Bacterial Cell Wall Structure and Function

• Microbiology studies microscopic living organisms.
• It is an interdisciplinary scientific study of microorganisms.
• It includes their taxonomy, physiology, genetics, ecology, and biochemistry.
• It examines complex interactions between microbes and their environment.
• This includes their role in health, disease, and biotechnological applications.
• Medical microbiology is about the causative agents of infectious disease in humans.
• It focuses on the host's response to infection and methods of diagnosis, treatment, and prevention.
• The term "microbe" was first used by Sedillot in 1878, now replaced by "microorganism."

Infection and Contagion

• Ancient people believed epidemic and endemic diseases were supernatural.
• Diseases were thought to be punishments from gods for human sins.
• Sacrifices and lustrations were used to appease gods for treatment and prevention.
• The concept of contagion existed before microbes were seen.
• Communicable disease observations led to the idea of disease spread by contact.
• This idea was present in early biblical laws against leprosy spread.

Discovery of Microorganisms

• Varro recorded the principle of contagion by invisible creatures in the 2nd century BC.
• Roger Bacon suggested invisible living creatures caused disease in the 13th century.
• Fracastorius concluded communicable diseases were caused by living agents in 1546.
• Kircher reported finding minute worms in plague victims' blood in 1659.
• Von Plenciz suggested each disease was caused by a separate agent in 1762.
• Lucretius and Girolamo Fracastoro suspected disease was caused by invisible living creatures.
• Direct microorganism observation awaited microscope development.
• Antoni van Leeuwenhoek (1632-1723) is credited for first observing and reporting bacteria.
• The Dutchman was a draper and haberdasher in Delft, Holland.
• He had little education, but great patience and curiosity.
• Leeuwenhoek was the amateur microscopist and first person to observe microorganisms.
• He first observed microorganisms in 1673.
• In 1683, he made accurate bacteria shape descriptions.
• He communicated these descriptions to the Royal Society of London.
• Their medical and biological importance was recognized two centuries later.

Conflict Over Spontaneous Generation

• Spontaneous generation believed living organisms could develop from nonliving matter.
• Aristotle thought animals could originate from soil.
• Francesco Redi proved gauze over meat jars prevented maggots.
• Others discredited the theory for larger organisms.
• Some proposed microorganisms arose by spontaneous generation while larger organisms did not.
• John Needham published experiments in 1745 supporting microorganism spontaneous generation in putrescible fluids.
• Felix Pouchet claimed experiments conclusively proved microbial growth occurred without air contamination in 1859.
• Louis Pasteur disproved this claim.
• Lazzaro Spallanzani opposed spontaneous generation by boiling and sealing beef broth.
• Franz Schulze, Theodore Schwann, Georg Friedrich Schroder, and Theodor von Dutch tried to counter this argument.
• Louis Pasteur proved all life forms, even microbes, arose only from their like in classic experiments.
• Pasteur filtered microorganisms from the air as the source of contamination.
• In 1859, in public controversy, prepared boiled broth in long, narrow gooseneck flasks open to air.
• Air could pass, but microorganisms settled in the gooseneck, preventing growth.
• Growth commenced immediately if the necks were broken.
• Pasteur resolved controversy by 1861 and showed how to keep solutions sterile.

Contributions of Antoni Von Leeuwenhoek

• He constructed the first microscope consisting of a single biconvex lens that magnified about x200.
• He was the first person to observe microorganisms.
• Antoni van Leeuwenhoek found microorganisms in water, mud, saliva, and intestinal contents in 1673.
• He recognized them as living creatures called animalcules.
• To Leeuwenhoek, the world of "little animalcules" was a curiosity of nature.
• He accurately described bacteria shapes like cocci (spheres), bacilli (rods), and spirochetes (spiral filaments).
• This information was sent to the Royal Society of London in 1683.

Contributions of Louis Pasteur in Microbiology

• Coined the term microbiology
• Proposed germ theory of disease
• Disapproved theory of spontaneous generation
• Developed sterilization techniques
• Developed methods and techniques for microorganism cultivation
• Studied pebrine, anthrax, chicken cholera, and hydrophobia
• Pasteurization
• Coined the term vaccine
• Discovery of attenuation and chicken cholera vaccine
• Developed live attenuated anthrax vaccine.
• Developed rabies vaccine.

Robert Koch

• Robert Koch (1843-1910) was a German physician.
• He provided first direct demonstration of bacteria in carrying disease by studying anthrax.
• Winner of the Nobel Prize in 1905.
• He is the "Father of bacteriology"

Contributions of Robert Koch

• He described easy microscopic examination methods for bacteria in dried films stained with aniline dyes in 1877
• He used the hanging drop method to study bacterial motility
• Devised method to isolate pure bacteria cultures by plating mixed material on solid medium
• Discovered causal agents of anthrax, tuberculosis, and cholera
• Koch (1890) observed guinea pig infected with bacillus responded with exaggerated response when injected with tubercle bacillus or its protein.
• This hypersensitivity reaction is known as Koch's phenomenon.

Koch's Postulates

• It was necessary to introduce criteria for proving claims that a microorganism from a disease was a causative agent; Robert Koch proved that microorganisms cause disease.
• Koch used criteria proposed by Jacob Henle to establish Bacillus anthracis and anthrax relationship which he published in 1876.
• They are used to prove a particular microorganism causes a particular disease.
• These state:
  • Must be present in all cases of disease absent from healthy persons
  • Must be isolated from diseased patients;
  • Must cause disease when reintroduced to healthy susceptible animal model
  • Then be isolated again from the new host.

Limitations of Koch's Postulates

• Utilized in many new infectious disease cases.
• Criteria are invaluable in identifying pathogens.
• Some organisms cannot be grown on artificial media.
• Some are pathogenic only for men.
• Mycobacterium leprae, causative agent of leprosy, not cultured on artificial medium, so not fulfilling Koch's postulates.

Paul Ehrlich

• Paul Ehrlich was an outstanding German Scientist.
• He was known as "Father of chemotherapy"

Contributions of Paul Ehrlich

• Stains to cells and tissues for the purpose of revealing their function
• reported the acid-fastness of tubercle bacillus
• Introduced methods of standardizing toxin and antitoxin and coined the term minimum lethal dose.
• Proposed side chain theory of antibody production, including antibody reaction leading to better understanding of the immune system.
• Introduced salvarsan, an arsenical compound, sometimes called the 'magic bullet'.
• Capable of destroying the spirochete of syphilis with moderate toxic effects
• Continued experimentation until 1912 when he announced the discovery of neosalvarsan.

Role of Microorganisms in Disease

• A firm basis for the casual nature of infectious disease was established only in the latter half of the nineteenth century.
• Fungi, being larger than bacteria, were the first agents to be recognized
• Agostino Bassi (1773-1856) demonstrated in 1835 that silkworm disease was due to a fungal infection.
• MJ Berkeley (1845) proved that the great potato blight of Ireland was caused by a fungus.
• Pasteur showed pebrine disease of silkworm was a protozoan parasite.
• Protozoan parasite was disrupting silk industry so was asked by French government to inverstigate
• Empirical Observations and the etiologic role of bacteria was first established with anthrax.
• Pollender (1849) and Davaine (1850) observed anthrax bacilli in blood of animals dying of the disease.

Type of Microorganisms

• Classification groups:
  • Bacteria
  • Archaea
  • Fungi
  • Protozoa
  • Algae
  • Viruses
• Carl Woese introduced the system with three domains (bacteria, archaea, and eukarya).
• Robert Whittaker created a five-kingdom classification system (Animalia, Plantae, Fungi, Protista, Monera).

Microscope

• Microscope is an optical instrument used to magnify enlarge small objects or microorganisms.
• Microscopy is for two basic purposes:
  • Initial detection of microbes
  • Definitive identification of them

Light Microscopy

• Uses visible light to observe specimens
• Types of light microscopy:
  • Bright-field microscope/Compound light microscope
  • Dark-field microscopy
  • Phase-contrast microscopy
  • Fluorescent microscopy
  • Confocal Microscopy
    • To obtain three-dimensional images of entire cells and cellular components
    • To evaluate cellular physiology by distributions and concentrations monitoring of substances such as ATP and calcium ions

Bright-field Microscope/Compound Light Microscope

• Uses light to illuminate a sample and create an image
• The sample is placed on a glass slide and illuminated by a light source, typically a halogen lamp or a light-emitting diode (LED) light.
• The sample appears dark against a bright background, hence the name “bright field.”

Dark-field Microscopy

• Frequently performed on same microscope on which bright-field microscopy is performed
• Uses a dark-field condenser with an opaque disk
• The disk blocks light that would enter the objective lens directly.
• Specimen appears as an area of light contrast against a dark background.
• Particularly important for observing organisms such as Treponema pallidum
• Treponema pallidum is a spirochete which is less than 0.2 mm in diameter and therefore cannot be observed with direct light.
• The disadvantage is that internal structure of organisms cannot be studied because light passes around rather than through organisms.

Phase-contrast microscopy

• Based on the wave nature of light rays, and the fact that light rays can be in phase or out of phase.
• In a phase-contrast microscope, one set of light rays comes directly from the light source, and the other comes from light that is reflected or diffracted from a particular structure in the specimen.
• Phase-contrast microscopy improves the contrast , makes internal cell structures defined, showing thickness differences.

Uses of Phase Contrast Microscope

• To study unstained living cell
• Detailed examination of internal structures in living microorganisms.
• To study flagellar movements and motility of bacteria and protozoans
• To study intestinal and other live protozoa such as amoebae and Trichomonas
• To examine fungi grown in culture.

Electron Microscopy

• It uses electromagnetic lenses, electrons, and a fluorescent screen.
• Electromagnetic lenses focus beam which produce magnified image (figure).
• The superior resolution due to electrons much shorter wavelength than white light photons.
• Image captured on photographic film that creates electron photomicrograph.
• Two types:
  • Transmission Electron Microscope or TEM
    • Electrons pass through specimen and focused on fluorescent viewing screen
  • Scanning Electron Microscope or SEM
    • Electrons sweep across specimen and knock other electrons from its surface

Magnification

• In microscopy magnification is the process of enlarging the appearance of the object with objective lens magnification power multiplied by ocular lens magnification (power).
• Three common objective lenses:
  • Low (10x) yielding 100x total magnification
  • High dry (40x)yielding 400x total magnification
  • Oil immersion (100x)yielding 1000x total magnification
• Most oculars magnify by 10x.

Resolution

• The limitation of bright-field microscopy is the resolution
• Resolution is also called resolving power: the ability to distinguish that two objects are separate and not one). The resolving power of a microscope is determined by the wavelength of light used to illuminate the subject and the angle of light entering the objective lens
• A general principle of microscopy is that better the resolution requires shorter the wavelength of light used in the instrument.

Immersion Oil

• White light in a compound light microscope has long wavelength resolving structures smaller than 0.2 µm.
• Objective lens must be short to achieve high magnification with high resolution and must be used with immersion oil and has same refractive index as glass so it becomes part of scope's glass.
• The oil enhances resolution by preventing light rays dispersing/changing wavelength so are designed for use with oil giving 100 x mag.

Comparison Between Prokaryotic and Eukaryotic Cells

• Two cell types are: prokaryotic and eukaryotic cells.
• This is based on cellular organization and biochemistry.
• Prokaryotes include Bacteria and Archaea.
• Eukaryotes include Eucarya.
• Prokaryotic (primitive nucleus) cells lack a membrane-bound nucleus.
• All bacteria and blue-green algae are prokaryotes.
• Bacteria lack chlorophyll, while blue-green algae possess it.
• Eukaryotic (true nucleus) cells have a membrane-bound nucleus.
• Other algae, fungi, slime molds, protozoa, higher plants, and animals are eukaryotic.
• Three features distinguish prokaryotes from eukaryotes:
  • Small size
  • Absence of a complex, organelle-containing cytoplasm
  • Absence of a nuclear membrane.

Differences Between Prokaryotic and Eukaryotic Cells:

• Prokaryotic and eukaryotic cells: Both are covered with the plasma membrane and located on top of cell membrane/mucous capsule.
• Table presents majors difference between the two cell types and despite their relative simplicity, prokaryotes are independent.

Bacteria Morphology

• Microorganisms are microscopic and simple, usually unicellular.
• Bacteria are single-celled and reproduce by binary fission.
• They contain genetic, energy-producing, and biosynthetic systems for growth.
• Microorganisms are a heterogeneous group and distinct classes.
• Whittaker recognizes five kingdoms of living things:
  • Monera (bacteria)
  • Protista
  • Fungi
  • Plantae
  • Animalia
• The Five kingdoms have been modified further by the development of three domains, or Super kingdoems system- the Bacteria, the Archaea, the Eucarya.

Size of Bacteria

• Unit of measurement in bacteriology micron/micrometer (µm).
• Medically important bacteria generally are 0.2-1.5µm in diameter and about 3-5 µm in length.
• Requires light microscope
• The electron microscope provides superior resolving power compared to the best light microscope (1000 o 2000 times magnification).

Shapes of Bacteria

• Classified into several categories:
  • Cocci
    • Spherical/nearly spherical.
    • Example: Streptococcus pyogenes, Staphylococcus aurus.
  • Bacilli
    • Relatively straight, rod-shaped.
    • Some exhibit length equals width: coccobacilli.
    • Example: Bacillus anthracis, Escherichia coli, Clostridium botulinum.
  • Vibrio
    • Curved/comma-shaped with vibratory motility.
    • Example: Vibrio cholera, Vibrio vulnificus.
  • Spirilla
    • Rigid spiral/helical.
    • Example: Helicobacter Pylori, Campylobacter jejuni.
  • Spirochetes
    • Flexuous spiral forms moving in twisted manner
    • Example: Treponema pallidium.
  • Mycoplasma
    • Cell wall-deficient bacteria with unstable morphology.
    • Occur as round/oval bodies and interlacing filaments.
    • Example: Mycoplasma hominis, Mycoplasma genitalium.

Anatomy of Bacterial Cell

• Divided into:
  • The outer layer or cell envelope consists of:
    • Cell wall
    • Cytoplasmic membrane
    • Plasma membrane beneath cell wall.
  • Cellular appendages
    • Capsule
      • Some bacteria make a protective gelatinous covering layer called a capsule Microcapsule
        • Thin capsule only seen with light microscope.
    • Loose slime:
      • Viscid dispersion of colloidal material in the environment.
    • Flagella
      • Appendages on cell wall and organs of locomotion.
    • Fimbriae
      • Organs of adhesion;
    • Pili
      • Involved in transfer of genetic material.

Bacterial Cell Interior

• Includes:
  • Cytoplasm: Bounded by cytoplasmic membrane
  • Cytoplasmic inclusions: Mesosomes, ribosomes, inclusion granules, vacuoles
  • Single circular chromosome of deoxyribonucleic acid (DNA).

Cell Envelope (Outer Layer or Cell)

• Cell Wall
  • Lies outside the plasma membrane, 10-25 nm thick, strong, rigid, elastic, and porous
  • Cell wall functions:
    • Imparts shape and rigidity
    • Supports weak cytoplasmic membrane against osmotic pressure in protoplasm
    • Maintains characteristics shape of Bacterium
    • Involved in cell division
    • Functions in interactions (e.g. adhesion) w/other bacteria and with mammalian cells
    • Provides specific carb/protein receptors for bacterial viruses.
  • Chemically
    • Composed of mucopeptide (peptidoglycan or murein) scaffolding with chains of alternating N -acetyl glucosamine and N-acetyl muramic acid with peptide bonds in crosslink
    • Has parts
      • composed of alternating N-acetyl glucosamine and N-acetylmuramic
      • side chains attached to N acetylmuramic acid
      • identical pentapeptide cross-bridges
    • Antibiotics with construction interference
    • Gram (+) are peptidoglycan and teichoic acids while gram (-) is more complex with thinner peptidoglycan and an outer membrane.

Staining

• Staining is to color a microorganism with a dye, to allow morphologic examination of organisms under a microscope. Types of stains:
• Simple Staining
• Differential Staining
• Types of Stains:
  • Gram's Stain
• Gram's Stain is used to differentiate similar organisms by categorizing them gram (+) or gram (-). In gram staining, positive organisms take up crystal violet dye while gram negative organisms take up the counter stain safarani red.
• Red/Pink specimen indicates gram (-), purple/blue indicates gram (+).
• Gram Positive Bacteria
  • Stains purple
• Gram Negative Bacteria
  • Stains red/pink

Difference between Cell Wall of Gram-positive and Gram-negative Bacteria

• Reside in the cell wall.
• In general, the walls of gram-positive bacteria simpler chemical nature than those of gram-negative bacteria as integrity is essential to viability of bacterium.
• Protoplasm may swell from osmotic inflow of water, rupturing resulting in lysis.
• Gram-positive cell wall about 80 nm thick w/layers of peptidoglycan in addition to Teichoic acid.
• Peptideglycan thicker and make up 50-90 dry weught of the wall
• Imbedded protein
• Gram-Negative Cell Wall.
• Gram-negative structurally different but composed of peptidoglycan + lipoprotein outer membrane, and lipopolysaccharide.
• Peptidoglycan layer:
  • Single unit constituting 5-10% of the weight of the negative bacteria & is covalently bonded in the membrane.
• Lipoprotein
  • Attaches peptidoglycan by lipid and or protein for function on membrane stabilization
• Outer membrane
• Attached by lipoprotein and phospholipid (inner) and lipopolysaccharide LPS is (outer)- forms barrier for salts and antibiotics.

Cytoplasmic (Plasma) Membrane Structure

• Cytoplasmic (plasma) membrane limits bacterial protoplast that is thin so observed w/ electron microscope composed of double layer with hydrophilic regions and lipoprotein (little carbohydrate) lacking sterols except Mycoplasma.
• Functions of Cytoplasmic Membrane in bacteria
  • Semipermeable for inflow and metabolite outflow.
  • Housing enzymes: Outer/cell wall synthesis for extra assemblies
  • Sensory/chemo taxis proteins monitoring chemical and environmental changes
  • Chemical ebergy generation
  • Mediation of Chromosomal segregation during replication

Functions of Capsule

• Virulence factor, protective barrier
• Protects from Cell all attacks
• For Identification and typing
  • It is specific for bacteria and to ID bacteria by cell wall type
• Flagella is what mobilizes spirochetes with 1+ unbranched filaments (organs of mobility)
• Formed with long helical 3 -20 um tip that originates through cell's w/ protein (flagellin) that are different but similar in composition to flagella antigens
• They are serodiagnostic on gram -/+ but cocci are immotile
• Basals consist of filament: Lengthy portion that extends from the cell surface, Hook
• They are the ones found on hooks in CM
• Some species consist of cell interiors such as cytoplasm with viscosity including organics as to solubles/ribosomers/polysomes
• Cytoplasm varies due to higher eukaryotic species in ER, micromomes, mitochondria, lysosomes, signs of motility- with granules or vacuoles that stains with dyes.

Intracytoplasmic Inclusions

• Synthesis+ accumulation in different conditions that reduces pressure by storage of volumin, glycogen with functions on DNA replication.
• Bact Nucleus structure has genetic material of bacterial cell with double helix for transcription
• Plasmids has not essential functions with antibiotics to create high tolerance in species
• Bacterial Spores generate high resistance from genes in Bacillus that when depleted form endospores (simple spore) with one cell that germinate in preservation
• Spores formations = sporogenesis when growth slows due to nutrient deficiency with new antigens.
• Germination in the transition of spores to vegetative cells
  • Activation (initiation and outgrowth) _Fully developed with structure but inactive _ wall and peptidoglycan germ cell _outer coat enclosed and lysosomes assist in germination

Bacteria Growth/Nutrition

• Described as an increase in size and the number of cells that will result in a biomass.
• Bacterial division described when cells enlarge and divide (binary fission) when the cell stretches.
• Generation/Doubling time - Size and time of bacteria increase exponentially with time where is necessary
• Growth by: -Lag (no increase)
  • Log (increase)
  • Stationary (growth and decay are equal)
  • Decline (death of number of cells)
• Colonies are clones with all phases of growth within growth mediums

Detection and Measurement of Bacterial Growth

• Two methods:
  • Direct Cell Counts
    • Measured on a slide of the bacterial suspension (Breed count on milk) or Cell Counter for fluids.
  • Viable Counts
    • Number of cells, that is in, cells able to replicate and multiply in a growth medium. Measured by:
    • Turbidity
    • Dry Weight
    • Chemical Constituents
  • Cell Products
    • Acid, gases, ATP

Bacteria Culture

• Requires a medium, which can be solid or liquid
• Components include water, agar and peptone plus growth factors
• 2 Phases of growth are liquids and solids -Liquid (medium) growth indicated by turbidity. -Solid (agar) - medium needs that solidifying agent nutrients and water.

Types of Media based on Nutrition

• Simple- Basal containing nutrients growing (water broth for other medium prep)
• Complex- unknown nutrients containing ingredients with distinct colony morphology with dyes.

Types of Media based on Classification

• Classified as having specific functions (Special),
• Liquid with a growing bacteria (broth)
• Solid state (agar cell culture)
• Semi Solid State
• Incolations occur with colonies that separate out for tests against sterilizations
  • Disinfectants

Culture

• Cultures are observed at 1st without microscopy and sufficient quantities.
• Types of bacteria Culture
• Batch Cultures - Systems in broth to which transferred continuous culture.
• Open Vs the turbid stat in Chemo (replinsh with water) and regulates density

Main Poses and Bacteria Cultivation

-Grow and isolate bacteria

_Differentiate infections contaminants _ID characteristics

• Factors with Carbon (sugars), chemical/organic composition
• Microbes often require additional compounds that must be obtained from the external environment They include:
  • Vitamins & amino acids
  • Purines and pyrimidines
  • Fatty acids and cholesterol

Physical Conditions

• Temperature - Each bacterial has best range

1. psychrophiles (cold 15 or less)
2. Mesophiles ( Moderate (20-45c [human])
3. Thermophiles (higher to 80)

• pH.Bacteria have specific pH ranges they can tolerate:
  • Neutrophiles(neutral pH) Acidophiles(Thrive acidic environments) .
  • Alkalophiles(alkaline conditions) _Oxygen or non toxic if able to grow, _ Aerobes with 02 like a virus. Moisture needs water as the vector, pressure like deep sea species will grow,

Osmotic Efcct

Light - Darkness needed with sensitivity by a variation due to cell resistance Mechanical Strength of cell wall and tonicity resistance Chemical- Carbon Dioxide all bacteria in trace elements. Energy- Obtain what we metabolize in our substances that have been described within bacterial cell-

Bacteria Metabolism

• Metabolic substance defined as the series/changes in substances (carbohydrates, proteins, or fat), that is within the cell from absorption to elimination
• Has Aerobic/anaerobic reactions where 3 critical processes can be seen -Aerobic/anaerobic reproduction/Fermentation
• Aspects of Energy Productions: Oxygen transfers during oxidations.

Explore the structure and function of bacterial cell walls, focusing on components like peptidoglycan and fimbriae. Differentiate between Gram-positive and Gram-negative bacteria based on cell wall composition. Understand how cell wall integrity relates to bacterial survival and Gram staining results.