as resistance to antibiotics and heavy metal salts, or the ability to produce pathogenicity factors and metabolic enzymes. A crucial role of extrachromosomal genetic elements is that they facilitate Horizontal Gene Transfer (between cells within a population) and drive prokaryotic evolution. Thus, extrachromosomal elements enable bacteria to thrive across a broader range of environmental conditions, acting as an adaptive factor.

Bacterial cytoplasm refers to the cellular contents enclosed by the CPM. The homogeneous fraction of the cytoplasm containing a mixture of soluble substances—such as proteins, RNA, and metabolic intermediates—is known as the Cytosol. The remaining portion of the cytoplasm consists of various structural elements, including the genetic apparatus, ribosomes, and inclusions that vary in Chemical Composition and function. The cytoplasm is characterized by high viscosity (comparable to glycerin or thick syrup). Its consistency depends on cell age, growth medium, and bacterial species; the cytoplasm of older bacteria becomes increasingly gel-like.

The cytoplasm contains small granules measuring 15–20 nm in diameter, composed of protein and Nucleic Acids (RNA), which are called ribosomes. They are essential and vital structures for bacterial life. A single cell typically contains anywhere from 5,000 to 90,000 ribosomes, and their total mass can constitute up to a quarter of the cell's dry weight. Ribosomes serve as the primary sites of protein synthesis.

Prokaryotic ribosomes have a sedimentation coefficient of 70S and are composed of two subunits: 30S and 50S. The 30S particle contains a single molecule of 16S rRNA and, in most cases, single copies of over 20 different proteins. The 50S subunit consists of two rRNA molecules (23S and 5S) along with over 30 distinct proteins, also present in single copies. The majority of ribosomal proteins perform structural roles.

Inclusions. Throughout The life cycle of a bacterial cell, its cytoplasm may accumulate morphologically distinct structures that vary in shape, size, and chemical composition across different species. These are known as inclusions. Bacterial inclusions include Glycogen and starch granules, lipid droplets, sulfur globules, oxalic acid crystals, volutin granules, calcium carbonate, gas vacuoles (gas vesicles), and others. In some cases, an inclusion represents an accumulation of Metabolic waste products, while in others it serves as a nutrient reserve. These diverse inclusions are not essential for cell viability and may be present or absent depending on the bacterial species and physiological state.

4.4. Spores and Sporulation

Microorganisms belonging to the genera Bacillus, Clostridium, Desulfotomaculum, Sporolactobacillus, Sporosarcina, and several others (encompassing around 20 genera in total) are capable of forming spores (endospores)—round or oval bodies. Sporulation is characteristic of both pathogenic species (such as the causative agents of anthrax, botulism, and tetanus) and saprophytic species inhabiting soil, water, and animal bodies. Spores are most commonly produced by rod-shaped microorganisms, whereas spore formation is relatively rare in cocci and spiral forms.

Spores occupy various positions within the cell. A spore's diameter may be roughly equal to or slightly exceed that of the mother cell. In some bacteria, the Spore Forms at the cell pole (terminal position), causing the cell to swell slightly and resemble a drumstick; in others, it forms centrally, with the cell either retaining its normal shape (genus Bacillus) or bulging in the center to take on a spindle shape (genus Clostridium) (Fig. 6).

Fig. 6. Localization of endospores within the bacterial cell: 1, 2 - central; 3, 4, 5 - terminal; 6 - lateral

Spores exhibit high light refractility and appear as shiny granules under unstained microscopic examination; they also take up stains with great difficulty. Due to a tough, multilayered, lamellar coat, a minimal free water content, and high levels of calcium and lipids, spores possess exceptional resistance to environmental stressors and can survive hostile conditions for extended periods (decades). The spores of certain bacilli can withstand boiling and exposure to high concentrations of disinfectants. They are destroyed in an autoclave by saturated steam at 115–125 °C (1.5–2 atm) after 20 minutes, or upon exposure to 150–170 °C in a dry-heat oven for 1 hour.

Spore production is a stage in the Life Cycle of certain microorganisms, evolved as an adaptation to ensure species survival. Spores represent a resting stage. When exposed to unfavorable environmental conditions, the cytoplasm containing the genetic material (one or more chromosomes) isolates itself from the rest of the cellular content through an invagination of the cytoplasmic membrane inward. The membrane grows from the periphery toward the center, where it fuses, leading to The formation of a spore septum. Next, a forespore is formed—a structure located inside the mother cell and completely separated from it by inner and outer membranes. Spore coats are then synthesized over the forespore's outer membrane. Following spore maturation, lysis (destruction) of the mother cell wall occurs, and the spore is released into the environment. The sporulation process takes 18–20 hours. Typically, a single spore is formed per cell, though instances of up to 5 spores forming within one cell are known. Sporulation should not be considered a mode of bacterial reproduction, as the formation of spores does not increase the number of microorganisms.

Upon encountering favorable conditions, spores germinate and transform back into vegetative cells. In the process, they swell, increase in size, their water content rises, and metabolic processes intensify. The First stage involves Enzyme Activation and active Protein and RNA synthesis, whereas DNA replication begins only 1–2 hours after germination starts. Subsequently, the thick layer of peptidoglycan molecules situated between the two forespore membranes is degraded, the spore coats rupture, and the resulting structure—known as a germ tube—emerges. This is followed by its elongation, the completion of the cell wall, and the division of the elongated cell. Germination generally proceeds much faster than sporulation, taking about 4–5 hours.

The ability to form spores is utilized in microbial Taxonomy; however, this trait can be lost as a result of frequent subculturing on nutrient media or cultivation at high temperatures.

In addition to endospores, certain other bacteria possess alternative resting forms—exospores and cysts. Exospores arise via budding of the mother cell and share similar properties with bacillary endospores. Some bacteria form spherical, thick-walled cells called cysts, which are characteristic of Azotobacter, spirochetes, myxobacteria, rickettsia, methylotrophs, and certain other bacteria. When nutrient resources are depleted, the entire rod-shaped vegetative cell transforms into a cyst, rather than just a part of it as occurs during endospore formation. Cysts are resistant to desiccation, mechanical stress, and radiation, but not to high temperatures.