Microbiology: History, Morphology, and Structures

History of Microbiology

• Micrographia

  • Robert Hooke

    • First report of cell structure in 1665.

    • Observed 'little boxes' in cork, which he termed "cells".

  • First illustrated book on microscopy.

Anton Van Leeuwenhoek (1678)

• First person to observe bacteria.

• Used a single-lens microscope.

• Microscope components:

  • Focus knob

  • Lens

  • Sample holder

  • Sample translator

Spontaneous Generation

• Aristotle (384 BC):

  • Proposed that living things arise from damp bodies.

  • Associated with "miasma" (bad air).

  • Referenced phoenix myths (rebirth).

  • Mentioned "Golam" (clay figure brought to life).

• Herodotus (c. 484 – c. 425 BC):

  • Suggested living organisms could arise from non-living matter.

  • Example: crocodiles from mud.

• Van Helmont (17th century):

  • Proposed small animals could arise spontaneously.

  • Examples: maggots from meat, mice from feed.

Challenging Spontaneous Generation

• The spontaneous generation claimed that living organisms could develop from nonliving or decomposing matter

• Francesco Redi (1626-1697):

  • Challenged spontaneous generation.

  • Demonstrated that maggots on decaying meat originated from fly eggs, not the meat itself.

• John Needham (1713-1781):

  • Experiment: Boiled mutton broth in flasks, sealed them, and observed microorganism growth.

  • Supported the theory of spontaneous generation.

• Lazzaro Spallanzani (1729-1799):

  • Experiment: Sealed flasks, then boiled them, resulting in no microorganism growth.

  • Proposed that air carries germs to the culture medium.

  • Suggested external air might be needed for the growth of organisms already in the medium, appealing to spontaneous generation supporters.

Louis Pasteur (1822-1895)

• Germ Theory:

  • States that microorganisms (pathogens or "germs") can cause disease.

  • These organisms invade humans, animals, and other living hosts.

• Fermentation:

  • Demonstrated that alcoholic fermentation results from microbial activity.

  • Identified both aerobic and anaerobic fermentations.

• Pasteurization:

  • Developed pasteurization to preserve wine during storage.

• Rabies Vaccine:

• Contributions: Streptococcus pneumoniae causes lobar pneumonia

Pasteur's Experiment

• Trapped airborne organisms in cotton.

• Used swan-necked flasks: heated necks into long curves, sterilized media, and left flasks open to air.

• No growth occurred as dust particles (carrying organisms) were trapped in the flask neck.

• Breaking the necks allowed dust to settle, and organisms grew.

• Disproved the theory of spontaneous generation.

Significance of Pasteur's Work

• Added 20 years to the average human lifespan.

• Improved the quality of life.

• Experiment embodies modern scientific inquiry:

  • Begins with a hypothesis.

  • Tests hypothesis using a controlled experiment.

  • This process has evolved into the scientific method.

Recognition of Microbial Role in Disease

• Robert Koch (1843-1910)

  • Confirmed germ theory

  • Discovered causes of:

    • Anthrax

    • Cholera

    • Tuberculosis

  • Developed:

    • Pure culture techniques

    • Staining techniques

    • Solid media

Koch's Postulates

• Rules to prove an organism causes a disease

  • Organism consistently isolated from diseased individuals

  • Organism cultivated in pure form

  • Signs and symptoms induced after inoculation

  • Same organism isolated from experimentally infected individual

Detailed Explanation of Koch's Postulates

1. The microorganism must be found in abundance in all organisms suffering from the disease, but should not be found in healthy organisms.

2. The microorganism must be isolated from a diseased organism and grown in pure culture.

3. The cultured microorganism should cause disease when introduced into a healthy organism.

4. The microorganism must be re-isolated from the inoculated, diseased experimental host and identified as being identical to the original specific causative agent.

Microbiology Definition

• Study of microorganisms (microscopic organisms).

• Too small to be clearly seen by the unaided eye (micrometer).

• Microbiology Types of microorganisms: Viruses, bacteria, protozoa, algae, and fungi.

• Some algae and fungi are large enough to be visible, but are included in the field of microbiology because they have similar properties and because similar techniques are employed to study them (isolation, sterilization, culture in artificial media).

Universal Tree of Life

• Three domains: Bacteria, Archaea, and Eukarya.

• Illustrates evolutionary relationships between different organisms.

Prokaryotic and Eukaryotic Cells

• Cells are divided into two types by how their DNA is stored.

  1. Prokaryotic cells lack a membrane covered nucleus; DNA located in the nucleoid.

  2. Eukaryotic cells have a membrane-covered nucleus storing the cell's DNA.

• Bacteria and archaea were once lumped together as prokaryotes.

• Since 1960s, biochemical, genetic and genomic analysis have shown that bacteria and archaea are distinct taxa.
  Norman Pace proposed the term "prokaryotes" should be deserted in 2006.

• Bacteria are a type of prokaryotic cell.

Quick Revision: Prokaryotic vs Eukaryotic Cells

• Comparison table highlighting differences:
  *Pilli/Flagella, Nucleoid/Nucleus, Cytoplasm, Cell Wall, Plasma membrane, Cytoskeleton and other organelles.

Principal Differences between Prokaryotic and Eukaryotic Cells

Bacteria Morphology and Structure

• Bacteria diverse but share some common features such as morphology and structure.

Bacteria Morphology (Shape, Size, Arrangement)

• Bacterial cells are small and relatively simple but they have considerable morphological variety.

• Two most common shapes: Cocci (spherical) and Rods (bacilli).

• Cocci can exist singly or in characteristic arrangements.

• Rods are sometimes called bacilli.

Arrangement of Cocci

• Cocci may be oval, elongated, or flattened on one side.

• Cell division patterns lead to characteristic groupings:

  • Streptococci: chains (cells adhere after repeated divisions in one plane).

  • Diplococci: pairs after dividing.

  • Tetrads: groups of four (divide in two planes).

  • Sarcinae: cube-like groups of eight (divide in three planes).

  • Staphylococci: grape-like clusters or sheets (divide in multiple planes).

Arrangement of Bacilli

• Bacilli divide across their short axis, resulting in fewer groupings.

• Bacillus refers to a shape (rod) and a genus name.

• Most bacilli appear as single rods.

  • Diplobacilli: pairs after division.

  • Streptobacilli: chains after division.

  • Coccobacilli: short and fat, resembling cocci.

Less Common Bacteria Shapes and Arrangements (Spiral Bacteria)

• One or more twists.

  • Vibrios: curved rods (comma-shaped).

  • Spirilla: helical shape (spiral-shaped) and rigid bodies.

  • Spirochetes: helical shape and flexible bodies (move by axial filaments).

  • Pleomorphic: Variable in shape, lacking a single characteristic form.

Multicellular Bacteria

• Some bacteria can be considered multicellular.

• Actinobacteria form long filaments called hyphae, which form a network called a mycelium.

• Many cyanobacteria (photosynthetic bacteria) are also filamentous.

Examples Shown in Images

• S. agalactide - cocci in chains.

• S. aureus - cocci in clusters.

• L. pneumophila - rods in chains.

• V. vulnificus - comma-shaped vibrios.

• C. jejuni - spiral-shaped.

• L. interrogans - a spirochete.

Size of Bacteria

• Escherichia coli: representative average-sized bacterium (1.1 to 1.5 $⅄m$ wide by 2.0 to 6.0 $⅄m$ long).

• Size range varies widely:

  • Mycoplasma: small (0.3 $⅄m$ in diameter).

  • Spirochetes: can reach 500 $⅄m$ in length.

  • Cyanobacterium Oscillatoria: about 7 $⅄m$ in diameter (same as a red blood cell).

• Size varies with bacteria type, age, and external environment.

Factors Influencing Bacterial Size and Shape

• Size and shape are related and selected for during evolution.

• Small size increases surface area-to-volume ratio (S/V ratio).

• Higher S/V ratio facilitates nutrient uptake and diffusion, leading to rapid growth rate.

• Shape affects S/V ratio.

  • Rod has higher S/V ratio than a coccus with the same volume.

Bacteria Structure (Cell Organization)

• Components: capsule, cell wall, plasma membrane, cytoplasm, ribosomes, nucleoid, chromosome (DNA), inclusion, flagellum, fimbriae.

• Note that no single bacterium has all of these structures at all times.

• Some are found only in certain cells, conditions, or life cycle phases.

Common Bacterial Structures and Their Functions

Plasma Membranes

• Bacterial Plasma Membranes Control What Enters and Leaves the Cell

• Cell envelope: plasma membrane and all surrounding layers external to it.

  • Many bacteria have a plasma membrane, cell wall, and additional layers (capsule or slime layer).

• Plasma membrane encompasses the cytoplasm and defines the cell.

• Selectively permeable barrier: allows passage of particular ions/molecules in or out of the cell.

• Prevents loss of essential components through leakage.

Structure of a Phospholipid

• Components:

  • Glycerol backbone

  • Two fatty acid chains (hydrophobic tails)

  • Phosphate group attached to a head group (polar, hydrophilic head)

Bacterial Plasma Membrane Functions

• Location of crucial metabolic processes: respiration, photosynthesis, synthesis of lipids and cell wall constituents.

• Fluid Mosaic Model of Membrane Structure: lipid bilayers within which proteins float.

• Bacterial membranes have roughly equal amounts of lipids and proteins.

• Cell membranes are very thin: about 2 to 3 nm thick.

• Composed of phospholipids (amphipathic).

  • Polar ends interact with water (hydrophilic).

  • Nonpolar hydrophobic ends associate with each other.

Membrane Proteins

• Peripheral membrane proteins:

  • Loosely connected; easily removed.

  • Soluble in aqueous solutions.

  • Make up 20-30% of total membrane protein.

• Integral membrane proteins:

  • Not easily extracted; insoluble in aqueous solutions when freed of lipids.

  • Amphipathic: hydrophobic regions buried, hydrophilic portions project from the membrane surface.

  • Carry out important functions:

    • Transport proteins.

    • Energy-conserving processes (e.g., electron transport chains).

    • Regions exposed to the outside enable cell interaction with the environment.

Fluid Mosaic Model of Bacterial Membrane Structure

• Lipids and proteins are dynamic and can move laterally within the membrane.

• Includes phospholipids, integral membrane proteins, peripheral membrane proteins, glycolipids, and oligosaccharides.

Bacterial Plasma Membranes Are Dynamic

• Lipid composition varies with environmental temperature, maintaining membrane fluidity during growth.

  • Bacteria at lower temperatures: more unsaturated fatty acids.

  • Bacteria at higher temperatures: more saturated fatty acids.

Nutrient Uptake

• Bacteria Use Many Mechanisms to Bring Nutrients into the Cell

• Obtaining nutrients is a key function of the bacterial plasma membrane.

• Macroelements (macronutrients): carbon, oxygen, hydrogen, nitrogen, sulfur, phosphorus (required in large amounts).

• Micronutrients (trace elements): manganese, zinc, nickel, copper (required in small amounts).

• Nutrient uptake mechanisms:

  1. Passive Diffusion

  2. Facilitated Diffusion

  3. Primary and Secondary Active Transport

  4. Group Translocation

Passive Diffusion

• Molecules move from high to low concentration (down the concentration gradient).

• Rate depends on concentration, temperature, and molecule size.

• Most substances cannot freely diffuse into a cell.

  • Water and some gases ($O<em>2$ and $CO</em>2$) easily cross the membrane.

  • Larger molecules, ions, and polar substances need transport proteins.

Facilitated Diffusion

• Substances move across the plasma membrane with assistance from transport proteins (channels or carriers).

• Aquaporins facilitate the diffusion of small polar molecules.

• Observed in all organisms.

Active Transport

• Solute molecules transported against a concentration gradient with the input of metabolic energy.

• Primary active transport:

  • Energy used by the breakdown of ATP (Adenosine triphosphate) to transfer molecules throughout the membrane against a concentration gradient.

• Secondary active transport:

  • Uses electrochemical energy.

  • A transporter protein combines the motion of an electrochemical ion (generally $Na^+$ or H^+}) down its electrochemical gradient to the upward movement of another molecule or an ion against a concentration or electrochemical gradient.

Example of Primary and Secondary Active Transport

Primary active transport:

• The ATP-driven $Na^+-K^+$ pump stores energy by creating a steep concentration gradient for $Na^+$ entry into the cell.

Secondary active transport

• As $Na^+$ diffuses back across the membrane through a membrane cotransporter protein, it drives glucose against its concentration gradient into the cell.

Group Translocation

• Molecule is chemically modified as it's brought into the cell.

• Phosphoenolpyruvate: sugar phosphotransferase system (PTS).

• PTS transports and phosphorylates various sugars, using phosphoenolpyruvate (PEP) as the phosphate donor.

Bacterial Cell Wall

• Cell wall surrounds the plasma membrane and protects from pressure changes.

• Consists of peptidoglycan (or murein):

  • Polymer of NAG (N-acetylglucosamine) and NAM (N-acetylmuramic acid).

  • Short chains of amino acids.

• Penicillin interferes with peptidoglycan synthesis.

Peptidoglycan Structure

• Repeating NAG-NAM disaccharides.

• Amino acid side chains cross-linked.

• Lysozyme cleaves the (ẞ1-4) glycosidic bond between NAG and NAM.

Gram-Positive Cell Wall

• Consists of many layers of peptidoglycan.

• Teichoic acids:

  • Bind and regulate cation movement.

  • Prevent cell lysis during growth.

  • Contribute to the cell wall's antigenicity.

Gram-Negative Cell Wall

• Lipopolysaccharide-lipoprotein-phospholipid outer membrane surrounding a thin peptidoglycan layer.

• No teichoic acids.

Gram Positive vs Gram Negative cell walls

Gram Positive

• thick peptidoglycan layer.

• Teichoic acid

• Lipoteichoic acid

Gram Negative

• Outer membrane layer

• Lipopolysaccharides

• Porins

• thin Peptidoglycan layer

• Lipoproteins

• Periplasmic space

Gram Staining

• Developed by Christian Gram in 1884.

• Differentiates between Gram-positive and Gram-negative cell walls.

• Remains important technique to this day.

Gram Stain Mechanism

1. Cells are stained with crystal violet for 1 minute.

2. Wash with water.

3. Mordant: Gram's iodine for 1 minute.

4. Wash with water

5. Decolorize with acetone-alcohol for 5-10 seconds.

6. Counterstain with safranin

Results

Gram-positive cells stain purple; Gram-negative cells stain pink.

Comparative Characters of Gram-positive and Gram-negative bacteria

• Gram Positive Cocci

• Gram Negative Bacilli

Atypical Cell Walls

• Bacteria that lack cell wall (Mycoplasma).

• Mycoplasma:

  • Genus that lacks cell walls.

  • Sterols in the plasma membrane protect from osmotic lysis.

• Mycobacterium:

  • Genus that has mycolic acids in its cell walls.

  • Waxy cell wall resistant to decolorization.

• Archaea:

  • Have pseudomurein.

  • Lack peptidoglycan.

Cell Wall Functions

• Protects from phagocytosis and chemicals.

• Porins permit small molecules to pass through the outer membrane.

• Channel proteins allow other molecules to move through the outer membrane.

• Lipopolysaccharide component:

  • Sugars (O polysaccharides) act as antigens.

  • Lipid A is an endotoxin, causing fever and shock.

Structures External to the Cell Wall

• Cell envelope include layers outside the cell wall.

• Layers outside the cell wall:

  1. Capsules and Slime Layers.

  2. S- Layers.

Capsules

• Well-organized; not easily washed off.

• Composed of polysaccharides.

• Visible with negative or specific capsule stains.

• Help pathogenic bacteria resist phagocytosis.

• Protect against dehydration.

• Exclude viruses and hydrophobic toxic materials.

Slime Layers

• Diffuse, unorganized material easily removed.

• Usually composed of polysaccharides.

• Not as easily observed by light microscopy.

• Facilitate motility.

Capsule Staining

• Example image: Klebsiella pneumoniae on MacConkey agar.

• Capsules appear as clear zones around the rods.

Bacterial Cytoplasm

• More Complex than Once Thought

• Cytoskeletal proteins in bacterial cells help organize the cytoplasm.

• Less complex than eukaryotes; still participates in cell division, protein localization, and cell shape determination.

Intra-cytoplasmic Membranes & Inclusions

• No complex organelles like mitochondria or chloroplasts are found in Bacteria members,

• Internal membranous structures (aggregates of spherical vesicles, flattened vesicles, or tubular membranes) are observed in some bacteria and are often connected to the plasma membrane
  *Inclusions: Storage Inclusions, Carboxysomes, The gas vacuole, Magnetosomes

Bacterial Inclusions with Figures Shown

• Sulfur globules

• Carboxysomes

• Gas vacuoles

• Magnetosomes

Bacterial Ribosomes

• Site of protein synthesis.

• Large numbers (10,000 to 20,000) in nearly all cells.

• Made of proteins and RNA molecules.

• Bacterial ribosomes are 70S ribosomes.

  • Composed of a 50S and a 30S subunit.

Antibiotic Targeting of Ribosomes

• Protein synthesis occurs at ribosomes; it can be inhibited by certain antibiotics.

• Prokaryotic (70S= 30 S+ 50S) vs eukaryotic (80S = 60 S + 40 S) ribosomes.

• Antibiotics selectively target prokaryotic ribosomes.

Nucleoid

• Ellipsoidal region containing the cell’s chromosome and numerous proteins.

• Not separated from the cytoplasm by a membrane.

• Chromosomes of most bacteria are circular double-stranded deoxyribonucleic acid (DNA).

Plasmids

• Small, circular, double-stranded DNA molecule.

• Distinct from chromosomal DNA.

• Naturally exist in bacterial cells and some eukaryotes.

• Have relatively few genes (less than 30).

• Not essential to the bacterium, but provide selective advantages:

  • Antibiotic resistance.

  • Inherited stably during cell division.

Structures External to the Cell Wall (Attachment and Motility)

• Many Bacteria Have External Structures Used for Attachment and Motility

• Many bacteria have structures extending beyond the cell envelope involved in attachment to surfaces or motility.

  • Bacterial Pili and Fimbriae

  • Bacterial flagella

Bacterial Pili and Fimbriae

• Short, thin appendages.

• Fimbriae: many per cell; help adhere to surfaces.

• Pili: only one or two per cell.

  • Join cells for DNA transfer (sex pili).

Flagella

• Relatively long filamentous appendages consisting of a filament, hook, and basal body.

• Prokaryotic flagella rotate to push the cell.

• Motile bacteria can exhibit taxis:

  • Positive taxis: movement toward an attractant.

  • Negative taxis: movement away from a repellent.

• Can respond to environmental signals (thermotaxis, phototaxis, aerotaxis, osmotaxis, and gravity).

Arrangement of Flagella

• Monotrichous: single flagellum.

• Lophotrichous: tuft of flagella at one end.

• Amphitrichous: flagella at both ends.

• Peritrichous: flagella distributed over the entire cell.

Flagellar Movement

*Bacteria move by Swimming or Swarming

• Bacterial flagellum is a rigid helix that rotates like a propeller.

• Flagellar rotation results in two types of movement:

  • Run: smooth swimming movement that moves the cell.

  • Tumble: reorients the cell.
    *Swarming : group behavior in which cells move in unison across the surface

Swarming growth

• Swarming Bacteria Often Produce Distinctive Patterns on a Solid Growth Medium.

• Proteus on blood agar.

Structure of Flagella

• Filament: hollow, rigid cylinder made of flagellin subunits.

• Flagellin can vary in structure and is used to identify some pathogenic bacteria serologically.

• Flagellar antigens are referred to as H antigens.

Bacterial Endospores

• Survival Strategy

• Dormant cells formed within a mother cell.

• Produced by certain members of Bacillus, Clostridium (rods), and Sporosarcina (cocci).

• Not a reproductive strategy.

• Extraordinarily resistant to environmental stresses:

  • Heat, ultraviolet radiation, gamma radiation, chemical disinfectants, and desiccation.

• Some endospores have remained viable for around 100,000 years.

• Species that form endospores are dangerous pathogens.

Significance of Endospores

• Several species of endospore-forming bacteria are dangerous pathogens (e.g., Clostridium botulinum, which causes botulism).

Endospore Structure and Resistance

• Core, inner membrane, germ cell wall, cortex, outer membrane, coat, exosporium.

Endospore Formation and Germination

• Process of endospore formation is called sporulation.

• Return of an endospore to its vegetative state is called germination.

Endospore Location and Size

• Endospore location (central, terminal, subterminal) and shape help in identification.

• Swollen sporangium

Cell Wall Synthesis

Targets of antibiotics:

• Cell Wall Integrity: D-cycloserine, Vancomycin, Bacitracin, Penicillins, Cephalosporins, Cephamycins

• DNA Synthesis: Metronidazole

• Translation/ Protein Synthesis:(50S Inhibitors:) Erythromycin, Choramphenicol, Cindamycin, Lincomycin, (30S Inhibitors:) Tetracyclines, Streptomycin, Spectro nycin, Kanamycin

• DNA Gyrase Quinolones/RNA Polymerase Rifampicin/Phospholipid Membranes Polymyxins