Untitled Quiz

Lecture Content

• Classification of bacteria
• Growth, Survival, and Death of Microorganisms
• Physiology
• Cultivation of microorganisms

Identification of Bacteria

• Understanding differences in bacteria is crucial.
• Each infectious agent adapts to specific transmission methods.
• The capacity to grow in a human host (colonization).
• Mechanisms to cause disease (pathology).

Bacterial Taxonomy

• Bacterial taxonomy (Gk. Taxon) is the arrangement and classification of organisms, indicating natural relationships.
• Identification, classification, and nomenclature are separate but interrelated aspects of bacterial taxonomy.

Universal Tree of Life

• Bacterias, Archaea, and Eukarya are shown in a branching diagram.
• Different types of bacteria are categorized.
• Chloroflexus, Purple bacteria, Chloroplast, Cyanobacteria, Flavobacteria, Thermotogales, Aquifex are among the examples.
• Entoamoebae, Slime molds, Diatoms, Animals, Fungi, Plants, Ciliates, Flagellates, Trichonomads, and other groups are shown.
• Methanothrermus, Halophiles, Methanococcus, Thermoproteus, Pyrodictium, Thermococcus are example archaea.

Identification

• Identification practically utilizes a classification scheme to:
  • Isolate and distinguish specific organisms in a microbial mixture.
  • Verify the authenticity and properties of cultures in clinical settings.
  • Isolate the causative agent of a disease.

Koch's Postulates

• Rules for proving that an organism causes a disease
• Organism consistently isolated from diseased individuals.
• Organism grown in pure form.
• Signs and symptoms induced after inoculation.
• Same organism isolated from experimentally infected individual.

Identification Schemes

• Identification schemes are not classification schemes.
• There may be superficial similarities.
• Escherichia coli is often reported in literature as the causative agent of hemolytic-uremic syndrome in infants.
• Hundreds of E. coli strains exist, but only a few are associated with the syndrome.
• Strains can be identified using antibody reactivity with O-, H-, and K-antigens.

Classification

• Classification categorizes organisms into taxonomic groups.
• Experimental and observational techniques are essential for taxonomic classification.
• Factors considered for classification include biochemical, physiologic, genetic, and morphologic properties.
• Microbiology is a dynamic field with evolving tools, including new microscopy techniques, biochemical analysis, and computational nucleic acid biology.

Nomenclature

• Nomenclature is the naming system used by scientific and medical professionals for microorganisms.
• Formal ranks include kingdom, phylum, class, order, family, genus, species, and subtype.
• Linnaean taxonomy is the system used for these ranks.

Classification of E. coli

• Different types of E. coli are classified. This encompasses commensal, intestinal pathogenic, pathogenic, and extraintestinal pathogenic E. coli strains.
• Strains are also categorized based on their roles in health and disease.

Criteria for Identification of Bacteria

• Growth on media:
  • Complex media (carbon source, hydrolysate, vitamins)
  • Nonselective media (blood, chocolate agar)
  • Selective media (eliminates irrelevant bacteria, containing inhibitory agents).
• Examples of selective media include McConkey agar for Gram-negative rods and Columbia CAN agar for Gram-positive cocci.
• Differential media (distinguishes bacteria based on differences in metabolism) are also shown.

Gram Staining and Microscopy

• Gram staining, developed by Christian Gram in 1884, differentiates between Gram-positive and Gram-negative cell walls.

Gram Stain Mechanism

• Primary stain (crystal violet)
• Mordant (Gram's iodine)
• Decolorizer (acetone-alcohol)
• Counter-stain (safranin)
• Gram-positive cells retain crystal violet-iodine complex, appearing purple.
• Gram-negative cells loose crystal violet and take up safranin, appearing pink.

Gram Positive and Gram Negative Cell Wall Structure

• Key differences in the structures of Gram-positive and Gram-negative bacterial cell walls.

Staining Methods

• Simple staining utilizes a single dye and doesn't differentiate bacterial types.
• Differential staining employs multiple dyes to differentiate between bacteria. This includes Gram staining and acid-fast staining.
• Special staining techniques highlight specific bacterial structures.

Acid-Fast Stain

• Used to stain organisms resistant to conventional staining.
• Specifically used to stain Mycobacterium tuberculosis.
• High lipid concentration in the cell wall prevents dye uptake.
• Cells are difficult to decolorize after staining.

Differential Staining Methods - Acid-Fast Staining

• Acid-fast bacteria retain the primary dye (carbol fuchsin)
• Non-acid-fast bacteria are decolorized in the process.

Special Stains

• Endospore stain (enhance endospores by heating), Capsule stain, and Flagella stain (increase flagella diameter for visibility).

Biochemical Tests (1)

• Catalase test differentiates between catalase-positive (staph) and catalase-negative (streptococci) Gram-positive cocci.

Biochemical tests (2)

• Oxidase Test distinguishes Enterobacteriaceae from other Gram-negative rods based on the presence or absence of the cytochrome C enzyme.

Immunologic Tests-Serotypes, Serogroups, and Serovars

• "sero" designation signifies the use of antibodies for identifying bacterial cell surface structures, like LPS (lipopolysaccharide), flagella, or capsular antigens.
• Serotypes, serogroups, and serovars classify bacterial strains based on antibody reactivity and are essential for identifying specific bacterial strains in outbreaks.
• Phenotypic and serologic methods are used for subtyping microorganisms.

Clonality

• Clonality helps characterize organisms from outbreaks.
• Organisms from a common source are often genetically identical.
• This concept is important for epidemiological analysis of infectious diseases.

Genetic Diversity

• Advances in DNA sequencing allow comparing genetic relatedness.
• Genetic instability can cause high variability within a taxonomic group.
• Traits like antibiotic resistance can be carried on plasmids or bacteriophages.
• DNA sequencing techniques are important for analyzing genetically diverse microorganisms.

Nucleic Acid-Based Taxonomy

• Methods based on nucleic acids for understanding microbial taxonomy include plasmid profile analysis, restriction fragment endonuclease analysis or digestion, repetitive sequence analysis, ribotyping, 16S ribosomal sequencing, and genomic sequencing.

Plasmid Analysis

• Plasmids are extrachromosomal genetic elements.
• Isolating and analyzing plasmids helps understand bacterial outbreaks.
• Plasmid analysis is especially useful when combined with other methods for identifying restricted outbreaks.

Restriction Endonuclease Analysis

• Restriction enzymes cut DNA at specific sequences.
• This analysis generates different-sized DNA fragments, which help characterize different microbial groups.
• Pulsed-field gel electrophoresis separates the digested DNA fragments.

Ribosomal RNA sequencing

• Ribosomal RNA sequences are analyzed to understand evolutionary relationships between organisms, revealing new classifications.

Classification of microorganisms according to nutrition type

• Autotrophic nutrition (manufacturing organic molecules from CO2; photosynthetic, chemosynthetic).
• Heterotrophic nutrition (obtaining nutrients from complex food molecules via digestion, saprotrophs, parasites/pathogens).

The Major Categories and Groups of Bacteria – The Eubacteria

• Gram-negative Eubacteria (phototrophic, nonphototrophic, aerobic, anaerobic, facultatively anaerobic, microaerophilic).
• Gram-positive Eubacteria (chemosynthetic heterotrophs, aerobic, anaerobic, facultatively anaerobic, simple asporogenous/sporogenous bacteria, structurally complex actinomycetes).

The Major Categories and Groups of Bacteria – The Achaebacteria

• Lacks peptidoglycan cell wall
• Characteristic rRNA sequence
• Chemolithotrophs, heterotrophs, facultative heterotrophs
• Extremophiles and/or mesophiles

Bacterial growth - The Measurement of Microbial Concentrations

• Viable cell count (titer) per unit volume of culture (cells per ml)
• Biomass concentration (dry weight per unit volume).
• Turbidity (measurement of cloudiness) correlates with cell mass.

Growth curve of bacteria

• Steps include lag phase, exponential phase, stationary phase, and death phase.

Lag phase

• Time before the start of multiplication occurring after inoculation/introduction.
• Bacteria adjust to the environment (temperature, nutrients) before reproduction.

2. Log Phase

• Exponential growth rates of bacteria in an ideal (favorable) environment.
• Microbes are sensitive.
• This phase ends when resources (nutrients) and/or space become limited.

3. Stationary phase

• Growth rate drops/reduces.
• Bacteria begin to die/reduce at the same rate that new cells grow.
• Waste products from bacterial metabolic activity accumulate.

4. Decline phase

• Death rate exceeds growth rate.
• Bacterial population decreases dramatically.
• Involution forms occur and are potentially more resistant microorganisms.
• Toxin production can occur.

Food Curve and The Chemostat

• Maintaining cells in the exponential phase through repeated transfer to fresh media with constant composition and continuous growth.
• This is also called balanced growth.

Growth in Biofilms

• Bacteria growing in groups on surfaces, creating biofilms.
• Microorganisms interact via communication signals (Quorum sensing).
• Biofilms are often composed of several different microbial species
• The glycocalyx supports biofilm structure.

Control of bacterial growth

• Sterilization kills all organisms (including spores) through varied means.
• Disinfection kills vegetative cells but not spores.
• Pasteurization inactivates pathogenic bacteria through heat application.
• Aseptic techniques maintain free of microorganisms.

Physical and chemical agents affecting bacterial growth

• Heat: Sterilization requires higher temperatures and longer exposure time.
• Radiation (Ultraviolet): Causes DNA damage, generally bactericidal but not spores.
• Alcohols: Dehydrate cells and inhibit metabolic functions.
• Aldehydes (formaldehyde, glutaraldehyde): Effective sporicides.
• Peroxygens (H₂O₂): Effective broad-spectrum disinfectant, also sporicidal at high concentrations.
• Phenols: Antiseptic and disinfectant, not sporicidal.

Sources of Metabolic Energy

• Fermentation (glycolysis, substrate phosphorylation)
• Respiration
• Photosynthesis

Movement of materials across the membrane

• Simple diffusion (high to low concentration)
• Facilitated diffusion (needs membrane protein for high to low)
• Osmosis (water across selective membrane; high to low concentration)
• Active transport (low to high concentration, energy required)
• Group translocation (energy required from chemical modifications preventing exit from cell)

Nutrition – carbon source

• Nutrients in growth media contain necessary elements for new organisms' synthesis.
• Some use photosynthetic energy to reduce carbon dioxide.
• Autotrophs don't require organic nutrients for growth.
• Chemolithotrophs use inorganic chemicals and carbon dioxide as energy sources.
• Heterotrophs require organic carbon.

Nitrogen Source

• Nitrogen is essential for various compounds, including proteins and nucleic acids.
• Two major processes for acquiring nitrogen are nitrogen fixation and ammonification.

Sulfur source

• Some autotrophs oxidize sulfur to sulfate.
• Many organisms use sulfate as a sulfur source.
• H₂S is produced; this compound can be toxic for certain organisms.

Phosphorus Source

• Phosphate is essential for ATP, nucleic acids, and coenzymes.
• It is present in many metabolites, cell wall components, lipids and even some proteins.

Environmental factors affecting growth

• Nutrients:
  • Hydrogen lon concentration (pH)
  • Neutralophiles (optimal pH 6.0–8.0)
  • Acidophiles (optimal pH <3.0)
  • Alkaliphiles (optimal pH >10.5)
• Temperature:
  • Psychrophilic (-5 to 15°C)
  • Psychrotrophs (20-30°C but adaptable to lower)
  • Mesophiles(30-37°C)
  • Thermophiles (50-60°C)
  • Hyperthermophiles (temperatures exceeding boiling)
• Aeration:
  • Aerobic (oxygen requirement)
  • Anaerobic (absence of oxygen needed)
  • Facultative anaerobes
  • Aerotolerant anaerobes
  • Microaerophiles (optimum at lower oxygen requirements)

Cultivation methods - Culture media

• Classification by consistency:

  • Liquid media
  • Solid media
  • Semi-solid media

• Based on functional or growth applications:

  • Basal media
  • Enrichment media
  • Differential media
  • Transport media
  • Anaerobic media

• Classification by nutrition components:

  • Simple media
  • Complex media
  • Synthetic media

Cultivation of bacteria - isolation of microorganisms in pure culture

• Streak culture
• Lawn culture
• Stroke culture
• Pour plate culture
• Liquid culture

Aerobic and Anaerobic Cultivation

• Gas pack method
• Evacuation replacement method

Dilution method

• Serial dilution techniques to determine bacterial counts.