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biol 251 unlv

biol 251 unlv


BIOL 251: Final Exam Study Guide 

What are the types of organisms?

Everything covered in class.

The exam is supposed to be 50% lectures and 50% diseases – don’t stress

about details too much, just big ideas.

Likely on the test. 

Lecture 1: Intro to Microbiology 

∙ Microorganisms (microbes): organisms too small to be  

observed by the unaided eye.

∙ Limit of resolution: an optical system’s capacity to  

resolve point objects separately.

∙ The human eye in its peak condition can see about .1mm  

or 100μm. 

o For visualizing details, you need a microscope.  Higher detail/resolution = lower limit of  


∙ Types of organisms: Prokaryotes (anucleic Bacteria and  Archaea), fungi (single/multicelled, eukaryotic), protozoa (singlecelled, euk, microscopic algae (single/multicelled,  

euk), viruses (acellular), and tiny parasites (euk).  Bacteria is the domain; “bacteria” –  

What are the importance of microbiology?

Don't forget about the age old question of sarah myruski

uncapitalized and unitalicized – is a generic  

term for microbes.

∙ Importance of Microbiology: In the News

o Public health issues, emerging diseases, newly discovered pathogens (EV-D68 and C105 do the  same thing as polio), White Nose Syndrome

(increased humidity grows particular fungi and kills

frogs and bats), immunizations, food allergies,  

antibiotic resistance (superbugs).

∙ We could not exist without microbes. They are important  


o Health: microbes help maintain life; only a few are  


 An adult human is made up of around 30 trillion  cells and another 40 trillion bacterial cells.  

These human microbes form the microbiome or microbiota, the normal amount called flora (the first line of defense). Bacteria, such as E.  

coli, aid digestion, synthesize vitamins, and  

Who is John Needham?

prevent the growth of pathogenic microbial  


1) Only a few microbes are pathogenic (disease

producing); most are beneficial and necessary. 2) Health products, like insulin, are made using


3) LECTURE-ONLY MATERIAL If you want to learn more check out uf majors quiz

∙ Poor oral health is coordinated to


∙ The bacterium Porphyromonas

gingivalis secretes a toxic protein called

gingipain found in the brain, spinal fluid,

and saliva of Alzheimer’s patients.

∙ Blocking this protein is shown to halt


o Environment

1) Regulates the atmosphere (~78% N2; 21% O2; 1%  

Ar & trace gases)

o O2 generated by photosynthesis comes  

mostly from microbes (photosynthetic algae)  

not plants.

o N2 is also generated by microbes, which  

recycle everything.

2) Decomposition of organic waste and detoxification  If you want to learn more check out douglas klutz ua

of other wastes

o Water quality: microbes purify water.

o Bioremediation: microbes eat oil spills and  


o Food Industry

1. Produce fermented foods (e.g. vinegar and cheese) 2. Food spoilage and food poisoning

o Economic impact: bad food chains can go out  of business; knowledge of this can save  


o You can save moldy maple syrup by  

scooping out the affected part and  

boiling the rest.

o Toxins that cause botulism can be so  

potent as to be absorbed immediately by

the body.

o Chemical Industry

1. Produce valuable chemical products.

∙ It’s important to study microbiology for health, the  

economy, and the environment.

1. Prevent diseases, epidemics, and pandemics  

(worldwide disease)

2. Prevent food spoilage (saves a lot of money) 3. Enhance positive traits of microbes for human use 4. Enhance degradation of pollution (bioremediation)

∙ The First Observations: History of Microbiology ∙ 1623-1673: Anton van Leeuwenhoek: The first

microbes are observed; “animalcules.”

∙ 1665: Robert Hooke: living things are composed of little boxes (cells); cell theory – all living things are

composed of cells.

o Spontaneous generation: the hypothesis that life arises from nonliving matter; “vital force” We also discuss several other topics like blanks plus

used for life.

o Biogenesis: the hypothesis the living cells can

come only from preexisting living cells.

∙ 1668: Francesco Redi: filled jars with decaying meet; maggots only appeared in the open jars (flies laid If you want to learn more check out what is a graphical link of competitive​ advantage, ksfs, and supporting​ activities?

eggs); biogenesis.

∙ 1745: John Needham: boiled nutrient broth then placed in covered flasks; microbial growth;

spontaneous generation.

∙ 1765: Lazzaro Spallanzani: boiled nutrient broth in sealed flasks; no microbial growth; biogenesis, but

deniers claimed air was necessary for spontaneous

generation to occur.

∙ 1861: Louis Pasteur: growth appears in nutrient broth heated in flask unsealed; S-shaped flasks let air in, kept microbes out, no growth in; biogenesis – We also discuss several other topics like ion defintion

microbes are present in the air.

∙ 1858: Rudolf Virchow: cell theory; cells arise from preexisting cells – against germ theory of disease (hypothesis that disease came from the cells of the


Golden Age of Microbiology (1857-1914) 

o 1861: Louis Pasteur shows microbes are present in

the air.

 Proof for biogenesis: s-shaped flasks keep microbes out and let air in. Broth showed no

signs of life and proves biogenesis.

o 1835: Agostino Bassi shows silkworm disease was

correlated to fungus.

o 1865: Pasteur shows silkworm disease was caused by

a protozoan.

o 1840s: Semmelweis advocates handwashing to prevent transmission of puerperal fever (childbed fever) from physicians not cleaning their hands between obstetrical patients.

o 1860s: Joseph Lister (namesake of Listerine) used chemical antiseptic (phenol) to prevent surgical

wound infections.

o 1876: Robert Koch discovered anthrax was caused by a bacterium and developed Koch’s Postulates (still used today) to determine if a specific microbe causes

a specific disease.

 The microbe must be:

∙ Found in abundance in all diseased

organisms and not in healthy ones.

∙ Isolated from the test organism and grown

in pure culture.

∙ Cause disease when introduced to a

healthy organism.

∙ Re-isolated from the inoculated, diseased

test host and identified as identical to the

original causative agent.

o 1796: Edward Jenner inoculated a person with cowpox virus and made them immune to small pox

(protection = immunity).

∙ Modern Chemotherapy

o Treatment of disease with chemicals =


o Chemotherapeutic agents = synthetic drugs or


 Antibiotics: chemicals produced by bacteria or fungi that inhibit/kill other microbes.

∙ 1928: Fleming accidentally discovers the

first true antibiotic; penicillium fungus

makes an antibiotic, penicillin, that killed

Staphylococcus aureus.

∙ 1940s: Penicillin was tested and mass

produced in time for WWII.

 First synthetic drugs: quinine from tree bark

was used to treat malaria.

∙ 1910: Paul Ehrlich’s “magic bullet” could destroy a pathogen without harming the

host; a synthetic arsenic drug, salvarsan,

treats syphilis.

∙ 1930s: sulfonamides (sulfa drugs) are

synthesized (first antibiotic) and cause

allergies in some.

∙ Microbes Classification and Nomenclature

o Classification: process by which scientists group

living organisms based on similarities.

o Taxonomy: science of defining groups of life. o Nomenclature: science of naming organisms. ∙ Classification of Biological Organisms

o 1964: Robert Whittaker designs 5 kingdom scheme ∙ Plants, animals, fungi, protists, monera –

no tree of life

o 1980s: Carl Woese develops three domains based on specific genetic information – phylogeny (evolutionary relationships).

 Bacteria, Archaea, and Eukarya

∙ Virus = possible 4th domain

 LUCA: Last Universal Common Ancestor 

 Node = last common ancestor between two branches 

∙ Biological Hierarchy 

∙ Naming microorganisms – common names don’t translate


o Binomial naming: Genus + specific (species) epithet  Species epithet can be used more than once,

but the genus can only be used once (e.g. Escherichia coli).

 First use, you must spell it out. Second use, you can abbreviate the genus (e.g. E. coli).

Lecture 2: The Microscope 

∙ Resolution (resolving power): the ability to distinguish  be two separate objects; the distance between the objects (e.g. a resolving power of .02 μm means can distinguish  

two points .02 μm apart.

∙ Know these units and be able to convert them (μm to nm  

to mm) 

1mm = 10-3m; 1μm = 10-6m.

1pm = 10-12m = 10-9mm

1nm = 10-9m = 10-6mm

1μm = 10-6m = 10-3mm

1000nm = 1μm

.001μm = 1nm

1. Light microscopy (LM): good for cells and bacteria, not  


2. Electron microscopy (EM): cells, cellular structures,  


3. Atomic force microscopy (AFM): good for even molecules. 4. Combinations and specialized microscopy

∙ Limit of resolution (resolving power): ability to distinguish  two separate objects with distance between them (e.g.  

resolving power of .2 μm means distance of .2 μm). ∙ Shorter wavelength of light means greater resolution. o Electrons have much lower wavelength than light,  

thus they produce images with lower resolution. ∙ Microscopes and Magnification

o Higher magnification/resolution = lower limit of  


∙ Anatomy of a Microscope

∙ Light Microscopy 

o Compound Light M. 

 Uses visual light and a 2 lens system

 Total magnification = ocular lens (10X) x  

objective lens (4X, 10X, 40X, or 100X)

 Light may refract after passing through a  specimen, stopping it from passing through the  

objective lens.  

∙ Refractive index: measure of a medium’s light

bending ability.

∙ Immersion oil keeps light from refracting; lowers  the refractive index because it has the same  

index as glass.

 Brightfield Illumination: objects are either  

colored or dark

∙ Requires staining; little detail; dark objects  are visible against a bright (grey)  


 Light reflected off specimen doesn’t enter the  

objective lens

o Darkfield m. 

 Light objects, dark background; internal  


 Opaque disk placed in condenser; only light  reflected off the specimen enters the objective  lens.

o Phase-Contrast M. 

 Brings two sets of light (direct and diffracted) to  form an image; grey background; living  

organisms, internal structures, and visible cilia.

o Differential Interference Contrast (DIC)  

M. – Nomarski Illumination – False Color  Similar to phase contrast; 3D image; uses two  

light beams + prisms to split  

beams; organelles; no  


o Fluorescence M. 

 Uses UV light: short wavelength = better  


 Fluorescent substances absorb UV light and  emit longer visible wavelengths; naturally or  

dyed fluorescent; dark background.

o Confocal m. 

 Fluorochromes; short wavelength (blue) light  excites a single plane; each plane

is illuminated; 3D image  

constructed to look at one plane  

at a time.

o Scanning Acoustic M. – False Color

 Computer-generated; measures  

soundwaves; studies biofilms; Rz =  

1 μm.  

∙ Electron M.

o Electrons have shorter wavelength, greater  

resolution, higher magnification; used for small  images (e.g. virus).  

o Transmission EM – False Color

 Beam of electrons passes through ultra-thin  specimen sections, an electromagnetic lens,  

then a projector lens.

 Specimens stained with toxic heavy-metal salts  for contrast – beam is dangerous (shown on a  


 10,000 – 100,000x magnification; Rz = 10 pm o Scanning EM 

 Beam of electrons scans surface of the  

specimen; secondary electrons produce a 3D  

image, false color

 1,000 – 10,000x magnification; Rz = 10 nm

∙ Scanning Tunneling M. 

o Tungsten probe has resolution of 1/100 of  an atom; scans details of a specimen

∙ AFM – 12 nm

o 3D images; metal-diamond probe

∙ Light Microscopy Procedures: Staining is Important o Staining: coloring microbes with dye to emphasize  

its structures

o Smear: thin film of material containing microbes o Fixed: attaching the microbes to the slide with heat,  

killing them  

∙ Prokaryotic staining: the cell wall is negative and can only  

be stained with basic stains (+ cation chromophore).  One dye = simple stain – highlights entire  

microbes to visualize structures.

 Mordant: chemical that makes the specimen  

look larger by keeping the stain in the cell.

∙ Differential stains: distinguishing prokaryote types o Gram stain: reflection of cell wall chemistry; Gram

positive (thick peptidoglycan cell walls) vs Gram negative (thin peptidoglycan cell walls and  


 Apply crystal violet (primary stain)

 Apply iodine (mordant)

 Alcohol wash (decolorization) – destroys LPS

 Apply safranin (counterstain)

 Gram-positive: purple 

 Gram-negative: pink 

o Acid-fast stain – Mycobacterium and Nocardia  Binds bacteria with waxy material in cell wall;  

acid-fast can’t be decolorized; IDs tuberculosis

 Carbolfuchsin (primary stain)

 Acid-alcohol (decolorization)

 Methylene blue (counterstain)

 Acid-fast: red 

 Non-acid-fast: blue 

∙ Structural Stains

o Capsule stain

 Capsule: uncharged jelly covering (no dyes).  Requires negative staining; acid dye (anion)  

stains the background black and creates a halo. o Endospore stain

 Endospores: resistant, dormant structures  

within cell.

∙ Primary stain: malachite green with heat

∙ Decolorize cells: water

∙ Counterstain: safranin

∙ Spores appear green within red/pink cells

o Flagella staining: difficult

 Flagella: structures of locomotion (motility)

 Use mordant and carbolfuchsin stain.

∙ Domains: Bacteria vs Archaea 

 o Bacteria 

 Prokaryotic (pre-nucleus), single-celled

 Peptidoglycan cell walls (rigidity and antibiotic  


 Binary fission or budding

 Organic/inorganic chemicals or photosynthesis for


o Archaea 

 Prokaryotic, single-celled

 No peptidoglycan cells walls

 Extremophiles (e.g. methanogens , halophiles,  

and thermophiles)

∙ Virology: Study of Viruses

o Iwanowski (1892) and Stanley (1935) discovered the  cause of mosaic disease to be a virus (aka a  

filterable agent).

∙ Mycology: Study of Fungi  

 o Eukaryotic with a distinct nucleus 

o Chitinaceous cell walls

o Absorbs organic chemicals for energy (can’t make  

their own)

o Yeast (unicellular) and molds and mushrooms  


 Molds consist of mycelia, composed of hyphae  


∙ Protozoology: Study of Protozoa

 o Eukaryotes 

o Absorb/ingest organic chemicals (not autotrophic like  


o May be motile via pseudopods (amoeba), cilia, or  


o Free-living or parasitic

∙ Phycology/Algology: Study of Microscopic Algae o Eukaryotic cells 

o Found in freshwater, saltwater, and soil 

o Cellulose cell walls 

o Use photosynthesis to produce oxygen, carbohydrates, 

and toxins (if in water) 

∙ Parasitology: Study of Multicellular Animal Parasites  o Eukaryotic/multicellular animals 

 o Parasitic flat worms and roundworms = helminths  Considered microbes because of microscopic life  


Lectures 3 & 4 

∙ Prokaryotic vs Eukaryotic Cells

 o Prokaryotic (pre nucleus) 

 One circular chromosome;  

no membrane

 No histones 

 No organelles

 Bacteria: peptidoglycan  


 Archaea: pseudomurein  


 Binary fission/budding  70S ribosomes (S =  

Svedberg unit; reflection of size)


 o Eukaryotic: true  


 Paired chromosomes in  

membrane-bound nucleus  Histones 

 Organelles 

 Polysaccharide cell walls  (plants) like chitin and  


 Divides by mitosis 

  80S ribosomes 

∙ Sizes, Shapes, and Arrangements of Prokaryotic Cells o Sizes: average is 0.2 – 2.0μm diameter x 2-8μm in  


o Shapes: monomorphic (a single shape) or pleomorphic

(showing many shapes).

 Bacillus (rod-shaped) or bacilli

 Coccus (spherical) or cocci

 Spiral (helical)

∙ Vibrio (comma-shaped)

∙ Spirillum or spirilla

∙ Spirochete

o Arrangements

 Pairs: diplo-

 Clusters: staphylo-

 Chains: strepto-

 Groups of four: tetrads

 Cube-like groups of eight: sarcinae

∙ Structure of Protists 

o Glycocalyx: external to cell wall; viscous, jelly-like,  


 Two types: capsule (firm, organized) and slime  

layer (loose, unorganized)

1. Capsule: provides virulence, prevents  


a. Compartmentalization: DNA tightly  

coiled in a nucleoid

b. Flagella (propels bacteria backward and  


c. Fimbrin (attaches to structures)

2. Cell wall

3. Plasma membrane

∙ Prokaryotic Flagella 

o Appendages external to cell that propel and rotate  


o Made of flagellin; three parts:

 Filament: Outermost region

 Hook: Attaches to filament (motor)

 Basal body: Embeds flagella into the wall

o Gram-positive bacterium do not have the outer  membrane of a gram-negative bacterium; gram negatives have much more anchored flagella (doubled  basal body).

o Allows bacteria to move toward or away from stimuli  


 Chemotaxis – run (to positive) and tumble

(from negative).

o Flagella proteins: H-antigens (anything that  stimulates antibiotic resistance) that distinguish  serovars (forms of bacterium).

∙ Arrangements of Flagellum 

o Peritrichous: covered in flagella

o Monotrichous/polar: one flagellum

o Lophotrichous/polar: bundled at one end

o Amphitrichous: flagella on both ends

∙ Specialized Flagella – Axial Filaments/Flagella o Endoflagella in spirochetes (only between  

membranes in gram-negatives); Anchored at one end  

of cell

o Moves more like a worm/corkscrew in rotation (e.g.  syphilis)

∙ Fimbriae: attachment with hair-like appendages ∙ Pili: proteinaceous, two types:

o Twitching motility pili (attaches, loosens, lifts,  

attaches again)  

o Conjugation pili (constant sex, intercellular DNA  


∙ The Cell Wall

o Prevents osmotic lysis, protects the cell membrane,  

contributes to pathogenicity

o In Bacteria, it is partially peptidoglycan and therefore  


 Peptidoglycan (rigid) is a polymer of NAG and  NAM in rows linked by polypeptides. Recognize:

∙ Gram-positive vs Gram-negative

 o Gram-positive 

 Thick peptidoglycan  Teichoic acids (rigidity,  

negative charge)

 NO outer membrane  Cell walls

 Lipoteichoic acids link wall

to the plasma membrane  Antigen specificity:  

polysaccharides and  

teichoic acids

 o Gram-negative 

 Thin peptidoglycan

 NO teichoic acids

 Outer membrane: protects against phagocytes and  


 Lipopolysaccharides (LPS)  O polysaccharide:  


 Lipid A: endotoxin in top  


 Porins (proteins) form  membrane channels

o Know the Gram

negative diagram.

o  Periplasm (not periplasmic space) in the middle ∙ Contains thin peptidoglycan layer

∙ The Gram Staining Mechanism 

o With crystal violet stain, iodine crystals form inside the

cell wall.

o Add decolorizing agent.

 Gram +: crystals can’t get out, cells stay purple.  Gram -: alcohol dissolves the LPS, crystal violet  

leaves through peptide holes, safranin stains cells red/pink.

 o Gram + Walls 

 2 rings in the basal body  Exotoxins (botulism)  Susceptibility to penicillin  Disrupted by lysosome  

(enzyme that destroys  peptidoglycan)

 o Gram – Walls 

 4 rings in basal body  Endotoxin (Lipid A) +  

exotoxin (secreted outside

the cell)

 Decreased penicillin  susceptibility

∙ Atypical Cell Walls 

o Acid-fast in Mycobacteria and Nocardia: use acid

alcohol to decolorize, stain with carbolfuschin.

 Contains a waxy lipid (mycolic acid) bound to  


o Mycoplasma: walking pneumonia

 Lacks cell walls; sterols in naked plasma  


o Archaea: wall-less, or walls of pseudomurein (no  

peptides (NAM or D-amino acids))

∙ Cell Wall Damage 

o Lysozyme hydrolyzes peptidoglycan bonds

o Penicillin inhibits peptide bridges in peptidoglycan  


o Two cells susceptible to osmotic lysis:

 Protoplast: wall-less gram +

 Spheroplast: wall-less gram -

o L forms: wall-less cell that swells into irregular shapes ∙ Cytoplasmic membrane: phospholipid bilayer o Types of proteins:

 Peripheral: weakly linked on the surface

 Integral: embedded, penetrates the membrane  Transmembrane: spans across the membrane ∙ Cell Membrane Structure: Mostly Lipid

o Fluid mosaic model: flexible, adaptive, and self


 Membrane is as viscous as olive oil

 Proteins move freely for various functions

 Phospholipids rotate and move laterally

 Self-sealing: HIV

∙ Cell Membrane Functions

o Selective permeability allows passage for and  

against some molecules.

o Contains ATP production enzymes: cellular respiration.

 Some have chromatophores, photosynthetic  pigments, on the folds of the membrane; no  

chromatophores = invagination for surface area. o Cell content leakage via by alcohols, quaternary  

ammonium (detergents), and polymyxin antibiotics. ∙ Antibiotics target: peptidoglycan, ribosomes, and the  


∙ Movement Across the Membrane

o Passive: high to low concentration; no energy  


 Simple diffusion: small molecules (e.g. O2)  travel until homeostasis or equilibrium is  


 Facilitated diffusion: ions/larger molecules use  a transporter protein; move with the  

concentration gradient.

 Osmosis: movement of water across the  

membrane from high water concentration (low  solute) to areas of low water concentration (high  


∙ Uses aquaporins to move through the lipid  


∙ Osmotic pressure: pressure needed to  

stop water movement across the membrane

(an increase in solute within – water follows  

solute – equals greater pressure).

o Isotonic solution: the solute  

concentration is equal inside and  

outside the cell; water is at equilibrium.

o Hypotonic: the solute concentration is

lower outside than within; water moves

into the cell.

o Hypertonic: the solute concentration  

is higher outside than within; water  

leaves the cell.

o Active: substances move from low to high  

concentration; energy expended.

 Active transport: requires a transport protein  

and ATP.

∙ Goes against the concentration gradient; the cell needs more of what it already has a lot  


 Group translocation: requires a transporter  protein and a phosphotransferase (phosph- = you

can get energy from it via the phosphate group) ∙ Substance is altered as it crosses the  


∙ Cytoplasm: not just a sac; the cell compartmentalizes o Specifically, the substance inside the membrane. o 80% water + proteins, carbs, lipids, and ions o Cytoskeleton: filaments (or microtubules for  


∙ Nucleoid: where the DNA is; no membrane or nucleus

o Bacterial chromosome: circular DNA (single, size  

varies) thread contains cell’s genetic information o Plasmids: extrachromosomal genetic elements (e.g.  

noncrucial genes like antibiotic resistance and toxin  


 Resistance can be accidentally passed on to  

pathogenic microbes during sex at the pili.  

∙ Ribosomes (70S): protein synthesis sites

o Made of protein and ribosomal RNA

o Svedberg units (S) are not additive – they are based on


 70S = 50S + 30S subunits

∙ Inclusions: structures within prokaryotes

o Metachromatic granules (volutin): polyphosphate  

stores glycogen

o Polysaccharide granules, lipid inclusions, and sulfur  

granules: energy reserves

o Carboxysomes: RuBisCo (most abundant protein in the body, autotrophic) enzyme, used in photosynthesis  

CO2 fixation

o Gas vacuoles: protein-covered cylinders maintain  


o Magnetosome: iron oxide (magnetic crystals)  

inclusions: destroys H2O2

∙ Endospores: produced by Bacillus and Clostridium.

o Resting, resistant cells of gram-positives for survival o Sporulation: six steps to formation

1. Spore septum isolates new DNA and part of  

the cytoplasm.

2. Plasma membrane surrounds DNA, cytoplasm,

and isolated membrane.

3. Spore septum surround isolated portion –  


4. Peptidoglycan layer forms between  


5. Spore coat forms.

6. Endospore is free from cell.

o Germination: the endospore returns to a vegetative  


∙ Eukaryotic Cell Structures 

o Cell Wall: found in plants, algae, and fungi

 Made of carbohydrates (plants – cellulose; fungi  

– chitin; yeasts – glucan and mannan)

o Flagella (long) and cilia (short) – proteins made of  


 Used for motility

 Made of microtubules made of tubulin,  

organized as nine pairs in a ring with two  

tubules in the center.

∙ Glycocalyx (animal cells): carbs bonded to proteins and  

lipids in the plasma membrane

∙ Plasma Membrane: similarities to prokaryotes o Phospholipid bilayer

o Integral and peripheral proteins

o Selective permeability

o Simple and facilitated diffusion, osmosis, and active  


o Differences from prokaryotes

 Sterols: complex lipids

 Carbohydrates: rigidity and recognition

 Endocytosis (white blood cells)

∙ Phagocytosis: pseudopods engulf  


∙ Pinocytosis: membrane folds in, vesicles  

around liquid

∙ Cytoplasm: inside the plasma, outside the nucleus o Cytosol: fluid portion

o Cytoskeleton: microfilaments and intermediate  

filaments – shape and support

o Cytoplasmic streaming: movement of cytoplasm  throughout he cell; how an amoeba moves (building  

and breaking up the cytoskeleton).

∙ Ribosomes: site of protein synthesis

o Eukaryotes: 80S (60S + 40S) – membrane-bound and free

 Has 70S ribosomes in chloroplasts and  


o Prokaryotes: 70S density

∙ Nucleus

o Nuclear envelope contains DNA

o Histones – chromatin protein complexes that stabilize


 Chromatin condenses into chromosomes

o Communication to the cytoplasm through pores and  


∙ Endoplasmic Reticulum (ER): transport network  

(Transmission EM) 

o Rough ER: studded with ribosomes, protein synthesis o Smooth ER: no ribosomes, synthesizes membranes,  

fats, and hormones

∙ Golgi Apparatus (GA): transport organelle (Transmission  


o Modifies ER proteins – changed from original mRNA  


o Transports proteins to the membrane via secretory  


o Cisternae: form secretory and transfer vesicles o Lysosomes: formed inside; contains digestive  


o Vacuoles (secretory vesicles): cavities in cell formed  from GA; brings food in (phagocytosis); provides  

shape and structure.

∙ Mitochondria – 70S ribosomes

o Double membrane, cellular respiration, ATP  production

o Inner folds (cristae) and fluid/material within (matrix)  Increases surface area for increases # of  


∙ Also in prokaryotes

∙ Chloroplasts – 70S ribosomes

o Double membrane, location of photosynthesis o Thylakoid: flattened membranes of chlorophyll  

(stacked = Granum)

∙ Other Organelles

o Peroxisomes: oxidize fatty acids, destroys hydrogen  


o Centrosomes: protein fibers and centrioles; mitotic  

spindle and chromosome segregation

∙ Eukaryote Evolution

o The earliest organism was prokaryotic.

o Life arose 3.5 – 4 billion years ago (byo).

o First eukaryotes: 2.5 byo

o Earth is the same age as the solar system: 4.54 byo o Universe is 13.8 byo.

∙ Origin of Eukaryotes – Endosymbiotic

o The early cell takes up 70S chloroplast (for  photosynthesis) from photosynthetic bacteria and  70S mitochondria (for respiration) from aerobic  


o Larger cell eats this smaller cell – forms the double  membrane.

 ∙    Lectures 5 & 6 

∙ The Study of Phylogenetic Relationships

o Taxonomy: science of classification – degree and  


o Phylogeny and systematics: study of evolutionary  


o 1735: Linnaeus – Kingdoms Plantae and Animalia. o 1800s: Nägeli – Bacteria and fungi (kingdom  


 Haeckel – Kingdom Protista proposed for  

bacteria, protozoa, algae, and fungi.

o 1937: The term “prokaryote” introduced to  

distinguish cells without a nucleus.

o 1968: Murray – Kingdom Prokaryotae

o 1969: Whittaker – five-kingdom system

o Early 1978: Carl Woese – 3 Domain System o No fossil evidence of prokaryotes

 3.5 byo structures of prokaryotes from South  


∙ Appearances deceive

 Stromatolites: cross section of fossilized  


∙ Three Domains – Carl Woese (1978)

o Based on nucleotide sequence in 16S rRNA;  

evolutionary chromometer.

o Eukarya: animals, plants, and fungi

o Bacteria

 Prokaryotic

 Peptidoglycan walls

 Membrane lipids: straight carbon chains linked  

to glycerol by ester linkage

 First AA in synthesis: Formylmethionine

o Archaea: methanogens, halophiles, and thermophiles  Prokaryotic

 Pseudomurein walls

 Membrane lipids: branched carbon chains  

attached to glycerol by ether linkage

 First AA in synthesis: Methionine

 No RNA loops or arms

o Recognize ester vs ether

o Phylogeny (Evidence of Endosymbiosis)

 Originated from infoldings of prokaryotes –  

double membrane

 Symbiosis: relationship between two organisms ∙ Bacteria within organism formed organelles

∙ Mitochondria has its own DNA that looks  

like a prokaryote’s (circular)

∙ Eukaryotic mitochondria and chloroplasts are 70S, have no

histones, and divide via binary fission

∙ Eukaryotic species: group of closely related organisms that

breed and produce non-sterile progeny.

∙ Prokaryotic species: population of cells with similar (not  

genetically identical) characteristics.

o Culture: bacteria grown in laboratory media  Mixed: more than one species

 Pure: one species (achieved with streak plates)

o Clone: populations of cells derived from a single  


o Strain: same species, but not the same parent cell. ∙ Archaea and Bacteria do not have Kingdom classifications. ∙ Virus classification: not part of any domain and not  

composed of cells; requires a host cell.

o Viral species: population of viruses with similar  

characteristics within a particular ecological niche. ∙ Methods of classifying and identifying microbes o Classification: placing organisms in groups of related  


o Identification: matching characteristics of an  unknown organism to lists of known organisms  

(clinical lab identification).

o Bergey’s Manual of Determinative Bacteriology  provides identification schemes for identifying  

species of Bacteria and Archaea.

o Approved Lists of Bacterial Names lists species of  known classification; peer review decides the names  

of bacteria.

o Transport media: used to collect and transport  

pathogens to a laboratory.

o Morphological characteristics: useful for  identifying eukaryotes and some prokaryotes; says  little about phylogenetic relationships.

o Differential staining: Gram staining, acid-fast  staining (e.g. for tuberculosis); not useful for wall-less

bacteria like mycoplasms.

o Biochemical tests: determines presence of  

bacterial enzymes.

o Polyphasic: a diverse approach to classification  

using multiple techniques.

∙ Biochemical tests: used to be difficult, now easy. o Rapid identification methods: several biochemical  

tests occur simultaneously using 20 tubes at a times,

with results assigned a number.

1. One tube with multiple tests.

2. Incubation with growth medium.

3. Assign each result its appropriate number.

4. Compare to a book of identification.

o Serology: science that studies serum and immune  

responses in serum

o Microorganisms are antigenic – they stimulate the  

body to form antibodies in the serum

o In an antiserum, a solution of antibodies is tested  

against an unknown bacterium

o Slide agglutination test: bacteria bind, or  agglutinate, when mixed with antibodies produced in  response to the bacteria. (Positive result =  


o Serological testing: differentiate between species  and strains – very specific.

o Enzyme-linked immunosorbent assay (ELISA):  Known antibodies and an unknown type of bacterium  are added to a well, where the reaction identifies the  

bacteria (via agglutination).

 Antibody is absorbed to the well. Sample is  added, antigen binds to antibody. Enzyme

linked antibody specific to the test antigen is  

added, binds to antigen and forms a sandwich.  Enzyme’s substrate is added, reaction produces  

a visible color change.

o Western blotting: identifies antibodies in a  

patient’s serum, confirms HIV infection.

 Super quick; for detecting Lyme disease; uses  lysing, gel, and electricity for separating  

proteins by weight; transfer to filter, add serum,  add antibodies; if it sticks, the bacteria is  


∙ Phage typing: virus that affects bacteria; tests for  determining which phages a bacterium is susceptible to;  specific; lysing and plagues (large clearings where the  

cells were killed) appear when the test is positive. ∙ Flow cytometry: uses differences in electrical  

conductivity between species or fluorescence or size.

∙ Fluorescence-activated cell sorter (FACS): separates  

cells that don’t react; two collection tubes.

∙ DNA base composition: separating and IDing by DNA;  guanine (G) pairs with cytosine (C); adenine (A) pairs with  thymine (T) (more common); two closely related  

organisms have similar amounts of various amounts. ∙ DNA fingerprinting: electrophoresis of restriction  

enzyme digests of an organism’s DNA; comparing  

fragment profiles to find patterns and similar cuts. ∙ Nucleic Acid Amplification Tests (NAATs): organism  

with few cells; use of PCR to amplify DNA (up to the entire  genome of a single cell) of an unknow microorganism that  

can’t be cultured

∙ Nucleic acid hybridization: measures the ability of DNA  strands from one organism to hybridize with DNA strands  of another organism; greater hybridization = greater  

degree of relatedness

o Southern blotting uses this and a DNA probe to  identify unknown organisms. Fluorescence = positive  


∙ DNA chips, or microarrays, contain DNA probes and  detect pathogens via hybridization between the probe and DNA, detected by fluorescence. Any gene; thousands of  bacteria at once.

o Manufactures thousands of single-stranded DNA  


o Fluorescent in situ hybridization (FISH): fluorescent  DNA or RNA probes stain the target microbes;  

determines identity, abundance, and relative activity. ∙ Putting classification methods together

o Node: similarity between rRNA sequences.

o Dichotomous keys (based on questions) and  cladograms (maps that show evolutionary  

relationships among organisms; based on rRNA  sequences; >/= 98% means same species).

 ∙    Lecture 7: Viruses 

∙ General characteristics of viruses

o Obligatory intracellular parasites (can’t grow alone) o Contain either DNA or RNA but never both; one  

nucleic acid.

o No ribosomes or ATP-generating mechanisms. ∙ Virus host range: spectrum of host cells a virus can  


o Most viruses infect only specific types of cells in one  


 Determined by attachment sites and receptors  20 nm to 1000 nm in length

∙ Bacteriophages: viruses that infect bacteria  ∙ Structure of viruses

o Virion: complete, fully developed viral particle  Nucleic acid: DNA or RNA can be single- or  

double-stranded; linear or circular.

 Capsid: protein coat made of capsomeres.

 Envelope: lipid, protein, and carbohydrate  coating on some viruses (generally a lipid  

bilayer from the host cell; not all virions have  


 Spikes: projections from the outer surface (like  


∙ Morphology of a nonenveloped polyhedral virus o Icosahedrons: Adenoviruses cause colds; 20  

equilateral triangle faces.

∙ General morphology

o Helical: hollow, cylindrical capsid

o Polyhedral: many-sided

o Enveloped: indefinite shape, surround by host  


∙ Morphology of complex viruses (phages)

o Most are made of proteins; act like syringes injecting  

DNA into the bacterial cell.

∙ Viral taxonomy

o Genus name: ends in -virus

o Family name: ends in -viridae

o Order name: ends in -ales

o Viral species: a group of viruses sharing the same  

genetic info and host, or what disease they cause  Descriptive common names for species;  

subspecies are designated by a number.

∙ Growing bacteriophages in the lab

o Viruses must be grown in living cells.

o Bacteriophages are grown in bacteria

 Form plaques on the surface of agar; each  

plaque corresponds to a single virus in plaque

forming units (PFUs).

∙ Growing animal viruses in the lab

o Complicated and time-consuming

o Done with living animals, embryonated eggs, and cell


∙ Cell cultures: tissue treated with enzymes, cells  suspended in culture medium, normal cells grow in a  single layer while transformed/continuous cell cultures  

grow in pile.

o Cancer cells don’t show contact inhibition. ∙ Viral identification

o Cytopathic effects (CPEs)

 Includes cell inclusions (e.g. Negri Bodies in  

the rabies virus)

 Syncytia: cell fusion; cells with many nuclei. o Serological tests

 Western blotting – reaction of the virus with  

antibodies (e.g. viral meningitis).

o Nucleic acids

 DNA or RNA and double- or single-stranded

∙ Viral multiplication

∙ Requires:

1. Must invade a host cell

2. Must take over the host’s metabolic machinery ∙ Two general tasks:

3. Replicate nucleic acid

4. Synthesize proteins for the capsid (DNA to mRNA to  protein)

∙ Viral one-step growth curve

o Bacteriophage enters the host and the eclipse  


o Virions are released from the host, triggering acute  

infection and peaking at the spike.

o Host dies.

∙ Bacteriophage multiplication

o Lytic cycle: Phage causes lysis and host cell death. o Lysogenic cycle: Phage DNA incorporated in the host  


 Phage conversion

 Specialized transduction

∙ T-even bacteriophages: the lytic cycle

o Attachment: phage attaches with tail fibers o Penetration: phage lysozyme opens the cell wall;  

tail sheath contracts to force the tail core and DNA  

into the cell

o Biosynthesis: production of phage DNA and proteins o Maturation: assembly of phage particles

o Release: phage lysozyme breaks the cell wall ∙ Bacteriophage Lambda (λ): The Lysogenic Cycle o Lysogeny: phage remains latent

o Phage DNA incorporates into host cell DNA

o Prophage: inserted phage DNA

o When the host cell replicates its chromosome, it also  

replicates prophage DNA – phage conversion ∙

 ∙    Lecture 8: Metabolism 

∙   Metabolism: the buildup and breakdown of nutrients o Chemical reactions provide energy and create life

sustaining substances.

o Catabolism: break down of complex molecules;  


o Anabolism: build-up of complex molecules;  


o Metabolic pathways: enzymatically catalyzed  chemical reaction sequences (determined by  

enzymes, encoded by genes).

∙   Collision theory: chemical reactions occur when atoms,  

ions, and molecules collide.

o Activation energy: collision energy required for a  

chemical reaction.

o Reaction rate: frequency of collisions containing  enough energy to bring about a reaction (increased  by enzymes or increased temperature, pressure, or  


∙   Catalysts: speed up chemical reactions without  


∙   Enzymes: bio-catalysts (specific substrate, lowers

activation energy)

o Substrate + enzyme’s active site = enzyme

substrate complex

o Substrate is transformed into products, enzyme is  


o Turnover number: number of substrates an enzyme

converts to a product per second (1 to 10,000) o Named by adding -ase to the end; grouped based on  


 ∙    Enzyme components 

o Apoenzyme: protein portion

o Cofactor: nonprotein component (metal ion)  Coenzyme: organic cofactor

o Holoenzyme = apoenzyme + cofactor

o Electron carriers assist enzymes.

∙   Factors of Enzyme Activity

o Extreme temperature and pH = denatured proteins o High substrate concentration (saturation) = enzyme

catalyzes at its max. rate

o Higher temperature and pH are good, but only up to  

a certain point (35-40 degrees Fahrenheit, 5 pH) ∙   Inhibitors 

o Competitive inhibitors: fill the active site of an  

enzyme, competes with the substrate.

o Noncompetitive inhibitors: interact with the  allosteric site (somewhere other than the active  

site) in allosteric inhibition.

o Feedback inhibition: a reaction’s end-product  allosterically inhibits the enzyme that made it;  

prevents waste of energy.

∙   Ribozymes: RNA that acts as catalysts by cutting and  

splicing RNA.

∙   OIL-RIG: Oxidation is Loss (of electrons), Reduction is  

Gain (of electrons)

o Redox reaction: oxidation paired with reduction o Dehydrogenation: biological oxidation

∙   Generation of ATP 

o 3 Main Pathways

 Fermentation

 Respiration

 Photosynthesis

o 3 Main Biochemical Mechanisms

 Substrate-Level Phosphorylation: Glycolysis ∙   High-energy phosphate is added to ADP  

forming ATP.


 Oxidative Phosphorylation

∙   Electrons are transferred from one electron

carrier to another in the electron  

transport chain (ETC) system, releasing

energy to generate ATP.

 Photophosphorylation: only in light-trapping  

photosynthetic cells

∙   Light converted to ATP when chlorophyll  

transfers electrons (oxidation) through an  


∙   Prokaryotes: no oxygen produced, one  


∙   Plants & Cyanobacteria: oxygen produced,  

two photosystems.

 ∙    Lecture 9: Microbial Growth

∙ Micronutrients are required for microbial growth, but you

don’t need much of them.

o Organic: vitamins, amino acids, purines, and  


 If you have to add these, it means the body  

doesn’t make it itself.

o Inorganic (trace) elements: required in small  

amounts, usually as enzyme cofactors (e.g. metal)  Includes iron, copper, molybdenum, zinc, etc. ∙ The Effects of Oxygen on Growth: Recognize Growth  


o Obligate aerobe: requires oxygen, grows near the  


o Facultative anaerobe: grows via  

fermentation/anaerobic respiration when oxygen isn’t available but prefers to have oxygen (enhanced  

growth near the top).

o Obligate anaerobes: can’t use and are harmed by  

oxygen (e.g. Clostridium tetani and botulin).

o Aerotolerant anaerobes: tolerate but can’t use  

oxygen – no enhanced growth (fermenters).

o Microaerophiles: require oxygen concentration  lower than air – hardest to grow.

∙ Toxic Byproducts of O2 Respiration

o Single oxygen (1O2-): charged, reactive

o Superoxide radicals: O2-

 Toxic thieves that convert normal molecules to  


 Neutralized to water and oxygen by SOD

o Peroxide anion: O22-

 Toxic, destroyed to water by…

∙ Catalase (bubbles)

∙ Peroxidase

o Hydroxyl radical: OH – ionizing radiation

 Attacks DNA

∙ Biofilms: non-pure microbial communities that form  slime/hydrogels (similar to glycocalyx) to adhere to  


o Quorum sensing: bacteria communicate through  the biofilm cell-to-cell; uses chemical called an  


o Share nutrients

o Protection: shelters bacteria from the environment;  1,000x resistant to microbiocides; more resistant to  


o Found in digestive and sewage treatment system;  

cleans water but can also clog pipes.

o Involved in 70% of infections, usually involving  implanted devices (e.g. catheters, heart valves,  

contact lenses).

∙ Culture Media

o Liquid: broth or broth culture.

o Solid/semi-solid: agar

 Complex polysaccharide used as a solidifying  

agent for culture media

 Not metabolized by microbes

 Liquifies at 100°C; solidifies at 40°C 

o Chemically-defined media: the exact chemical  

composition is known. 

 Fastidious organisms that require many  

growth factors.

o Complex media: chemical compositive varies, uses  extracts and digest of plants or meat (e.g. nutrient  

broth or agar). 

∙ Composition of Mediums

o Chemically Defined: Chemoheterotroph

∙ Constituent

∙ Amount

∙ Glucose (C source)

∙ 5g

∙ Ammonium phosphate, monobasic  (nitrogen for protein)

∙ 1g

∙ Sodium chloride

∙ 5g

∙ Magnesium sulfate (for DNA)

∙ .2g

∙ Potassium phosphate, dibasic (for  phosphates and buffer to prevent end product poisoning)

∙ 1g

∙ Water

∙ 1L – always  add 1L of water

o Defined: Leuconostoc mesenteroides

∙ Carbon/Energy

∙ Glucose

∙ Salts

∙ e.g. NaCl

∙ Amino Acids

∙ e.g. alanine, glycine

∙ Purines and


∙ e.g. adenine, uracil

∙ Vitamins

∙ e.g. folate

∙ Trace Elements

∙ Fe, Co, Mn, etc.

∙ Buffer, pH 7

∙ Sodium acetate

∙ 1 L Distilled


∙ Also serves as


o Complex for heterotrophic

∙ Constituent

∙ Amount

∙ Peptone (N, S, and  C)

∙ 5g

∙ Beef extract (N, S,  and C)

∙ 3g

∙ Sodium chloride

∙ 8g

∙ Agar (solidifying  agent)

∙ 15g

∙ Water

∙ 1L

∙ Anaerobic Growth

o Reducing media: removes oxygen and reduces  


 Cultivates anaerobic bacteria

 Contains chemicals (sodium thioglycolate) that  combine with O2 to deplete it (antioxidants,  

reducing agents)

 Heated: drives off O2

 In an anaerobic jar, the catalyst palladium  

combines hydrogen and oxygen to form water. A colorless methylene blue indicator means  

the environment is anaerobic.

∙ Special Culture Techniques

o Capnophiles: microbes that require lots of CO2;  

candle jar or CO2 packet.

∙ Selective Media

o Suppress unwanted microbes, encourage desired  microbes.

o Contains inhibitors to suppress growth

∙ Differential Media

o Distinguishes colonies of different microbes on the  

same plate by measuring reactions.

∙ Selective and Differential: MacConkey’s Agar o Selective: crystal violet and bile salts only grow  


o Differential: shows which ones ferment lactose (pink)  

and which ones don’t (colorless).

∙ Selective and Differential: Mannitol-Salt Agar o Selective: high salt grows only some microbes. o Differential: ferments mannitol (yellow), doesn’t  

ferment mannitol (phenol red stays red).

∙ Differential: Blood Agar

o Tests for virulence, not species; pathogenic = uses  


o Hemolysis:

 Alpha: incomplete destruction of RBCs – E. coli  Beta: complete destruction of RBCs – S.  


 Gamma: no hemolysis at all

∙ Enrichment Culture

o Encourages the growth of a desired microbe by  increasing small numbers to detectable levels; first,  

enhance; second, inhibit; detects salmonella.

∙ Culture Media Summary

∙ Biosafety Levels

o BSL-1: no special precautions, basic teaching labs.

o BSL-2: lab coat, gloves, eye protection.

o BSL-3: biosafety cabinets prevent airborne  


o BSL-4: sealed, negative pressure, hot zone –  

exhaust air is filtered twice, sterilize with UV light. ∙ Obtaining Pure Cultures

o Contains only one species/strain

o Colony: population of cells arising from single  


 AKA colony-forming unit (CFU) with viable  

cell count.

o Streak plate method: used to isolate CFUs. ∙ Preserving Bacterial Cultures (Theoretically Suspended  


o Deep-freezing: -50 to -95 Celsius (generally -80C). o Lyophilization (freeze-drying): frozen (-54 to  

-72C) and dehydrated in a vacuum.

∙ Bacterial Growth (Division)

o Increase in number not size

o Binary fission: 1 cell = 2 cells

o Budding: the parent cell is bigger than its single  


o Conidiospores (actinomyocyetes)

o Fragmentation of filaments: mold

∙ Generation Time 

o Time required for a cell to divide (20 mins to 24  


o Binary fission doubles the number of cells each  


o Total number of cells = 2number of generations 

 e.g. if you are five generations out, the total  number of cells will be 25 = 32, log1032 =1.51

 Take the log10 of the total number for your  


∙ Growth curves are represented  

logarithmically to make the lines linear and  


∙ Phases of Growth (Know the Growth Curve)

1. Lag phase: not growing – shocked/had adapted to  

old culture

2. Log phase: linear upward

3. Stationary phase: run out of nutrients, slow down  

and stop growing b/c dividing = dying.

4. Death phase: population declines due to decrease  

in nutrients or increase in toxins; difficult to place.  ∙ Direct Measurement

1. Direct microscopic count

 Volume of a bacterial suspension is placed on  

a slide

 Average number of bacteria per viewing field  Petroff-Hausser bacterial cell counter (grid  

squares of diluted microbes)

 Total microbes = number counted / volume of  


 Depends on dilution (for decreasing  

cloudiness or killing motile microbes) and  


 Disadvantage: viable (living) cells aren’t  


2. Plate count: determines living cell number  Dilute the original inoculum via serial  


o Need number of 30-300 CFUs to give a  

statistically significant count.

o Find dilution (D): D = amount added /  

(amount added + volume of tube)

 Total dilution = D x D x D x …

o Find concentration of CFUs (C): C = #  colonies x inverse dilution; for two or  

more tubes, [#colonies (1) + #colonies  

(2)] / 2 (x inv. dil.)

o Count colonies: going forward, multiply  your starting tube’s colonies by its  

dilution; going backward, divide the  

starting tube’s colonies by its dilution.

 Bacteria are mixed into an agar dish (pour  plate method) or spread on a plate surface  

(spread plate method).

3. Filtration: metallic sheen, differential and selective.  Solution passed through a filter that collects  


 Pore size = 0.22 micrometers 

 Filter is transferred to a Petri dish; grows CFUs

on the surface. Used for water and coliforms. o Coliforms: aerobic/facultative anaerobic,  

Gram-negative, non-spore-forming rod-

shaped bacteria that ferment lactose  

with gas formation within 48 hours at  


 E.g. Enterobacteriacea found in the  


4. Most probable number (MPN) method

 Multiple tubes: dilution through decreasing  

amounts of inoculum.

 Count the positive tubes and compare with a  statistical table (goal is to have more  

negative tubes) to find number of viable cells.

∙ Indirect Measurement: Growth Curves, Not CFUs 1) Turbidity: measurement of cloudiness with a  


 Optical density (OD) increases with an  

increase in time and light. Increased OD  

means a decrease in light.

 A blank is set at 100, allowing all light, as the  


2) Metabolic activity: amount of metabolic product  

proportional to number of bacteria.

3) Dry weight: filtered, dried, and weighed  

(filamentous organisms which grow in length).

 ∙    Lecture 10: Control of Microbial Growth 

∙ Microbial Control 

o Sepsis: bacterial contamination (usually in blood).

o Asepsis: absence of significant contamination (e.g.  

aseptic technique).

o Sterilization: removing and destroying all microbial  

life (including spores).

o Commercial sterilization: killing C. botulinum 

endospores in canned goods.

o Disinfection: destroying harmful organisms on inanimate objects – not too worried about toxicity.  

Not sterilization.

o Antisepsis: destroying harmful microbes from living  

tissue – worry about toxicity. Not sterilization.

o Degerming: the removal of microbes from a limited  

area (e.g. washing hands).

o Sanitization: lowering microbial counts on eating  


o Biocide (germicide): treatments that kill microbes  Bacteriocidal – applies to antibiotic drugs and  


 Fungicide – applies to fungi.

o Bacteriostasis: inhibits growth in microbes  Bacteriostatic – applies to antibiotic drugs and  


 Fungistasis – applies to fungi.

∙ Rate of Microbial Death: The Microbial Death Curve o When microbes die, it’s not all at once. Occurs  


o One log decrease = 90% of population killed o Treatment effectiveness depends on:

1) Number of microbes (closer to zero is best)

2) Environment (organic matter – increased  

amount = increased difficulty; temperature;  

biofilms = resistant)

3) Time of exposure

4) Microbial characteristics

o Gram positive versus Gram negative –  

Gram-positives are harder to kill. 

o Spore formation

∙ Microbial Control Agents

o Alteration of membrane permeability

o Damage to proteins (enzymes – denaturation) o Damage to nucleic acids (radiation)

∙ Heat: denatures enzymes

o Thermal death point (TDP): lowest temp. at which  

all cells in liquid culture are killed in 10 min.

o Thermal death time (TDT): minimal time for all  bacteria in liquid culture to be killed at a particular  


o Decimal reduction time (DRT): minutes to kill 90%

of population at a given temp.

∙ Moist Heat Sterilization

o Moist heat (boiling, free-flowing steam) denatures  


o Autoclave: steam under pressure contacts the  item’s surface; heat, steam, and pressure in a double


o 121C at 15 psi for 15 min must reach the core of  

the item.

o Kills everything; larger item = larger sterilization  time.

o Test strips indicate sterility: black = positive. ∙ Heat to Reduce Numbers of Organisms

o Pasteurization: reduce spoilage and pathogens, but  

can affect texture/taste.

o Equivalent treatment: 63C for 30min

 High-temp short-time (HTST): 72C for 15 sec  Ultra-high-temp (UHT): 140C for 4 sec

o Thermoduric: survive; don’t cause disease. ∙ Dry Heat Sterilization

o No autoclave; kills by oxidation.

 Flaming, incineration, hot-air sterilization in  

oven, etc.

 Either 160C for 2 hours or 170C for 1 hour.

 Instruments should be dry since water  


∙ Filtration: no dry heat; use for proteins, insulin. o Passage of substance through a screen-like material. o Used for heat-sensitive liquids.

o High-efficiency particulate air (HEPA) filters

remove microbes (>0.3 micrometer pores).

o Membrane filters remove microbes (>.22  


∙ Physical Microbial Control

o Low-temp = bacteriostatic effect

 Refrigeration, deep-freezing, and lyophilization. o High pressure denatures proteins

o Desiccation: absence of water prevents metabolism  

(decreases AW).

o Osmotic pressure uses salts/sugars to create  hypertonic environment; causes plasmolysis  

(decreases AW).

∙ Radiation

o Ionizing (X-rays, gamma rays, e-beams): ionizes  water to create reactive hydroxyl radicals; damages  

DNA by causing lethal mutations.

o Nonionizing (UV light): damages by creating  

thymine dimers.

o Microwaves: kills by heat, not very antimicrobial. o Radiant Energy Spectrum: increase the  

wavelength, decrease the energy.

∙ Effective Disinfection: concentration of disinfectant,  

amount of organic matter, pH, and time.

∙ Use-Dilution Tests

o Metal cylinders are dipped in test bacteria and dried. o Cylinders are disinfected for 10 minutes at 20C. o Cylinders are transferred to culture media to  

determine if the bacteria survived.

∙ The Disk Diffusion Method

o Evaluates efficacy of chemical agents to kill bacteria o Filter paper disks are soaked in a chemical, placed on

a culture

o That concentration of disinfectant works if there is a  

zone of inhibition.

 Undiluted works very well but has toxic residue. ∙ Phenol and Phenolics

o Injures lipids of membranes, causes leakage.

o Joseph Lister used phenol/carbolic acid in aseptic  

surgery which created phenol toxicity.

o Phenol was modified to be O-phenylphenol to be less  


o Recognize: 

∙ Bisphenols: two phenol groups connected by a bridge. o E.g. hexachlorophene and triclosan – disrupt plasma  

membranes and don’t burn people.

∙ Biguanides: antiseptic and disinfectant; pre-surgery;  

disrupts plasma membrane.

∙ Halogens (Disinfectants and Antiseptics)

o Iodine (tincture, iodophor): impairs protein  

synthesis, alters membranes.

o Chlorine (bleach, chloramine) : ozidizing agents shut

down cellular enzyme systems; WWI.

∙ Alcohols: denature proteins, dissolve lipids o No effect on endospores and nonenveloped viruses o Require water – 100% doesn’t inhibit growth, prefer  


 Ethanol and isopropanol

∙ Heavy Metals: nasty; be careful with the concentration.

o Oligodynamic action/effect: very small amounts  

exert antimicrobial activity.

o Denatures proteins.

o Silver nitrate (ophthalmia neonatorum), mercuric  chloride (milder in paint), copper sulfate (algicide),  

zinc chloride (mouthwash).

∙ Surface-Active Agents: removes oils (e.g. acne); just  


o Soap: degerming.

o Acid-anionic sanitizers: anions react with the  


o Quaternary ammonium compounds: toxic;  

bactericidal, denature proteins, disrupt membranes. o Recognize 

∙ Chemical Food Preservatives

o Sulfur dioxide prevents wine spoilage.

o Organic acids inhibit metabolism and prevent molds  

in acidic foods.

o Nitrites and nitrates prevent endospore germination,  

adds red color to meats.

∙ Antibiotics: bacteria that produce compounds to inhibit  

other bacteria

o Bacteriocins: proteins that inhibit a bacterium (e.g.  

nisin and natamycin – prevent cheese spoilage). ∙ Aldehydes: inactivate proteins by cross-linking with  

functional groups (e.g. -NH2)

o Used to preserve specimens and medical  equipment.

 Formaldehyde and glutaraldehyde (toxic  

fixatives that preserve tissues).

∙ Chemical Sterilization: Gaseous sterilants cause  alkylation (hydrogen atoms are replaced with a free  


o Cross-links nucleic acids and proteins

o “Cold” systems: used for heat-sensitive material  Ethylene oxide and formaldehyde

∙ Plasma: fourth state of matter consisting of electrically  

excited gas.

o Free radicals destroy microbes; used for hands and  

tubular instruments.

∙ Supercritical Fluids (SCF): CO2 with gaseous and liquid  


o Any substance at a temperature and pressure above  its critical point where liquid and gas phases do not  


o Effuses through solids like a gas, dissolves material  

like a liquid.

o Cleans medical implants

∙ Peroxygens and Others

o Oxidizing agents used for contaminated surfaces and

food packaging.

 O3 (Ozone) and H2O2 (hydrogen peroxide)

 Peracetic acid 

∙ Depending on what we sterilize, our methods change. For  instance, glutaraldehyde is fair against endospores, but  good against mycobacteria (which is acid fast).  ∙    Lecture 11: Genetics 

∙   Big Picture

o Genetics: science of heredity

o Central dogma of molecular biology:

 DNA -> mRNA -> protein -> function 

o Mutations drive evolution (mutated DNA = altered  


o Gene expression is controlled by operons (e.g.  

differentiates kidney from blood cell.)

o Sex: exchange and recombination of genetic  


∙   Types of Mutations

o Base-substitution mutation: a single DNA base  

pair is altered.

o Frame-shift mutation: DNA base pairs are added or


∙   Importance of Microbial Genetics

o Alteration of genes and gene expression in bacteria  


 Find causes of diseases.

 Prevent/treat disease (gene therapy).

 Manipulate them for human benefit (insulin and  

E. coli).

∙   Structure and Function of Genetical Material o Genetics: the study of genes, how they carry and  

express information, and how genes are replicated.

o Chromosomes: structures containing DNA that carry hereditary info; containing genes and non-coding  


o Genes: self-contained segments of DNA that encode  


o Genome: all the genetic info in a cell (prokaryote =  


o Genetic code: set of rules determines how a  

nucleotide sequence is converted to proteins.

∙   Genotype: genetic makeup of an organism (not  


∙   Phenotype: expression of a gene that can be observed as

a trait.

∙   DNA and Chromosomes

o Bacteria: singular chromosome made of DNA and  


o Eukaryotes: >1 chromosome and histones

o Chromosomes contain genes and short tandem  

repeats (STRs)

 STRs: short repeating sequences of noncoding  


∙   Small and specific; forensics.

∙   Flow of Genetic Information

o Vertical gene transfer: flow of genetic info from  one generation to the next with daughter cells and  

replication (eukaryotes).

o Horizontal gene transfer: flow of genetic info  

without cell division (bacteria).

∙   DNA Replication

o Describe DNA:

 Double helix

 Deoxyribose-phosphate backbone

 Nucleotides are held together by H-bonds  between A-T and C-G (requires more energy to  


 Antiparallel strands (5’ 3’, 3’ 5’)

 Sense and antisense strands

o The order of nitrogen-containing bases forms the  

genetic instructions of the organism.

o One strand is the template for the production of a  second – original strands are relaxed by  topoisomerase and gyrase (H-bonds are relaxed,  

and the activation energy is lowered).

o Helicase separates the strands, and a replication fork is created – bacterial DNA replication occurs at  

both forks.

o DNA polymerase (aka DNA-dependent DNA  


 Adds nucleotides to the growing strand. ∙   ALWAYS in the 5’ -> 3’ direction

∙   Initiated by an RNA primer

∙   Leading strand: continuous synthesis ∙   Lagging strand: discontinuous synthesis o Okazaki fragments

 Removes RNA primers.

 Uses DNA ligase to join Okazaki fragments  


o Energy for DNA replication: hydrolysis of two  phosphate groups (pyrophosphate).

o Semiconservative replication: each offspring cell  

receives one strand of the original.

o Proofreading: DNA polymerase makes replication  

highly accurate. 


∙   Transcription: RNA Synthesis

o RNA: single stranded nucleotide with 5-carbon ribose  


 Uracil (U) instead of thymine (T); A-U and G-C. o Transcription: DNA -> mRNA

 Sense strand: 3’ to 5’, not transcribed into  mRNA, contains codons, same nucleotide  

sequence as mRNA.

 Antisense strand: 5’ to 3’, transcriber to  

mRNA, anticodons.

1) Ribosomal RNA (rRNA): part of ribosomal  


2) Transfer RNA (tRNA): transports amino acids  

during protein synthesis.

3) Messenger RNA (mRNA): carries coded info from

DNA to ribosomes.

o Prokaryotes Transcription

 Antisense strand: template for mRNA

 Begins when RNA polymerase (DNA-dependent  RNA polymerase) binds to the promoter  

sequence on DNA.

 Proceeds in 5’ -> 3’ direction.

 Stops when it reaches the DNA terminator  


 Operon: unit of DNA with >1 gene.

∙   Translation: Protein Synthesis

o Translation: mRNA -> protein

o mRNA is translate into protein language; codons are  groups of three mRNA nucleotides that code for an  

amino acid.

o 61 sense codons encode the 20 essential amino  


o Genetic code: degeneracy means many amino acids are coded by several codons (the 3rd position is

flexible enough to enable silent mutations, which is a base pair transfer that results in the same amino  


o Begins at a start codon: AUG (Methionine)

o Ends at nonsense codons: UAA, UAG, UGA

o Codons are read sequentially

o tRNA transport amino acids to the ribosome to make  

polypeptide chains (joined by peptide bonds).

 An anticodon base-pairs with the codons  


∙   Process of Translation

1) AUG starts translation; peptide bond begins at P  


2) A site: acceptor site that attaches the next amino  

acid. E site: where the tRNA exits.

3) The tRNA is released and the ribosome moves along  

the mRNA.

4) UAG stops translation. The last tRNA is released, the  ribosome comes apart, and the polypeptide forms a  

new protein.

∙   In prokaryotes, translation can begin before transcription is completed, and multiple transcriptions can occur  

simultaneously; no wasting; efficient.

∙   In eukaryotes, transcription occurs in the nucleus, and  

translation occurs in the cytoplasm.

o Exons: regions that code for proteins

o Introns: DNA regions that don’t code for proteins. o Small nuclear ribonucleoproteins (snRNPs)  

remove introns to splice exons together.

∙   Bacterial Gene Expression Regulation

o Carefully controlled so energy is not wasted: gene  


 Constitutive genes: expressed at a constant,  

fixed rate.

 Others: expressed only as needed: inducible,  repressible, and catabolite repression –  

eukaryotes will not make more of what it’s given

in a medium.

∙   Pre-transcriptional Control

o Repression: inhibits transcription using repressors  Default position is on. 

o Induction: initiates transcription using inducers  Default position is off. 

∙   Operon Model

o Promoter: segment of operon where RNA  

polymerase initiates transcription of structural genes. o Operator: segment of DNA that controls  

transcription of structural genes.

o Operon: group of operatory and promoter sites and  

the structural genes they control

o Polycistronic mRNA: eukaryotes have mRNA for  

every gene.

o Inducible operon: structural gene transcription  does not occur until an inducer is present, the lac  


 Repressor active, operon off.

∙   The repressor protein binds to the operator

and prevents transcription from the  


 Repressor inactive, operon on.

∙   The inducer binds to the repressor protein  

and structural genes are transcribed,  

producing enzymes needed for lactose  


o Repressible operon: structural gene transcription  

occurs until a repressor is present, the trp operon.  Repressor inactive, operon on.

∙   Transcription and translation proceed,  

leading to tryptophan synthesis.

 Repressor active, operon off.

∙   The corepressor tryptophan bines to the  

repressor protein and binds the operator  

and prevents transcription.

∙   Positive Regulation

o Catabolite repression inhibits cells from using  

anything other than glucose as their carbon source. o Cyclic AMP (cAMP) builds up when glucose is not  


 cAMP binds to the lac promoter, initiating  transcription during a lag time and allowing the  

cell to use lactose.

∙   Epigenetic Control

o Epigenetics: heritable changes in gene  


o Methylating nucleotides turn genes off.

o Methylated (off) genes are passed to offspring. o Not permanent, reversible through specific enzymes. ∙   Post-Transcriptional Control (zero waste in  


o MicroRNAs (miRNAs) base pair with mRNA to make  

it double-stranded.

o Double-stranded RNA is enzymatically destroyed. o Some mRNA is recycled for nutrients.

∙   Changes in Genetic Material

o Mutation: a permanent change in the DNA base  


 Can be neutral, harmful, or beneficial.

o Mutagens: agents that cause mutations (e.g.  


o Spontaneous mutations: occur in absence of  

mutagens (increased with age).

∙   Types of Mutations (Part 2)

1) Base substitution: change in one base in DNA,  silent mutation doesn’t always change the amino  


2) Missense mutation: base substitution results in a


3) Nonsense mutation: base substitution results in  

a nonsense, or stop, codon.

4) Frameshift mutation: insertion/deletion of one or more nucleotide pairs shifts the translational  

reading frame.

∙   Chemical Mutagens

o Nitrous acid: causes adenine to bind with cytosine  

instead of thymine (A-C not A-T).

o Nucleoside analog: incorporates into DNA instead  

of a normal base, causing mistakes in pairing.

o Oxidation of a nucleotides create a mutagen. ∙   Raditation

o Ionizing radiation (X-rays and the sun’s gamma rays)  destroy DNA by forming ions that oxidize nucleotides  

and break deoxyribose-phosphate backbones.

o UV radiation causes thymine dimers (2 thymines bind on the same strand); know DNA ligase joins old and  

new DNA.

o Lethal mutation = cell death.

∙   Mutation Frequency

o Spontaneous mutation rate: one in a billion replicated

base pairs or one in a million replicated genes. o Mutagens increase the mutation rate to 10-5 or 10-3 

per replicated gene.

∙   Mutation Identification

o Positive (direct) selection: mutant cells grow or  

appear different from the normal cells.

o Negative (indirect) selection: mutant cells cannot  

grow or perform a certain function.

o Auxtotroph: mutant that has a nutritional  

requirement absent in the parent (replica plating). ∙   Identifying Chemical Carcinogens

o The Ames test: exposes mutant bacteria to  mutagenic substances to measure the rate of

reversal (indicates how mutagenic a substance is) –  

spontaneous mutation.

∙   Genetic Transfer and Recombination

o Genetic recombination: exchange of genes  between two DNA molecules; creates genetic  


o Crossing over: two chromosomes break and rejoin,  

resulting in foreign DNA insertion to the chromosome. o RecA protein catalyzes the joining of two strands. o Vertical gene transfer: genes from an organism to its  


o Horizontal gene transfer: genes between cells of the  

same generation (no daughter cells)

 Responsible for antibiotic resistance

 1) Transduction 

 2) Transformation: genes are taken up by one  

bacterium from another as naked DNA.

 a. Competent cells can transform/uptake

DNA – usually nonencapsulated.

 3) Conjugation: horizontal gene transfer.

∙   Conjugation in Bacteria

o Conjugation: plasmids transferred from one  

bacterium to another.

o Requires cell-to-cell contact via sex (F) pili.

o The F pilus pulls cells together for the mating bridge  

(not reproduction, just DNA transfer).

 Donor = F+

∙   Carries the plasmid ( Fertility factor) and  

make the F pilus.

 Recipient = F

o Mating F+ and F- = 2 F+ cells

o Hfr cells contain the F factor on their chromosomes o When an Hfr donor mates with an F- cell, the  

recombinant F- cell gets DNA from Hfr cells, but  

doesn’t have enough F factor.

∙ Plasmids: self-replicating, extrachromosomal, circular DNA. ∙ 1-5% the size of a bacterial chromosome (pretty  

small, but megaplasmids exist).

∙ Code for proteins that enhance a bacterium’s  


o Conjugative plasmid: F-factor; carries genes for  

sex pili and transfer of the plasmid.

o Dissimilation plasmid: encodes enzymes for the  catabolism of unusual compounds (e.g. certain  

sugars, hydrocarbons).

o Resistance factor: R-factor; multiple genes for  

antibiotic resistance.

o Copy number: number of copies of a specific  

plasmid per cell.

o Transposons, or jumping genes, are DNA segments that move from one region of DNA to another and may or may  

not replicate.

o Insertion sequences (IS) code for transposase that cut and reseal DNA (does this by itself, no other  


∙ Complex transposons can carry other genes  

(antibiotic resistance).

∙ BIG IDEA: The IS is the simplest transposon, and it  carries the transposase gene, an enzyme that  catalyzes transposition (horizontal gene transfer; a  chromosomal segment is transferred to a new  position on the same or another chromosome). The  transposase gene is bound at both ends by inverted  

repeat sequences, or transposon recognition sites. o Genes and Evolution: mutations and recombination create  

cell diversity; natural selection acts of populations to  ensure the survival of the fittest (e.g. antibiotic  resistance).

 ∙    Lecture 12: Principles of Disease 

o Pathology: the study of a disease.

o Etiology: the cause of a disease.

o Pathogenesis: the development of a disease. o Infection: the invasion or colonization by pathogens in  

the body.

o Disease: an abnormal state where the body is not  

performing normal functions.

o Normal microbiota: microbes that permanently colonize  the host and do not cause disease normally (first line of  


o Transient microbiota: not necessarily harmful;  present for days, weeks, or months.

o Human Microbiome Project: analyzes the  relationship of the microbial communities between  

human health and the body.

o Distribution and Composition Factors of the Human  


∙ Nutrients (in the gut and on the skin)

∙ Physical/chemical factors (lysosomes of saliva and  

tears inhibit microbes)

∙ Host defenses

∙ Mechanical factors (same as physical)

o The skin, eyes, and nose/throat regions are made up  of many different types of microbes. Keratin, low pH,  

tears, mucus, and cilia help inhibit pathogens. o Relationships Between the Host and the Normal Microbiota o Microbial antagonism (competitive exclusion):  

competition between microbes.

o Symbiosis: relationship between normal microbiota  and host; varies over time depending on the  


 Commensalism: one organism benefits, the  

other is unaffected.

 Mutualism: both organisms benefit (normal  


 Parasitism: one organism benefits at the  

expense of the other (all viruses).

o Opportunistic pathogens: the microbe goes where

it’s not supposed to be.

o Normal microbiota protects the host via:

o Competing for nutrients (e.g. a large amount of E.  coli in our microbiome protects against food  


o Producing substances harmful to invaders

o Affecting pH and O2 (sweat = acidic skin = dead  

microbes, low oxygen = no competition)

o Koch’s Postulates: used to prove the cause of an  

infectious disease.

1. The same pathogen must be present in every case of

the disease.

2. The pathogen must be isolated from the diseased  

host and grown in pure culture.

3. The pathogen from the pure culture must cause the  disease when it's inoculated into a healthy,  

susceptible laboratory animal.

4. The pathogen must be isolated from the inoculated  animal and must be shown to be the original  


o Koch’s Postulates: Problems

o Some pathogens cause several diseases.

o Some pathogens cause disease only in humans. o Some microbes have never been cultured.

o Classifying Infectious Diseases

o Symptoms: changes in the body felt due to the  

disease (pain).

o Signs: changes in the body that can be measured  

(qualitative/quantitative) due to the disease.

o Syndrome: a specific group of signs and symptoms  for a disease.

o Communicable disease: a disease that is spread 

from one host to another

o Contagious diseases: easily and rapidly spread. o Noncommunicable disease: a disease that is not  


o Disease Occurrence

o Incidence: number of people who develop a disease  

during a specific time period (only new cases). o Prevalence: number of people who develop a  

disease at a specified time, no matter when it first  

appeared (new and old).

o Sporadic disease: disease that only occurs  

occasionally (typhoid fever).

o Endemic disease: disease is constantly present  

(common cold).

o Epidemic disease: disease acquired by many  

people in a given area in a short time (influenza). o Pandemic disease: worldwide epidemic (AIDS). o AIDS data: education and data (expansion of surveillance)  

increased the number of cases, followed by a decline in  number due to treatment and education. It is believed we  

know Patient X.

o Severity/Duration of a Disease

o Acute disease: symptoms develop rapidly, disease  

is short.

o Chronic disease: symptoms develop slowly, last a  long time.

o Subacute disease: intermediate between acute and


o Latent disease: causative agent is inactive, but  

then activates with symptoms.

o Herd immunity: immunity in most of a population  (80% immunity is necessary to protect the  


o Extent of Host Involvement

o Local infection: pathogens are limited to a small  

area of the body (can become systemic).

o Systemic (generalized) infection: infection  

throughout the body.

o Focal infection: a systemic infection that started as  


o Sepsis: toxic inflammation from the spread of  

microbes from a focus of infection.

o Bacteremia: presence of bacteria in the blood. o Septicemia: aka blood poisoning; growth of bacteria

in the blood that causes infection/inflammation. o Toxemia: toxins in the blood.

o Viremia: viruses in the blood.

o Primary infection: acute infection that creates the  

initial illness.

o Secondary infection: opportunistic infection after  

the primary or predisposing infection.

o Subclinical disease: no noticeable signs/symptoms  

(inapparent infection).

o Predisposing Factors to Disease: What Makes the Body  Susceptible

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