Biology 1

Viruses need host cell machinery.

True

Bacteria

• Living cells

• Free living

• Genetic Information = DNA

Viruses

• Not a cell

• Dependent on host replication machinery

• Genetic information = DNA or RNA

Three domains of life

• Bacteria

• Archaea

• Eucarya

Microbes are Everywhere

• With little or no oxygen

• Under high pressure

• In extreme temperatures

• On radioactive waste

Microbiology

The study of very small living organisms (microbes).

Taxon

A group or “level” of classification

Hierarchical

Large groups are divided into smaller ones

Nomenclature

• Binomial System of Nomenclature:

  • Genus name + species name

• Names should be italicized or underlined

• Genus name is Capitalized and may be abbreviated

• Species name is never capitalized or abbreviated

Phenetic Classification System

• Groups do not necessarily reflect genetic similarity or evolutionary relatedness (although in some instances may coincide).

• Instead, groups are based on convenient, observable characteristics (e.g. Gram staining, shape of cell, etc.)

Phylogenetic Classification System

• Groups reflect genetic similarity and evolutionary relatedness

Bergey’s Manual of Determinative Bacteriology

• Manual that groups bacteria into phenetic groups, used in identification of unknowns.

  • Not necessarily based on genetic similarity or evolutionary relatedness

  • Groups based on convenient, observable characteristics

• It has been revised and updated several times (work still in progress)

Bergey’s Manual of Systematic Bacteriology

• Groups organisms based on ribosomal RNA sequences

Bergey’s Manual of Determinative Bacteriology and Bergey’s Manual of Systematic Bacteriology are both commonly referred to as “Bergey’s Manual”.

True

16sRNA

• Nucleic acid sequence

• Found in all living things (everything can be compared)

• Part of ribosomes (same function in all organisms)

• Relatively constant regions (compare ancient regions)

• Highly variable regions (compare recent relatives)

Robert Hooke uses compound microscope to describe mold.

True

Antony van Leeuwenhook was the first person to describe in detail microbial organisms.

True

Spontaneous Generation

Belief in spontaneous creation of simple life forms from non-living matter.

Pasteurization

Heating to high temperature and pressure

Edward Jenner developed smallpox vaccine.

True

Robert Koch

Developed methods for pure culture and aseptic technique.

If you can satisfy all of Koch’s Postulates you can determine that one particular organism is causing a particular disease.

• The microbe must be present in every case of the disease

• The microbe must be isolated from the diseased host and grown in pure culture.

• The specific disease must be reproduced when a pure culture of the microbe is inoculated into a healthy, susceptible host.

• The microbe must be recoverable from the experimentally infected host.

Limitations of Koch’s Postulates

• Some microorganisms cannot be grown in pure culture in the laboratory.

• There is no animal model of infection for some microorganisms.

• Not all hosts react the same way to every infectious agent.

Ignaz Semmelweis

• Doctor who introduced antiseptic practice to medicine

• Implemented hand washing after autopsy (before obstetric practice).

Fanny Hesse

Pioneered the use of agar in lab plates

Julius Petri

Invented the petri dish.

Flemming discovers penicillin.

True

Light Microscopy

• Simple

  • Contain a single magnifying lens

  • Similar to magnifying glass

  • Leeuwenhoek’s scopes

• Compound

  • Series of lenses for magnification

Total magnification

Objective lens mag. X Ocular lens mag.

Magnification

• Increase in apparent size of an object

• Results from beam of light refracting (bending) as it passes through a lens

• You see an enlarged, inverted image of the specimen

Light travels slowly through a thick medium

True

Refraction

• Bending of light as it passes through a lens

• Lights travels more slowly through denser medium

• Refractive index = measurement of how much the light is bent.

Refractive index

Measurement of how much the light is bent.

Light travels more slowly if it goes through thick medium.

True

Resolution

Ability to distinguish two adjacent points.

Resolution is dependent on

• Wavelength of radiation

• Numerical aperture of the lens

Contrast

Difference in intensity between 2 objects, or between an object and the background.

Staining increases contrast.

True

Dark-Field Microscopy

• Light is scattered by specimen

• Bright specimen against dark background

• Increases contrast

• Cells are not dead since you don’t need to stain

• Enables observation of details not visible in bright-field

• Do not need to stain or heat fix it

Phase-Contrast Microscopy

• Light is scattered by specimen

• Bright specimen against dark background

• Increases contrast

• Enables observations of details not visible in bright-field

• Living cells do not need to stain or heat fix

• Phase shifts treat rays of light differently

Fluorescence Microscopy (UV light source)

• Fluorescent molecules absorb energy from radiation (UV) light source.

• Emit energy as longer, visible wavelength

• Can be used to label molecules of interest within cell

  • GFP molecule from jellyfish commonly used

Electron Microscopy

• Resolution of light microscopes is limited by the wavelength of visible light

  • Cannot distinguish structures closer than -200nm

• EM uses electrons instead of visible light

  • Shorter wavelength = higher resolution

  • Can magnify image > 100,000X

  • Uses magnetic fields as lenses

• Can visualize smallest bacteria, viruses, molecules

Transmission Electron Microscopy (TEM)

• Electrons rather than light pass through specimen

• Dense areas of specimen block electrons, resulting in dark areas in image

• Sample must be very thin

• Can see into

Scanning Electron Microscopy

• Rapidly focuses electrons back and forth across the surface of the specimen

• Specimen coated with metal

• Scattered electrons pass through detector, producing signal

• Lower resolutions than TEM, but whole specimen can be observed

Capsule/slime layer (Glycocalyx)

• Found in almost all bacteria to some degree

• Loose polysaccharide and/or protein layer surrounding bacteria

• Can be thicker than the diameter of the cell

• Barrier to toxic molecules (i.e. detergents)

• Unnecessary for growth, but important in cell survival

  • Impedes ingestion by host’s immune cells

• Promotes adherence to other bacteria or host tissue

  • Ex: Streptococcus species use capsule ton adhere to teeth

What does peptidoglycan do for the cell?

• Maintains cell structure and rigidity

• Contributes to cell shape

Breaking the PG layer increases cell susceptibility to osmosis and cell lysis.

True

Gram negative bacteria has outer membrane and inner membrane and the peptidoglycan will be in the middle of the outer membrane and inner membrane.

True

Gram positive bacteria does not have outer membrane and has thicker peptidoglycan layer sitting about the inner membrane.

True

Lipopolysaccharide (a.k.a endotoxin) also known as LPS is a major component of the outer membrane.

True

Plasma membrane, inner membrane, cytoplasmic membrane

True

Cytoplasmic inner membrane

• Permeability barrier

• Protein anchor

• Energy generation

Plasmids

Most common in gram negative bacteria.

Prokaryotic cells have a nucleoid and 70S Ribosome while eukaryotic cels have a nucleus and 80S Ribosome.

True

Prokaryotic cells have a peptidoglycan and Eukaryotic cells do not have a peptiodoglycan.

True

Prokaryotic has circular DNA Eukaryotic has linear DNA.

True

Gram positive stains purple.

True

Gram negative stains pink.

True

Gram positive has lipoteichoic acid and teichoic acid.

True

Alcohol causes gram negative to be colorless.

True

Safranin causes gram negative to be pink or red.

True

Gram positive is purple throughout the staining of crystal violet, Iodine, Alcohol, and Safranin.

True

Swimming

Flagella

Swarming

Flagella

Twitching

Type IV Pili

Pushing

Host Cell Actin

Swimming Motility

• Movement through liquid

• Individual Movement

• Powered by rotating flagella

Flagella (sing. flagellum)

• Provide motility for bacteria

• Synthesis of flagella is complex (many genes involved)

• Movement driven by rotary protein engine and powered by proton motive force (i.e., flow of protons across the bacterial cell membrane)

  • Passage of protons cause rotation of motor complex

  • Motor complex is connected to flagellar filament, which is also rotated

Flagella

Location and number of flagella varies among different bacteria

Swarming Motility

• Surface motility

• Social or group behavior

• Requires flagella

• Requires surfactant to reduce tension

• Thought to be triggered by surface contact

• Cells become hyperflagellated

Twitching Motility

• Surface motility

• Mediated by type IV pili

• Pilus extends and attaches to surface

• Pilus retracts and pulls cell along

• Jerky movement over surfaces “twitching”

Pushing Motility

• Host cell actin polymerizations forms actin tail at bacterial pole.

• Bacteria is pushed by growing actin tail.

Chemotaxis

• In response to chemical gradients

• Can have chemoattractants and chemorepellants

Aerotaxis

• In response to oxygen concentration

Photoaxis

• In response to light intensity

Magnetotaxis

• In response to magnetic fields

Chemotaxis

• Bacteria move in response to chemical stimuli

• Swim toward food & away from toxins

• Cell undergoes random swim and tumble movements

• Direction of flagellar spinning determines swim or tumble

Longer swimming and running when you have chemoattracant than tumbling.

True

Longer tumbling when you have chemorepellent than running or swimming.

True

Swimming period becomes longer as concentration of chemoattractant increases or shorter for chemorepellents.

True

Phosphorylation of CheY allows it to interact with the flagellar motor.

True

Phosphorylated CheY binds to flagellar motor and favors clockwise rotation which leads to increased tumbling.

True

Anticlockwise rotation

CheY is not phosphorylated

More swimming

(receptors bound to attractant)

Clockwise rotation

CheY is phosphorylated

More tumbling

(receptors bound to repellent)

Spores

• Dehydrated, multishelled protective structure

• Response to adverse conditions

• Resistant to

  • Heat

  • Chemicals

  • Radiation

  • Dessication

• Not produced by all bacteria

• Have complete chromosome, but minimum # of proteins

• Hard to get rid of using standard disinfectants

• Survival is main goal, not reproduction

• when favorable conditions return it leads to 1 spore = 1 bacterial cell, no net gain

Sporulation is a process of cellular differentiation.

True

Metabolic Requirements of Bacteria

• Carbon source

  • Organic source or inorganic source

  • Used as building blocks

• Energy source

  • ATP generation

    • Substrate level phosphorylation (ADP → ATP)

    • Proton motive force and membrane-bound ATPase

• Electron source

  • Drives ions into, out of cells

  • Used to create ATP

Carbon Requirements of Bacteria

• Autotrophs

  • Derive energy from inorganic substrates (CO₂) or sunlight

  • “Feed themselves” by making organic compounds from CO₂

  • Need only H₂O, inorganic salts, and CO₂

    • Classified as photoautotroph or chemoautotroph

• Heterotrophs

  • Catabolize acquired organic molecules

    • Proteins

    • Carbohydrates

    • Amino acids

    • Fatty acids

      • Classified as photoheterotroph or chemoheterotroph

Oxygen Requirements of Bacteria

• Poisonous for some bacteria (obligate anaerobes)

• Required by others (obligate aerobes)

  • superoxide dismutase & catalase enzymes detoxify byproducts of aerobic metabolism

• Most grow with or without oxygen (facultative anaerobes)

Obligate anaerobes

Poisonous for some bacteria

Obligate aerobes

Oxygen is required

Facultative anaerobes

Most grow with or without oxygen

Nitrogen Requirements of Bacteria

Nitrogen source (unless capable of N₂ fixation)

• Must be fixed from the environment by certain bacteria and converted to a useable form, such as NH₃

Metabolic Requirements of Bacteria

• Iron

  • Important co-factor for enzymes

  • Often limiting

• Water

• Various ions necessary for protein function

  • Mg²⁺, Ca²⁺, K, P, S, Mn, Cu

Requirements for Bacterial Growth

• Physical conditions need to be conducive to growth

  • Temperature

  • pH

  • Salt

Obtaining Pure Cultures

• Dilution in liquid culture

  • Reduces number of cells in each tube

  • Spread liquid on plate to see single colonies

Defined Culture Media

Defined → Precise chemical composition is known

• Often minimal media

  • contains minimum nutritional requirements for growth

Complex Culture Media

• Complex → Composed of digests of chemically undefined substances (e.g., yeast and meat extracts).

  • Often nutrient or general growth media (supports the growth of many organisms).

Enriched Culture Media

• Enriched → Complex media enriched with components that support growth of fastidious organisms (organisms with complex and undefined requirements)

  • Ex: Sheep’s Blood

Selective Culture Media

• Selective → Kills (selective against) some organisms while allowing others to grow (selects for).

• MacConkey’s Agar

  • Crystal violet and bile salts select against Gram-positive organisms (Gm⁺ organisms do not grow on MacConkey’s)