Microbiology

Top 2 Respiratory Disease

• Wine/beer
• Bread
• Cheese
• Yogurt

Degrading of harmful chemicals by living organisms

Example: Pseudomonas in marine oil spills

• Synthesized by bacterial metabolism
• Ethanol, amino acid
• Insulin, vaccines, antibiotics by g.e.

• “Conquered” disease: smallpox
• Production of vaccines and antibiotics

• Model organisms to study
• Emerging diseases
• • Swine flu, avian flu, SARS
• • Changing lifestyles, population expansion, increased global travel

• Prokaryotes
• Escherichia coli
• Single cell
• Cell walls contain peptidoglycan

• Prokaryotes
• Pyrolobus fumari
• Similar in size and shape of bacteria
• Does not have peptidoglycan

• Microbial
• Algae
• Fungi
• Protozoa - Giardia

• Viruses
• Viroids
• Prions

Nucleic acid with a protein coat

RNA causing plant disease

Infectious protein causing mad cow disease

Sizes of cells biggest to smallest

• Water
• pH – concentration of H+ moles per liter
• Organic and inorganic molecules
• Macromolecules
• - Proteins
• - Polysaccharides
• - Nucleic acids
• - lipids

Large molecules formed from subunits (monomers)

Addition of monomers- dehydration

Withdrawal of water molecule - hydrolysis

• Catalyze reactions
• Cell movement
• Uptake of nutrients
• Gene regulation
• Regulating cell structure

• Each contain a unique side chain, 20 total
• Grouped according to side chain property
• - Hydrophilic
• - Hydrophobic

Linkage of a carboxyl group to an amino group of two amino acids

Single chain of amino acids connected by peptide bonds

Arranged according to hydrogen bonds between side chains of amino acids

Some organization; loosely arranged

Includes more than one unit of protein structure held by hydrogen bonds

Fear of water

Love of water

Some proteins require assistance by chaperones

• Protein shape is changed by broken bonds
• Effected by:
• • High temperature
• • High or low pH
• • Certain solvents

• Carbon (1):Hydrogen (2):Oxygen (1)
• Monosaccharides
• Disaccharides
• Polysaccharides

• Ribose, deoxyribose
• 5- or 6- carbon sugars found in DNA and RNA

Food sources: lactose, sucrose

Part of cell structure: Cellulose, glycogen

• Nucleotides
• Genetic Information: DNA, RNA

• Carry chemical energy - ATP
• Part of enzymes – coenzyme A
• Act as signaling molecule – cyclic AMP

• Long, double stranded helix with thymine
• Contains A, G, T, C
• Anti Parallel
• Complementary pairing between:
• - adenine:thymine
• - guanine:cytosine

• Short, single strand with uracil replacing thymine
• Contains, A, U, G, C

• Purines
• Pyrimidines

Components of RNA and DNA; the two major ones are adenine and guanine

Components of RNA and DNA; the three major ones are thymine, cytosine, and uracil

Ester bond between phosphate of 5’ carbon and 3’ carbon

• Major component of membrane
• Simple Lipids
• Compound Lipids

• Only carbon, oxygen and hydrogen
• Fats= fatty acid + glycerol
• Saturated- no double bonds
• Unsaturated- double bonds

Phospholipids- a lipid that has a phosphate molecules as part of it's structure

• Ocular lens magnifies image from objective lens
• Allows viewing at comfortable distance

• Enlargement of image of a specimen
• Maximum for light microscopes is 10X for ocular lens & 100X for objective lens = 1000X total

• The ability to distinguish between small objects close together
• Resolving power of light microscope 200 nm

The number of visible shades in a specimen

Light passes through

• Light bounces off
• We see the color of an object based on wavelengths of light reflected by its surface

Absorbs light

• Light of one wavelength is absorbed
• Triggers emission of lower energy light of a different wavelength
• Specimen is stained with fluorescent dye
• Expose specimen to ultraviolet, violet, or blue light then emits bright blue, orange, green or red

Light bends

• Bright field
• Phase contrast
• Dark-field

• Evenly illuminated field of view
• Usually requires dye or stain i.e. gram stain, acid fast

Using differences of refracted light

• Hollow cone of light is used to illuminate a specimen
• Object appears bright against a dark background

• Adhere to negatively charge cell parts
• Methylene blue, crystal violet, safranin

• Stains background; leaves cells colorless
• Ex: Capsule stain

• Gram stain
• Acid-fast staining

Based on membrane structure using crystal violet and safranin

• Stains Mycobacterium
• Based on high lipid content in membrane
• Carbol fusion applied over steam bath

• Specimens stained with flourescent dye
• Laser scans regions and planes to create 3D image
• Slices specimen into thin layers

• Light is replaced by a beam of electrons from tungsten filament in electron gun
• Sample preparation is complex – viewed in a vacuum
• Condenser magnet focuses beam on sample
• Final image focused on fluorescent screen or photographic film

• Observe fine cell structure
• Thin sectioning or freeze fracturing

Observe surface details

• Leading cause of pneumonia in college students
• Fever, headache, fatigue, followed with dry cough progressing to production of mucoid sputum
• Transmitted by aerosolized droplets; symptoms arise after 1 week

• Round
• Example: Staphylococcus aureus

• Rod shaped
• Example: Escherichia coli

• Looks like a bead
• Example: Haemophilus influenzae

Clusters

• Boomerang shaped
• Example: Vibrio cholerae

• Wave shaped
• Example: Helicobacter pylori

• Spiral shaped
• Example: Treponema pallidum

• Lipid bilayer embedded with proteins
• Selectively permeable
• Hydrophobic interior and hydrophilic exterior
• Proteins move around lipid layers
• • Fluid mosaic model

• Simple Diffusion
• Facilitated Diffusion
• Active Transport

• Small molecules move freely in and out of cell
• Creates large amount of osmotic pressure on membrane

Carrier protein helps movement from higher to lower concentrations

Energy used to transport against concentration gradient

• Required to withstand osmotic pressure from within and prevent cell from bursting
• - Approx. 75 psi- constantly in aqueous environment with low [solute]
• Confers cell shape
• Targeted by penicillin and lysosyme

• Two groups: gram + and gram -
• Peptidoglycan
• - Nacetylmuramic acid (NAM)
• - N-acetylglucosamine (NAG)
• Deviants:
• - Mycoplasma lack a cell wall
• - Archea do not contain peptidoglycan

• Very thick layer of peptidoglycan
• Contains
• - Teichoic acid
• - Often some proteins
• Retains crystal violet dye of Gram stain even after addition of alcohol
• Stains purple

• Peptidoglycan is much thinner
• Covered by an outer membrane
• • Porins, which provide channels for smaller molecules to pass through
• • High density of lipopolysaccharide
• Periplasmic space between cell wall & cell membrane
• Crystal violet stain easily washed off with alcohol
• Counterstain with safranin
• Stains red

• Confer swimming motility in liquids
• Long, rigid, threadlike propellers extending from bacterial surface

• Short and thin
• Used for attachment (fimbriae)
• - Conjugation

• Cell pool for all biosynthetic functions
• Mixture of sugars, amino acids & salts
• 70-80% water
• Packed with enzymes

• Irregular mass in nucleoid region
• Single circular ds DNA
• Supercoiled
• Contains essential genes

• Smaller circular ds DNA outside chromosome
• - Contain genes that enhance survivability but are not necessary

• Sites of protein synthesis
• - Thousands per cell
• Free in cytoplasm or tethered to membrane
• rRNA + protein
• Difference in prokaryotic (70S) and eukaryotic (80S) ribosome allows antibiotic targeting

• For surviving environmental extremes
• • Heat, drying, radiation, pH extremes
• • Conditions common to soil and rock environments, aquatic sediments, mud, deserts-where long periods without water are common
• All Gram+ to date
• Most common genera:
• • Clostridium (tetanus, botulism, gangrene)
• • Bacillus (anthrax)
• Have a VERY low water content

• External stimulus
• Extremes such as heat, dryness, etc.
• Differentiated gene expression
• Asymmetrical cell division

• Bacillus anthracis responsible for anthrax scare after 9/11
• Commonly found in soil

• Resemble fungi in form – mycelium
• Commonly found in soil
• At tips of filaments are conidia spores

• Size:
• Cell Wall:
• Chromosomes:
• Ribosomes:
• Organelles:

• Size:
• Cell Wall:
• Chromosomes:
• Ribosomes:
• Organelles:

Animalia and Plantae

• The 3rd kingdom added in the classification in 1866
• Includes protozoans, algae, fungi, and bacteria

5 Kingdoms Today

3 Domains Today

• Size and shape
• Gram stain

Fermentation of sugars

• Identification of bacterial surface proteins or polysaccharides
• Identify bacterial surface protein or polysaccharide
• Gram-negative bacteria like E. coli vary in lipopolysaccharide structure
• • O antigen
• Flagella can also vary in structure
• • H antigen

• Pattern of fragment size is restriction fragment length polymorphism (RFLP)
• Identifying a unique DNA pattern of a particular
• bacterial strain
• Using an enzyme that cuts ds DNA at specific
• points to create several fragments
• Separate fragments by gel electrophoresis

• Defined as an increase in number
• Achieved by binary fission
• Generation or doubling time
• Nt=N0 x 2n

• A community of microbes
• Established by planktonic cells and a slime layer to create a strategic system of cellular signaling and
• nutrient distribution
• Examples of location:
• - Teeth
• - Sinks
• - Rocks in streams

• 65% human infections involve these
• Treatment of them is difficult
• Often resistant to antibiotics
• Causes problems in industry
• Clogging or damaging pipes or equipment

• Extremely helpful inwastewater treatment
• Helps to breakdown organic products

• Taking samples from nature and isolating one
• species
• Descended from one cell
• Sterile instruments, aseptic technique
• Using streak-plate method, spread culture to single cells
• Culture medium:
• - Nutrients in liquid or solid form
• - Single cell grows into a colony
• Growth on agar plate
• - Polysaccharide from marine algae
• Maintain culture
• - Restreak cultures regularly
• - Store long term at -70˚C

• Pushing bacterial cells across a plate until they are
• separated individually from each other
• Individual cells grow into colony of thousands

• Culture Media
• Complex – unknown content
• •Meat juices, digested proteins
• Chemically defined – precise measured chemicals
• •Salts, sugar, selected growth factors

• Selective
• Differential

• Inhibits growth of some bacteria while allow others to grow
• MacConkey – inhibits Gram +

• Distinguishes related organisms based on metabolism or biochemical basis
• • Blood agar –production of hemolysin

• Closed or batch system
• - Nutrients are not added
• - Waste is not removed
• - Growth curve observed

4 Stages of Growth

• Metabolically active/no increase in number of cells
• Adaptation; induce enzymes needed
• Length varies w/ species & conditions

• Population doubles each generation
• Primary metabolites synthesized
• Balanced growth- all cellular constituents made at constant rates
• Most susceptible to antibiotics

• Primary
• Secondary

• Amino Acids
• Nucleic Acids
• Simple Lipids

Antibiotics

• Growth curve horizontal
• Population growth ceases
• New cells made at same rate as old cells die (growth rate = death rate)
• Secondary metabolites are made

• 99% of population dies
• Prolonged decline – 1% population mutates according to environment

Which growth stage(s) would synthesize the most DNA?

Which growth stage(s) would have the most active ribosomes?

Which growth stage(s) would be the most resistant to antibiotics?

Arctic regions

Refrigerator

Soil, human body

Compost heaps

Hydrothermal vents

• Archaea
• Volcanic vents; fissures; hydrothermal vents
• Pyrolobus fumarii grows between 90° and 113° C in deep sea vents
• • Makes H2S during metabolism

Must have O2

Require small amount of O2

• Grow better if O2 is present
• • Makes more ATP
• Can grow without as well

Exclusively ferment; O2 does not harm or help

O2 is toxic

• Hypertonic environment may cause plasmolysis
• • Higher concentration of salt
• Moderate halophiles
• • Marine enivironment (requires 3% salt)
• Osmotolerant
• • Skin surface (as much as 10% salt)- Staphylococcus sp.
• Extreme halophiles
• • Dead Sea (requires 9% or more – some archaea

• Autotrophs use CO2 to make carbon products for cell
• Heterotrophs break down organic carbon to make carbon products
• • Simple sugars such as glucose

• Some bacteria make all of their own:
• • Amino acids, vitamins, purines, pyrimindines
• Others require them from the environment
• • Fastidious- require additional factors to grow

• Phototrophs capture energy from sunlight
• • Cyanobacteria
• • Purple & green sulfur bacteria
• Chemotrophs use organic or inorganic molecules for energy
• - Organic- glucose
• - Inorganic:
• • H2 – Hydrogenomonas
• • H2S; FeS2 – Thiobacillus
• • NO2 - Nitrobacter

• Energy Source: sunlight
• Carbon Source: CO2

• Energy Source: sunlight
• Carbon Source: organic compounds

• Energy Source: inorganic chemicals (H2, NH3, NO2-, Fe^2+, H2S)
• Carbon Source: CO2

• Energy Source: organic compounds (sugars, amino acids, etc)
• Carbon Source: organic compounds

• Enzymes
• ATP
• Electron carriers
• Chemical energy source
• Precursor metabolites

• Biological catalysts
• Active site
• Substrate complex
• Specificity
• “Lock and key”
• Lower the activation energy

• Cofactors
• Coenzymes

Enzymes that need help

• Who help enzymes
• - FAD
• - NAD+
• - NADP+
• - Coenzyme A

• Non-competitive inhibition
• Competitive inhibition

• Allosteric
• • Feedback inhibition
• Non-reversible inhibitor damages enzyme

• Obstructs substrate
• Blocking the active site
• Mimics substrate
• Sulfa drugs and Folic Acid Synthesis

Changes active site meaning the substrate no longer fits

Excess end product of enzymatic reactions becomes allosteric inhibitor

• Immediate donor of free energy
• Substrate level, oxidative and photo phosphorylation

• Reducing power
• Lose of electron= oxidized
• Gain of electron= reduced