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-Substance that consists of a single type of atom.
*99% of all living matter by weight made up of four.
**Phosphorus and Sulfur make up additional 0.5%
-Basic unit of all matter.
-Made up of 3 major components
*Protons, Electrons, and Neutrons
-Acheive stability through the sharing of electrons between atoms.
-Creates a strong bond
-Difficult to break
-Bonds can be polar or non-polar.
-Covalent Bonds have an equal attraction for the shared electrons
-Bonds formed between identical atoms or between atoms that have similar attraction for electrons
*H-H or C-H
Polar Covalent Bonds
-One atom has a greater attraction to the electrons than the other (greater electronegativity)
-Produces a slight charge difference within the molecule (polarity)
-One part of the molecule will be slightly negatively charged and one molecule will be slightly positively charged. (dipole moment is formed)
-Formed by gaining or losing electrons
-Electrons completely leave first atoms and become part of outer orbital of second atom.
-Loss and gain of electrons leads to charged atoms (ions)
*Atom that loses electrons becomes positively charged
*Atom that gains electron becomes negatively charged
-Ionic bonds are weaker than covalent bonds.
*Bonds dissociate in water
*Easily broken at room temperature
*Approximately 100 times weaker than covalent bonds in biological systems.
-Important among weak forces holding biological molecules together
-Weak bonds formed from the attraction of positively charged hydrogen atoms or molecules.
*Most commonly oxygen or nitrogen.
-Weak biological bonds
-Constantly being formed and broken at room temperature
-At room temperature the average lifetime of a single hydrogen bond is a fraction of a second
-Large numbers of hydrogen bonds can hold molecules together firmly
-Hydrogen bonds form between positively charged hydrogen of one molecule and negatively charged oxygen of another.
-Hydrogen bonding produces a polar molecule.
-Acidity is measured as pH.
-Defined as the -log of the concentration of H+ ions (-log[H+])
-Measured on a logarithmic scale of 0 (acidic) to 14 (basic)
*High H+ = acid
*High OH- = base
3. Nucleic Acids
-Large molecules formed by joining smaller subunits together
*Joining subunits together involves dehydration reaction.
*H20 is removed during chemical reaction
-Macromolecules are broken downinto smaller subunits.
-instead of removing H2O, a molecule of H2O is added
*Reaction termed hydrolytic reaction or hydrolysis.
Proteins and Their Functions
-Constitute over 50% of cell dry weight
-Made up of amino acid subunits
-Some responsibilities include: Catalyzing reactions, Composition and shape of certain bacterial structures, Gene Regulation, and Nutrient Procurement
-Proteins are composed of numerous combinations of 20 amino acids
-Protein shape and ultimately its function depends on the shape of the sequence of amino acids.
-All amino acids have the following shared features
-A carboxyl group (COO-)
-An amino group (NHv2+)
-A central carbon
-A side chain (differentiates the amino acids)
*Amino acids are subdivided based on similarities of the side chain
-The amino acids that form proteins are held together by peptide bonds.
-Unique type of covalent bond
*formed between the interaction of the carboxyl group of one amino acid and the amino acid of the following amino acid.
*reaction causes the release of water and the formation of a peptide bond
-Sequence of amino acids
-In large part determines other protein features
-3 dimensional structure
-2 major shapes
-Becomes Functional protein
-Proteins must have specific shape to have proper function
*environmental conditions can break bonds within the protein and causes shape change.
*Shape change causes protein to stop functioning (denaturation)
-Denaturation can be reversible or irreversible.
-Light passes through specimen, then through series of magnifying lenses.
-Most common and easiest to use is the bright field microscope.
-Important factors: Magnification, resolution, and contrast.
-Compound microscope has 2 magnifying lenses. (ocular and objective)
-Most have 4 magnifications of objective lenses (4x, 10x, 40x, and 100x)
*multiply the lenses together to find the magnification.
-Bright field scopes have a condenser lens whichis used to focus illumination
-Usefulness of microscope depends on its ability to resolve 2 objects that are very close together.
-Minimum distance existing between 2 objects where they still appear as seperate objects.
-Determines how much detail can be seen
0.2 μm (2 x 10^-7m)
-Significant to see most bacterial structures
-Too low to see viruses
-Enhanced with lenses of higher magnification (100x) by the use of immersion oil.
-Oil reduces light refraction.
-Light bends as it moves from glass to air.
oil bridges the gap between specimen slide and lens and reduces refraction.
*Immersion oil has nearly the same refractive index as glass.
-Reflects the number of visible shades in a specimen.
-Higher contrast acheived for microscopy through specimen staining.
-Confocal Scanning Laser Microscope
-Amplifies differences between refractive indexes of cells and surrounding medium.
-Uses set of rings and diaphrams to acheive resolution
-Causes specimen to appear 3-dimensional.
-Depends on differenses in refractive index
-Most frequently used interference scope is Nomarski differential interference contrast.
-Specimen appears bright on a dark background
-Image acheived through a modified condeser
-Used to observe organisms that are naturally fluorescent or are flagged with fluorescent dye.
-Fluorescent molecule absorbs ultraviolet light and emits visible light.
-Uses electromagnetic lenses, electrons and fluorescent phosphorescent screen to produce image
-Resolution increased 1000 fold over brightfield microscope (to about 0.3 nm (1 x 10^-10m)
-Magnification increased to 100,000x
-2 Types: Transmission and Scanning
-Used to observe fine detail.
-Directs beam of electrons at specimen. (Electrons pass through or scatter at surface)
-Specimen preparation through thin sectioning, freeze fracturing or etching.
-Used to observe surface detail
-Beam of electrons scan surface of specimen. (specimen coated with metal, usually gold)
-Electrons are released and reflected into viewing chamber.
-Some atomic microscopes capable of seeing single atoms.
-Cells are frequently stained to observe organisms
-Stains are made of organic salts
*Dyes carry (+) or (-) charge on the molecule
*Molecule binds to certain cell structures
-Dyes divided into basic or acidic based on charge.
carry positive charge and are repelled by cell structures that carry negative charge (commonly stain the background)
Carry positive charge and bond to cell structures that carry negative charge (commonly stain the cell)
-Simple stain uses one basic stain to stain the cell
*Allows for increased contrast between cell and background
*All cells stained the same color
*No differentiation between cell types
-Used to distinguish one bacterial group from another.
-Uses a series of reagents
-2 most common differential stains:
-Most widely used procedure for staining bacteria
-Developed over a century ago by Dr. Hans Christian Gram
-Bacteria seperated into 2 major groups
*Gram Positive - Stained Purple
*Gram Negative - Stained red or pink
-Counter or Secondary Stain
-Stains the cell
-Holds primary dye onto cell
-Removes primary dye from gram negative cell
-Recolors the cells that lose stain through decolorization
-Used to stain organisms that resist conventional methods.
Ex: Mycobacterium has high lipid concentration in cell wall which prevents uptake of dye
-Can be used for presumptive identification in diagnosis of clinical specimens.
-Colors acid-fast bacterias red
-Generally acid alcohol
-Removes stains from non acid-fast bacteria
-Colors non acid-fast bacteria blue
-Example of negative stain
-Allows capsule to stand out around organism.
-Staining enhances endospore
-Uses heat to facilitate staining
-Staining increases diameter of flagella
-Makes more visible
-Coccus - Spherical
-Bacillus - Rod shaped
*Cell shape not to be confused with Bacillus genus.
-Cells adhere together after cell division for characteristic arrangements.
-Pairs or chains of cells.
-Pairs = diplococci
-Chains = streptococci
-Some bacteria live in groups with other bacterial cells.
Ex: myxobacteria - These organisms form a swarm of cells.
-Allows for the release of enzymes which degrade organic material
-in the absence of nutrients cells form fruiting bodies.
-Formation allows for changes in cellular activity.
Ex: On teeth, In catheters, on rocks in streams.
-Contains open channels for movement of nutrients and waste.
-Plasmids can be acquired by sex pilus mediated transduction.-If it doesn't contribute to the fitness of the cell, daughter cells may replicate without the plasmid.
Large = 50sSmall = 30sTotal = 80s
-Resistant to damaging conditions-Produced thru sporulation-Theoretically remain dormant for 100 years.
-Heat, dessication,chemicals and UV light
-Can be killed by autoclaving-Vegetative cell can be produced thru germination.
-Germination Occurs after exposure to heat or chemicals
-Low G+C Gram positive bacteria, Bacillus species and others with this Phylum.-Complex, ordered sequence
1. organism can be isolated from a host suffering the disease
2. organism can be cultivated in the lab
3. organism causes the same disease when introduced into another host
4. organism can be re-isolated from the host
*If true, then the organism is the cause of the disease and is infectious
– A particular enzyme will only act with one or a limited number of substrates
– Enzymes do not alter the reactants or products of a chemical reaction
• Enzymes are not altered by the chemical reaction they catalyze
– Enzymes are usually named for the substrate they act on and end in the suffix –ase
-Modified side chains create derivatives-Penicillin drugs include:
-Natural penicilins-Penicllinase-resistant penicillin-Broad-spectrum penicillins-Extended spectrum penicillins-Penicillins + β-lactamase inhibitor
-Augmentin® amoxicillin plus calvulinic acid
-Solid at room temp.-Made of glycerol and fatty acids
-Allows for the binding of 3 fatty acids to one glycerol (Triglyceride).
-Monosaturated - 1 double bond-Polysaturated - 1 double bond
-Classified as a lipid because steroids are insoluble in water.-Structure consists of 4-membered ring
-helped disprove spontaneous generation
-3 jars of meat experiment
(or normal flora)
cell type that does not contain membrane bound nucleus or any other membrane-bound organelles.
-dna resides in the nucleoid
-(Nt) = Number of cells in population.-(N0) = Original # of cells in the population- (n) = Number of division
-Nt = 10 x 212*4 hours assuming 20 min. generation time.
-Synthesize compounds useful for growth.-Cells produce multicellular associations to increase survivability.
Ex: biofilms and slime layers
-Bioremediation is a beneficial use of biofilm-Architecture of the biofilm resists immune response and antimicrobials
-Used for growing large numbers of bacteria
-Agar marine algae extract.-Liquefies at temperatures above 95 degrees C.-Solidifies at 45 degrees C.-Vessel called petri dish
-Object is to reduce number of cells being spread.
-Solid surface dilution-Each successive spread decreases number of cells per streak.
-Death rate slower than growth in log phase-Decrease at constant rate
-Continually drips fresh nutrients in-Releases same amount of waste product
-Import and export
-Within range lies optimal growth temperature
-Use for energy production
-May cause death
-In laboratory cultures-In bodies of water-In drinking water-In food
-Direct microscopic count-Use of cell counting instruments
-Viewed under microscope-Counting Chamber
-Must have at least 10 million cells per ml to gain accurate estimate
-Flow cytometer-Detects changes in electrical resistance
-Use laser and fluorochrome
-Cells able to replicate
-Methods include:-Often used when cell counts are too low for other methods-Valuable in monitoring bacterial growth.
-Plate counts-Membrane filtration-Most probable numbers
-A goal of the method is to inoculate agar plates with concentration of bacteria so that counting is reasonable (30 to 300 colonies)-# of colonies = # of living cells in sample
-Pour plate - 1.0 mL of dilution is mixed with melted agar (50°C)-Spread Plate - 0.1 - 0.2 mL of dilution is spread on top of solidified agar
-For 10-3 dilution, 350 colonies were counted-Dilution Factor = Reciprocal of Dilution
-For 10-4 dilution, 35 colonies were counted-(350cfu/0.1mL)(103) = 3.5 x 106 CFU/mL
-(35cfu/0.1mL)(104) = 3.5 x 106 CFU/mL
-Growth parameters may select for growth of some organisms and retard growth for others.
-Used with relatively low numbers-Known volume of liquid passed through membrane filter
-Filter pore size retains organism-Filter is placed on appropriate growth medium and incubated
-Cells are counted
-Statistical assay-Series of dilution sets created
-Sets incubated and results noted-Each set inoculated with 10-fold less sample than previous set
-Results compared to the MPN table-Table gives statistical estimation of cell concentration
-Need to verify or confirm estimate-Detects intestinal bacteria based on gas production when lactose is present in medium
-Plate on special medium such as EMB; E. coli colonies will have green sheen
-Green colonies are used to inoculate broth for further verification
-Turbidity-Total weight-Amounts of cellular chemical constituents
-Measures light transmitted through sample-Measurement is inversely proportional to cell concentration
-Limitation = must have a high number of cells*Must be used in conjunction with another test once to determine cell numbers
-Wet weight-Not routinely used-Useful in measuring filamentous organisms
-Dry weight-Cells centrifuged down and liquid growth medium removed-Packed cells weighed
-Packed cells allowed to dry at 100°C for 8 - 12 hrs-Cells weighed
-Gas Production-pH indicator detects acids that result from sugar breakdown
-Gas production monitored using Durham tube
-ATP (detected by use of luciferase)*Tube traps gas produced by bacteria
-Enzyme catalyzes ATP dependent reaction*If reaction occurs ATP present→bacteria present
-Discovered penicilin while working with Staphylococcus.- Noticed there were noStaphylococcus growing near a mold contaminant
-Mass production began in WWII-Full Recovery
-Broadened Spectrum of antimicrobial killing.
-Lowest dose toxic to patient divided by dose typically used for treatment.
-High therapeutic index = less toxicity to patient
-Inhibit = bacteriostatic-Kill = bacteriocidal
-Allergies to penicillin
-Aplastic anemia - chloramphenicol
-Antibiotic associated colitis
-Microorganisms have innate or adaptive resistance to antibiotics
-Prokaryotes have easiest targets - more antimicrobials-Eukaryotic microbes are more difficult to treat
-Antimicrobials of this class:-Low toxicity with high effectiveness
-β lactam drugs-Vancomycin-Bacitracin
-Interfere with the formation of the peptide side chains between adjacent strands of peptidoglycan by inhibiting penicillin-binding proteins.
-Binds to the amino acids in the side chain of NAM molecules interfering with peptidoglycan synthesis
-Interferes with the transport of peptidoglycan precursors across the cytoplasmic membrane
-Tend to have low affinity to penicillin-binding proteins of Gram + bacteria-Chemically modified to produce family of related compounds
-First, second, third, and fourth generation cephalosporins
-Very resistant to β-lactamases-Carbapenems and monobactams
-Acquired resistance most often due to alterations in side chain of NAM molecule.
• Blocks attachment of tRNA to ribosome
• Allows for less frequent dosing
• Prevents continuation of protein synthesis
– Prevents peptide bonds from forming and
blocks protein synthesis
– Prevents continuation of protein synthesis
• Very effective against Bacterioides fragilis
– C. difficile-associated colitis
– Manages supercoiling of DNA during DNA
• Ciprofloxacin and ofloxacin
– Block initiation of transcription
– Primarily used to treat tuberculosis and Hansen’s disease as well as preventing meningitis after exposure to N. meningitis
– Mode of actions to inhibit the production of
– Collectively called sulfa drugs
• Through competitive inhibition of enzyme that aids in production of folic acid
-Plasmid codes for enzyme that has lower affinity to the drug
– Interferes with activity of enzyme that catalyzes
formation of tetrahydrofolate from dihydrofolate
– Common ingredient in first-aid skin ointments
– Alters permeability
– Leads to leakage of cell and cell death
– Limits use to topical application
– MIC = Minimum Inhibitory Concentration
• Quantitative test to determine lowest concentration of specific antimicrobial drug needed to prevent growth of specific organism
– Growth determined by turbidity of growth medium
• From highest concentration to lowest
• Zone is tear-drop shaped
• Zone will intersect strip at inhibitory concentration
• Hundreds of tons used each year
• Impact on cost, complications, and outcomes of treatment
• Penicillinase, chloramphenicol acetyltransferase
• Minor structural changes can prevent binding
• E.g., changes in porin proteins of outer membrane of Gram-negatives
– Resistance of this type particularly worrisome; might allow resistance to multiple drugs
– In a population of 109 cells, at least one likely has that mutation; if streptomycin is added, only that cell and progeny will replicate, yielding resistant population
– May also originate from the soil microbes that naturally produce the antibiotic
– Gene coding for enzyme that modifies aminoglycoside likely originated from the Streptomyces species that produces the drug
-Community acquired (CA-MRSA) currently treatable with drugs other than β-lactams\– Hospitals have reported resistant isolates; strict guidelines have halted spread of these vancomycin-intermediate S. aureus (VISA) and vancomycin-resistant S. aureus (VRSA) strains
-Combination therapy is therefore required
– Starting compound is converted to intermediatemolecules and end products– Intermediates and end products can be used asprecursor metabolites– Pathways can be linear, branched, cyclic
• Accelerate conversion of substrate to product
– Energy required to initiate a chemical reaction
– A substrate is the specific substance on which the enzyme acts
– Enzymes are regulated to prevent over production of product.
– Act as carriers for molecules or electrons
» NAD+, FAD and NADP+ are coenzymes
– May act with numerous enzymes
–Increasing temperature increases speed of reaction
» But extremely high temperature makes enzyme nonfunctional
–Many enzymes function best at pH just above 7
–Low salt concentration are most desired
• Alters affinity of enzyme to substrate
• Regulation controls metabolic activity
• Shuts pathway down
• Inhibitor and substrate act on different enzyme sites
– Allosteric inhibition
– Feedback inhibition
– Sulfa drugs compete with PABA for active site on enzyme that produces folic acid
• Energy currency of cell
• ATP consists of a base, in this case adenine (red), a ribose (magenta) and a phosphate chain.
• Negative charges of phosphate repel
• Create unstable bond that is easily broken releasing energy
– Uses chemical energy to add phosphate ion to ADP
– Uses energy from proton motive force to add
phosphate ion to ADP
– Utilizes radiant energy from sun to add phosphate ion to ADP
-There are a variety of compounds available
-Glucose is the most common organic molecule used.
– Compounds that LOSE electrons are oxidized
– Termed electron donor
– Compounds that GAIN electrons are reduced
– Termed electron carrier
• NAD+ + 2H → NADH + H+
• FAD + 2H → FADH2
• NADP+ + 2H → NADPH + H+
– Serve as raw materials for construction of
– How energy is produced (and how much energy
– How electron carriers are recycled
– Terminal electron acceptor
– Reducing power
– Precursor metabolites
• Modified to 2 carbon compound-acetyl CoA; CO2 is released
– NADH and FADH2 transfer electrons to produce
proton motive force
– Allows for recycling of electron carriers
• Oxygen in aerobic respiration
• Anaerobic respiration uses another inorganic molecule
• Two 3-C pyruvate molecules
• Net gain of two ATP
– 2 ATP expended to break glucose
– 4 ATP harvested
• Two molecules reducing power (2NADH +2H+)
• 6 different precursor metabolites
-5 intermediates and pyruvate
• Glucose + ATP→ Glucose-6-phosphate + ADP
• Phosphoglucose isomerase
-Glyceraldehyde 3-phosphate ↔ dihydroxyacetone phoshphate and Aldolase
– 1,3-bisphosphoglycerate +ADP → 3- phosphoglycerate + ATP
– Phosphoenolpyruvate + ADP→ pyruvate +
– carbohydrates via gluconeogenesis,
– fatty acids or energy through acetyl-CoA,
– the amino acid alanine
– to ethanol
• Also produces glyceraldehyde 3-phosphate
– Can go into glycolysis for further breakdown
• Produces reducing power in NADPH + H+
• Two precursor metabolites
– Ribulose 5-phosphate and erythrose 4-phosphate
– Oxygen: terminal electron acceptor
• CO2 is removed through decarboxylation
• NAD+ is reduced to NADH + H+
• Reaction occurs twice for one glucose
• Reducing power
– NADH + H+ and Acetyl CoA
– Two turns for each glucose molecule
– 2 ATP
– 6 NADH + 6H+
– 2 FADH2
– 2 precursor metabolites