Microbiology

Microbiology/Definition
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Science that study microorganisms Microorganisms – single cell particle that need a light or electronic microcope to be seen

Type of cells
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Eukariotic cells: fungi and protozoa Prokariotic cells: bacteria and blue-green algae Viruses: infective particle that need a viable cell for their replication and can be seen using EM

Microbiology
Bacteria structures and functions

Definition

Bacteria are living forms that are microscopical in size (1-10 µ m) and relatively simple, unicellular, in structure a microscope is therefore necessary for their observation.

Structure

Each cell consists of a body of protoplasm, the protoplast, enclosed by a thin, semipermeable membrane, the cytoplasmic membrane and also, in most cases, by an outer, relatively rigid cell wall.

The bacterial structures may be divided into three categories:
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1. Essential structures, present in all bacteria 2. Structures present in some species (primary taxonomic characters) 3. Structures present in some strains of some species

Essential structures, present in all bacteria
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Protoplast (cytoplasm and nuclear body) Cytoplasmic membrane Cell wall

Bacteria’structure

2. Structures present in some species (primary taxonomic characters):
 flagella;  spores;  inclusion granules.

Flagellum

3. Structures present in some strains of some species:
 fimbriae;  sex pili;  glicocalix (capsule, microcapsule, loose slime).

Fimbriae (pili), sex pili, capsule

Structure

 

The protoplast is differentiated into a major part, the cytoplasm, and an inner body, the nuclear body, which contains the hereditary determinants of character, the genes, borne on chromosome.

Schematic presentation

Bacteria = prokaryotic cells
 

The bacteria are prokaryotic cells. It is useful to draw a clear distinction between relatively primitive (prokaryotic) and more advanced (eukaryotic) cells.

The main distinguishing features of the prokaryotic cell are:

Its nucleus appears as a simple, homogeneous body, containing a single chromosome, not possessing a nuclear membrane separating it from the cytoplasm, nor a nucleolus, nor a spindle, nor a number of separate non-identical chromosomes. It reproduces by binary fission, not by mitotic division.

Nucleus - DNA

The main distinguishing features of the prokaryotic cell

It lacks the internal membranes isolating the respiratory and photosynthetic enzyme systems in specific organelles. Thus, the respiratory enzymes in bacteria are located mainly in the peripheral cytoplasmic membrane. The cytoplasm is a soft gel and has no internal mobility. Prokaryotes have 70 Svedberg unit ribosomes.

The main distinguishing features of the prokaryotic cell

Its rigid cell wall contains as its main strengthening element a specific peptidoglycan (substance not found in eukaryotic organisms). Steroids are absent in the procaryotic cell wall (exception, mycoplasmas).

PROKARYOTIC CELL STRUCTURE

The protoplast is bounded peripherally by a very thin, elastic and semipermeable cytoplasmic membrane. Outside, and closely covering this, lies the rigid, supporting cell wall, which is porous and relatively permeable.

Cell membrane and cell wall

1. Essential structures, present in all bacteria
 the cytoplasm containing ribosomes;  the cytoplasmic membrane (or plasma membrane);  the rigid cell wall (exception, mycoplasmas).

2. Structures present in some species (primary taxonomic characters):
 flagella;  spores;  inclusion granules.

Flagella (electron microscopy)

Spores (Clostridium tetani)

Granules (inslusion bodies)

3. Structures present in some strains of some species:
 fimbriae;  sex pili;  glicocalix (capsule, microcapsule, loose slime).

Fimbriae, sex pili, capsule

Bacterial nuclear body (DNA)

BACTERIAL DNA

The bacterial cell lacks a nuclear membrane; instead, the DNA is concentrated in the cytoplasm as a nuclear body. The nuclear body consists of a single chromosome (1 mm length) of doublestranded, circular, covalently closed, supercoiled DNA.

Size

The cell solves the problem of packaging this enormous DNA molecule (2-3 x 109 kDa) by condensing and looping it into the supercoiled state.

DNA molecule and plasmid

Plasmids

In many bacteria, a small portion of DNA persists as extrachromosomal elements referred to as plasmids, which are also circular, but are much smaller than bacterial chromosomes.

Plasmids - extrachromosomal elements

Plasmids’ functions

Plasmids encode variable numbers of genes (mainly responsible for the bacterial survival in the environment) and often determine virulent behavior or antibiotic resistance.

Nuclear body

The nuclear body is constantly present in all cells and under all conditions of culture. It replicates by growth and simple fission, and not by mitosis. Electron micrographs reveal the absence of the outer nuclear membrane separating it from the cytoplasm, and of the nucleolus. Bacterial DNA has a similar structure to that of eukaryotic chromatin (see genetic).

Nuclear body’s observation

The nuclear body can’t be seen with the light microscope in bacteria stained by usual methods because it is covered by the multitude of RNA molecules from the bacterial cytoplasm; it is observed on stained films after RNA hydrolysis.

Functions

The bacterial DNA stores the genetic information. It is involved in autoreplication (by which copies of genetic information are transmitted to daughter cells) and heteroreplication (by which the information is copied in mRNA sequences).

Replication (relation of bacteria’s structure and antibiotics)

CYTOPLASM OF BACTERIA

The cytoplasm of bacteria is a viscous watery solution, or soft gel, containing a variety of organic and inorganic solutes, and numerous small granules called ribosomes. The variety of organelles seen in eukaryotic cells is missing in bacteria because the cytoplasmic membrane performs many complex functions carried out by these organelles. The cytoplasm of bacteria also differs from that of the eukaryotic organisms in not showing signs of internal mobility.

RIBOSOMES

Ribosomes are complex globular structures composed of several RNA molecules and many associated proteins; they function as the active centers for protein synthesis. Bacterial ribosomes are slightly smaller (10-20 nm) than those of eukaryotic cells and they have a sedimentation constant of 70S (Svedberg units), being composed of a 30S (1 RNA molecule and 21 proteins, S1-S21) and a 50S subunit (2 RNA molecules and 34 proteins, L1-L34).

Ribosomes

Ribosomes

They may be seen with the electron microscope in number aprox. 20.000 per cell. They are strung together on strands of messenger RNA (mRNA) to form polysomes and it is at this site that the code of the mRNA is translated into peptide sequences.

INCLUSION GRANULES

In many species of bacteria, round granules are observed in the cytoplasm. These are not permanent or essential structures, and may be absent under certain conditions of growth. They appear to be aggregates of substances concerned with cell metabolism, e.g. an excess metabolite stored as a nutrient reserve.

INCLUSION GRANULES (starch, glycogen, etc.)

INCLUSION GRANULES (exemples, functions)
 

They consist of volutin (polyphosphate), lipid, glycogen, starch or sulfur. Their demonstration may assist in the identification of certain organisms (primary taxonomic character); Ex.: the diphtheria bacillus (Corynebacterium diphteriae) may be distinguished from related bacilli found in the throat by its content of volutin granules.

Corynebacterium diphteriae (volutine granules - dark)

MESOSOMES

They are convoluted or multilaminated membranous bodies visible in electron microscope. They develop by complex invagination of the cytoplasmic membrane into the cytoplasm, sometimes in relation to the nuclear body and often from the sites of cross-wall formation during cell division. Mesosomes are thought to be involved in the mechanisms responsible for the compartmenting of DNA at cell division and sporulation.

MESOSOMES

CYTOPLASMIC MEMBRANE

The cytoplasmic membrane is the physical and metabolic barrier between the interior and exterior of the bacterial cell. It is 5-10 nm thick, consists mainly of lipoprotein and is visible in some ultrathin sections examined with the electron microscope (EM).

Cell (Cytoplasmic) Membrane
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Flexible, phospholipid bilayer sheet Hydrophobic tails Hydrophillic heads May contain steroid- like molecules: hopanoids to stabilize structure

Cell (Cytoplasmic) Membrane

The lipid molecules are arranged in a double layer with their hydrophilic polar regions externally aligned and in contact with a layer of protein at each surface.

Cytoplasmic membrane

Cytoplasmic membrane

Cell (Cytoplasmic) Membrane
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Boundary between a cell and its environment Dynamic interface Changes with temperature, age, environment

Functions of the procaryotic plasma membrane
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Osmotic or permeability barrier (separate “outside” (s from “inside”). Location of transport systems for specific solutes (nutrients and ions) Mesosomes (internal invaginations of cytoplasmic membrane). Energy generating functions, involving respiratory and photosynthetic electron transport systems, establishment of proton motive force, and transmembranous, ATP-synthesizing ATPase Synthesis of membrane lipids (including lipopolysaccharide in Gram-negative cells)

Functions of the procaryotic plasma membrane
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Synthesis of murein (cell wall peptidoglycan) Assembly and secretion of extracytoplasmic proteins Coordination of DNA replication and segregation with septum formation and cell division Chemotaxis (both motility per se and sensing functions) Location of specialized enzyme system

CELL WALL

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The cell wall encases protoplast and lies immediately external to the cytoplasmic membrane. It is 10-25 nm thick, strong and rigid. It supports the weak cytoplasmic membrane against the high internal osmotic pressure (5 – 20 atms) of the protoplasm and maintains the characteristic shape of the bacterium in its coccal, bacillary, filamentous or spiral form. The rigid cell wall is entirely absent in a few unusual bacteria (e.g., mycoplasmas, genus Mycoplasma).

Bacteria’s shapes/arrangement

CELL WALL

The integrity of the cell wall is essential to the viability of the bacterium. If the wall is weakened or ruptured, the protoplasm may swell from osmotic inflow of water and burst the weak cytoplasmic membrane (lysis).

Bacterial rigid cell wall is made up of peptidoglycan and special structures

Peptidoglycan (murein) is the principal structural component of the cell wall. This compound is found in both Gram-positive and Gram-negative bacteria, although is more abundant in Gram-positive organisms.

Peptidoglican of Gram positive bacteria

Peptidoglican of Gram negative bacteria

Gram (+), (-) and acid fast bacterial cell wall (comparison)

Gram (+) and Gram (-) bacterial cell wall (comparison)

Peptidoglycan

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Peptidoglycan polymers consist of repeating dissacharides formed by: N-acetyl-glucosamine (NAG) and N-acetylmuramic acid (NAM).

Peptidoglycan

The N-acetylmuramic (NAM) acid links the dissacharides to an oligopeptide chain consisting of four aminoacids (usually Lalanine, D-glutamic acid, either mesodiaminopimelic acid – in Gram-negative bacteria – or L-lysine – in Gram-positive bacteria and D-alanine).

Peptidoglycan

Lysozyme hydrolyses peptidoglycan by cleaving the glycosil bonds between N-acetylmuramic acid and the N-acetylglucosamine.

Cell division

Cell division occurs by the development, from the periphery inwards, of a transverse cytoplasmic membrane and a transverse cell wall, or cross wall. The cell wall plays an important part in cell division. As the protoplast increases in mass, the cell wall is elongated by the intercalation of newly synthesized subunits into the various wall layers.

Division of cell

Cell wall

The cell wall can be demonstrated by special staining methods, but most clearly by electron microscope. It is not seen in conventionally stained smears examined with the light microscope, but its chemical structure is responsible for the staining of bacteria in differential stainings.

Cell wall

Gram staining divides bacteria in grampositive (violet) and gram-negative (red); Ziehl-Neelsen staining divides bacteria in acid-fast (red) and non-acid-fast (blue).

Gram-staining

Gram-staining, the most common criterion for grouping medically important bacteria, is a simple differential staining technique that employs crystal violet (an aniline dye) as the primary stain and fuchsin (a red dye) as a counter-stain. Gram-positive bacteria appear violet because they retain the crystal violet and resist alcohol decoloration. Gram-negative bacteria appear red because they are decolorized completely by ethanol and they take up fuchsin, the counter-stain.

Gram staining it is influenced by cell wall structure

Staphylococcus (Gram positive cocci arranged in clusters)

Gram negative bacilli

Acid fast bacilli (ex. Mycobacterium tuberculosis)

Gram-positive bacteria

Gram-positive bacteria have a simpler, but thicker cell wall, consisting primarily of tridimensional peptidoglycan with teichoic acid polymers (ribitol or glycerol phosphate complexed with sugar residues) dispersed throughout; some of this material (lipoteichoic acid) is linked to lipids buried in the cell membrane.

Gram-positive bacteria

Teichoic acids

The teichoic acids are major surface antigens (see immunology) in the Gram-positive species that possess them. They bind magnesium ion and play a role in the function of the cytoplasmic membrane’s enzymes. They bind autolytic enzymes, being thus involved in the cell’s growth and division. They are receptors for bacteriophages (viruses of bacteria).

Gram-positive bacteria

Function of peptidoglycan

Prevents osmotic lysis of cell protoplast and confers rigidity and shape on cells

Gram-negative bacteria

Gram-negative bacteria have a cell wall that is thinner than that of Gram-positive bacteria, with bi-dimensional peptidoglycan and no teichoic acids. An additional membrane, the outer membrane, lies above the peptidoglycan layer. The outer membrane is much thicker than the single peptidoglycan layer.

The outer membrane
   

The outer membrane is composed of: a bilipid layer, proteins and lipopolysaccharide (LPS, endotoxin).

Diagrams of the cell wall structure of Gram-negative (left) and Gram-positive bacteria. Key: peptidoglycan layer (yellow); protein (purple); teichoic acid (green); phospholipid ( brown); lipopolysaccharide (orange).

The outer membrane

Cell wall of Gram (-) bacteria
The bilipid layer is attached to the peptidoglycan by lipoproteins that cross the periplasmic space (Braun’s lipoproteins: anchors the outer membrane to peptidoglycan – murein - sheet ).  The proteins include porins, which form transmembrane channels involved in the • transport of ions and • hydrophilic compounds from the extracellular compartment to the periplasm.

Porin

Functions of porins

Omp C and Omp F porins: proteins that form pores or channels through outer membrane for passage of hydrophilic molecules Omp A: provides receptor for some viruses and bacteriocins (antibiotics produce by some bacteria); stabilizes mating cells during conjugation (see genetic chapter).

Gram-negative bacteria are surrounded by two membranes. The outer membrane functions as an efficient permeability barrier because it contains lipopolysaccharides (LPS) and porins.

Lipoproteins

Lipopolysaccharide (LPS, endotoxin)
LPS is composed of a lipid portion (lipid A), a polysaccharide rich core, and a polysaccharide side chain.  the lipid portion is heat-stable and responsible for the biologic effects of endotoxin;  the polysaccharide rich core is antigen R of gram-negative bacteria;

Lipopolysaccharide (LPS, endotoxin)

the polysaccharide portion of LPS is antigenic and is designed as the antigen O; antigen O determines electronegativity and hydrophylia of the bacteria, thus confers smooth culture characters, stability in suspensions and antiphagocytic effects; antigen O has group specificity, being important for the identification of bacteria.

1. LPS; 2 Braun lipoproteines; 3 Fosfolipide 4 outer membrane; 5 Peptidoglican layer

LPS

The outer membrane
 -

-

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The outer membrane confers several important properties on Gram-negative bacteria: it protects the peptidoglycan from the effects of lysozyme; it impedes the ingress of many antibiotics that are thus rendered impotent; it alows the access of low molecular weight nutrients; components of the LPS, in particular the lipid A, form endotoxin, which when released in the blood stream, may give rise to the endotoxic shock.

The outer membrane

The periplasmic space

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-

The periplasmic space is placed between the outer membrane and the cytoplasmic membrane. It contains the peptidoglycan, lipoproteins and enzymes involved in: the extracellular digestion of gram-negative bacteria or in the inactivation of antibiotics.

The periplasmic space

The periplasmic space

The periplasmic space

Function of peptidoglycan

 

Peptidoglycan prevents osmotic lysis and confers rigidity and shape; outer membrane is permeability barrier; associated LPS and proteins have various functions

Functions of the cell wall

The cell walls of bacteria deserve special attention for several reasons: 1. They are an essential structure for viability, as described above. 2. They are composed of unique components (peptidoglycan) found nowhere else in nature. 3. They are one of the most important sites for attack by antibiotics. 4. They provide ligands for adherence and receptor sites for drugs or viruses. 5. They cause symptoms of disease in animals. 6. They provide for immunological distinction and immunological variation among strains of bacteria.

Acid - fast bacilli

Acid-fast bacilli (mycobacteria, but also other filamentous bacteria such as Nocardia) resist destaining with acid-alcohol after staining with carbol-fuchsin in Ziehl-Neelsen staining, so they stain red.

Acid - fast bacilli (red)

Acid - fast bacilli

The basis for the staining is the presence of unique fatty acids (e.g., mycolic acids) in the cell wall. The high content of wax and lipids of the cell wall confers the acid-fast bacilli resistance to antimicrobial agents, heat, dryness, chemical agents. They are facultative intracellular organisms and they survive in macrophage cells.

Acid - fast bacilli (mycolic acids, arabinogalactan)

Cell Wall-less Forms

A few bacteria are able to live or exist without a cell wall. There are two groups of bacteria that lack the protective cell wall peptidoglycan structure, the Mycoplasma species, one of which causes atypical pneumonia and some genitourinary tract infections and the L-forms, which originate from Gram-positive or Gram-negative bacteria and are so designated because of their discovery and description at the Lister Institute, London.

Cell Wall-less Forms

The mycoplasmas and L-forms are all Gramnegative and insensitive to penicillin and are bounded by a surface membrane structure. Lforms arising "spontaneously" in cultures or isolated from infections are structurally related to protoplasts and spheroplasts; all three forms (protoplasts, spheroplasts, and Lforms) revert infrequently and only under special conditions.

Cell Wall-less Forms

The mycoplasmas are a group of bacteria that lack a cell wall. Mycoplasmas have sterol-like molecules incorporated into their membranes and they are usually inhabitants of osmotically-protected environments. Mycoplasma pneumoniae is the cause of primary atypical bacterial pneumonia, known in the vernacular as "walking pneumonia".

Cell Wall-less Forms

For obvious reasons, penicillin is ineffective in treatment of this type of pneumonia. Sometimes, under the pressure of antibiotic therapy, pathogenic streptococci can revert to cell wall-less forms (called spheroplasts) and persist or survive in osmotically-protected tissues. When the antibiotic is withdrawn from therapy the organisms may regrow their cell walls and reinfect unprotected tissues.

CAPSULES, MICROCAPSULES AND LOOSE SLIME

Many bacteria, including several pathogenic species, are surrounded by a discrete covering layer of a relatively firm gelatinous material that lies outside and immediately in contact with the cell wall (glicocalix).

Microcapsule

When this layer, in the wet state, is wide enough (0.2 µ m or more) to be resolved with the light microscope, it is called a capsule. When it is narrower, and detectable only by indirect, serological means, or by electron microscopy, it may be termed a microcapsule.

Microcapsule

The capsular gel consists largely of water and it has only a small content of solids (e.g. 2%). In most species, the solid material is a complex polysaccharide, though in some species its main constituent is polypeptide or protein. Extracellular polymer is synthesized by enzymes located at the surface of the bacterial cell.

Loose slime

Loose slime, or free slime, is an amorphous, viscid colloidal material that is secreted extracellularly by some non-capsulate bacteria and also, outside their capsules, by many capsulate bacteria. In capsulate bacteria the slime is generally similar in chemical composition and antigenic character to the capsular substance.

Slime forming bacteria. Capsular slime surrounding bacterial cell is clearly visible

Capsule’s functions

It is probable that the principal action of capsules and microcapsules is to protect the cell wall against attack by various kinds of antibacterial agents, e.g. bacteriophages, colicines, complement, lysozyme and other lytic enzymes, that otherwise would more readily damage and destroy it.

Capsule’s functions

In the case of certain capsulate pathogenic organisms (e.g. pneumococ-cus, pyogenic streptococci, anthrax bacillus) good evidence has been obtained to show that the capsule protects the bacteria against ingestion by the phagocytes of the host. The capsule is thus an important agent determining virulence, and non-capsulate mutants of these bacteria are found to be non-virulent. The capsule is also involved in the adherence of bacteria to different surfaces, including host’s cells.

Capsule’s functions

The capsular substance is usually antigenic and the capsular antigens play a very important part in determining the antigenic specificity of bacteria. When capsulate pneumococci are treated with type-specific antiserum, the sharpness of outline of the capsule is greatly enhanced. This is referred to as the " capsule-swelling reaction ". The capsule is colorless in usual stainings.

20th

"Capsule-swelling reaction"

FLAGELLA

Motile bacteria possess filamentous appendages known as flagella, which act as organs of

locomotion.
 

The flagellum is a long, thin filament, twisted spirally in a wave form. It is about 0.02 µ m thick and is usual several times the length of the bacterial cell. It originates in the bacterial protoplasm and is extruded through the cell wall. According to the species, there may be one to several (e.g. 1-20) flagella per cell.

FLAGELLA

The arrangement of flagella

The arrangement of flagella may be monotrichus – a single flagellum at one end of the bacterium, amphitrichous – two flagella, each at one end of the bacterium, lofotrichous – a group of flagella at one end of the bacterium, or peritrichous, when they originate over the surface of the cell. The presence, number and position of the flagella are primary taxonomic

Flagella

Flagella consist largely or entirely of a protein, flagellin, and are driven by the rotary action of a swivel-like basal hook. They can be demonstrated easily and clearly with the electron microscope, ussually appearing as simple fibrils without internal differentiation.

Flagella

They are invisible by the light microscope, but may be shown by the use of special staining methods (e.g., silver staining), and in special circumstances by dark-ground illumination. Because of the difficulties of these methods, the presence of flagella is commonly inferred from the observation of motility.

Lofotrichous flagella

Bacterial Flagellum Structure

Motility

Motility may be observed either microscopically or by noting the occurence of spreading growth in semi-solid agar medium. On microscopical observation of wet films, motile bacteria are seen swimming in different directions across the field, with a darting, wriggling or tumbling movement.

Semisolid agar

Motility

Among the spirochete, motility appears to be a function of the cell body, since flagella do not occur. The most characteristic movement is a fast spiral rotation on the long axis with slow progression in the axial line; movements of flexion and lashing movements may be observed. Some spirochetes possess an axial filament and others a band of fibrils wound around their surface from pole to pole.

Spirochete’s flagella

Spirochete’s flagella

Treponema and Borrelia have numerous thin axial filaments, disposed around the protoplast, while Leptospira has only two thicker filaments, forming an axistil, surrounded by the protoplast. It has been suggested that these structures may contribute to motility, either through being themselves contractile or by acting as stiffeners for recoil against the contractile protoplast.

Motility

Motility may be beneficial in increasing the rate of uptake of nutrient solutes by continuously changing the environmental fluid in contact with the bacterial cell surface. Random movement and dispersion through the environment may be beneficial ensuring that at least some cells of the strain reach every locality suitable for colonization.

Motility

Bacteria tend to migrate towards regions where there is a higher concentration of nutrient solutes (a process known as chemotaxis) and away from regions containing higher concentrations of disinfectant substances (negative chemotaxis).

Motility

It might be supposed that the power of active locomotion would assist pathogenic bacteria in penetrating through viscid mucous secretions and epithelial barriers, and in spreading throughout the body fluids and tissue, but it must be noted that many non-motile pathogens (e.g. brucellae and streptococci) are no less invasive than motile ones.

PROTOPLASTS, SPHEROPLASTS AND L-FORMS

Weakening, removal or defective formation of the cell wall is involved in the production of the various abnormal forms called spheroplasts, protoplasts and L-forms. Complete removal of the bacterial cell wall of a Gram-positive bacterium results in the formation of protoplasts, which are constituted by the cytoplasmic membrane and the bacterial contents. Protoplasts require an isotonic medium in order to assume a spherical configuration; they cannot maintain integrity when placed in a hypo- or hypertonic medium.

PROTOPLAST

SPHEROPLASTS

The complexity of the Gram-negative cell wall results in innate resistance to enzymatic destruction of the cell wall. Gram-negative cells with damaged cell walls become spheroplasts (i.e., they assume a spherical shape) even in a nonisosmotic medium (i.e., they are resistant to differences in osmotic pressure between the extracellular and intracellular compartments). Spheroplasts commonly revert to normal bacterial morphology when transferred to culture medium lacking the cell wall inhibitor.

L-FORMS

L-forms (wall-less organisms) may emerge during antibiotic therapy. These aberrant organisms can cause persistent infection, resisting the effects of antibiotics whose mechanism of action involves interference with cell wall formation. They differ from the parent bacteria in lacking a rigid cell wall and, in consequence, regular size and shape, but they are nevertheless viable and capable of growing and multiplying on a suitable nutrient medium.

Spore

Some species, notably those of the genera Bacillus and Clostridium, develop a highly resistant structure or endospore, whereby the organism can survive in a dormant state through a long period of starvation or other adverse environmental condition.

Sporulation versus germination
 

The process does not involve multiplication: in sporulation, each vegetative cell forms only one spore, and in subsequent germination each spore gives rise to a single vegetative cell. Certain specific antigens develop in the spore that are not found in the vegetative cells. Spores are involved in the transmission of certain diseases.

Sporulation
 

Sporulation occurs as a response to starvation or, at least, the exhaustion of a limiting substance. In certain species, sporulation may be induced by depletion of nutrients necessary for vegetative growth; at the same time, the process requires a continued supply of other minerals (potassium, magnesium, manganese and calcium salts), and favorable conditions of moisture, temperature, pH, oxygen tension, etc.

Sporulation
 

The spore is formed inside the parent vegetative cell (hence the name “endospore”). It develops from a portion of the protoplasm near one end of the cell (“the forespore”), incorporates part of the nuclear material (equivalent to one genome) of the cell and aquires a thick covering layer, the “cortex”, and a thin, but tough, outer “spore coat” consisting of several layers. Spores of some species have an additional, apparently rather loose covering known as the “exosporium”.

Spore’s structure

Sporulation

Sporulation

Shape, dispozition

The appearance of the mature spore varies according to the species, being spherical, ovoid or elongated, occupying a terminal, subterminal or central position, and being narrower than the cell, or broader and bulging it. Finally, the remainder of the parent cell disintegrates and the spore is freed.

Clostridium spp.

C. tetani (culture smear)

Bacillus spp. (culture smear)

Bacillus anthracis (the lack of spore in vivo – capsule)

Bacillus (culture smear- special staining for spore)

Spore’s dispozition

Application

Spores are much more resistant than the vegetative forms to injurious chemical and physical influences, including exposure to disinfectants, drying and heating. Thus, application of moist heat at 100-120°C for a period of 10-20 minutes may be needed to kill spores, whereas heating at 60°C suffices to kill vegetative cells; spores can be used in the control of sterilization.

Spore = resistance
 

-

Spores may remain viable for many years, either in the dry state or in moist conditions unfavorable to growth. The marked resistance of spores has been attributed to several factors in which they differ from vegetative forms: the impermeability of their cortex and outer coat, their high content of calcium and dipicolinic acid, their low content of water and their very low metabolic and enzymatic-activity.

Germination

Germination of the spore occurs when the external conditions become favorable to growth by access to moisture and nutrients. It is irreversible and involves rapid degradative changes.

Germination

The spore successively loses its heat resistance and its dipicolinic acid; it loses calcium, it becomes permeable to dyes and its refractility changes. In the process of germination, the spore swells, its cortex disintegrates, its coat is broken and a single vegetative cell emerges.

Germination

Germination

When mature, the spore resists coloration by simple stains, appearing as a clear space within the stained cell protoplasm. Spores are slightly acid-fast and may be stained differentially using special staining.

Spore

The presence, shape and position of the spores are primary taxonomic characters of bacteria.

Spore’s dispozition

Bacterial reproduction

Among bacteria, multiplication takes place by simple binary fission. The cell grows in size, usually elongating to twice its original length, and the protoplasm becomes divided into two approximately equal parts by the ingrowth of a transverse septum from the plasma membrane and cell wall. In some species, the cell wall septum, or cross-wall, splits in two and the daughter cells separate almost immediately.

Bacterial reproduction

In others, the cell wall of the daughter cells remain continuous for some time after cell division and the organisms grow adhering in pairs, clusters, chains or filaments. If cross-wall splitting is thus delayed in an organism in which the cross-walls of successive cell divisions are all formed in parallel planes, the cells will be grouped in chains. If it is delayed in an organism that forms successive cross-walls in different planes, e.g. ones at right angles to each other, the cells will be grouped in pairs, forming either cubic or irregular clusters.

Cells’ division

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