MicroBio-Mid#1

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What's the diff b/w Procaryotes and Eukaryotes?: -Procaryotes don't have a true nucleus (genetic material is not enclosed within a membrane)

-Eukaryotes are cells with internal membrane bound structures
What 5 things does the scientific name of an organism tell us?: 1) describes the organism
2) habitat
3) disease
4) discoverer
5) special features
What are the 4 parts to a microscope?: 1) illuminator
2) condenser
3) objective lens(4x, 10x, 40x, 100x)
4) ocular lens (10x)
What are the 2 ways to improve resolution?: 1) oil immersion
2) use shorter wavelength of light
What's the purpose to using staining techs?: -to increase contrast and for preservation
In general, do bacteria have +'ve or -'ve charged surfaces?: -bacteria have -'ve charged surfaces, therefore +'ve charged dyes bind to their surfaces
How do you perform a Gram Stain?: -flood heat fixed slide with crystal violet for 1min
-rinse and flood with Grams Iodine
-rinse and rinse with acetone. Quickly rinse with water
-counter stain with Saffranin for a min
rinse and observe

Gram +'ve=blue, Gram -'ve=red
How do you perform an Acid-Fast Stain?: -flood with Carbol Fuchsin (red)
-apply heat(helps the dye to penetrate the surface)
-decolorize with acid alcohol(ie:HCl)
-counterstain with methylene blue or malachite green

-Acid-Fast bacteria=red
-non Acid-Fast bacteria=blue or green(depends on the counter stain used)
-used to diagnose tuberculosis
What is a species?: -groups of bacteria sharing many common properties
What are/is a strain?: -members of same species having small genetic differences
What are/is a clone?: a group of bacteria that have arisen (by reproduction) from a single "mother" cell.
What are/is a stain?: -a charged dye used to increase contrast
How do you perform a negative stain?: -add sample to slide
-add drop of -'ve charged dye
-drag across
-dye stains to slide, not to bacteria
How do you perform a Wirtz spore stain?: -apply heat to drive the stain into the spore
How do you perform a Liefson flagella stain?: -apply multiple doses of stain to buld layers on flagella, making it more visible
What are/is aseptic techniques?: -techniques used to avoid contamination
What are/is 'sterile'?: - no living microbes present
What are/is 'pure culture'?: -pop of bacteria made up of one species
What are/is 'mixed culture'?: -pop of bacteria made up of more than one species
What are/is a medium(media)?: -a supply of nutrients needed for bacteria to grow
-liquid(broth)
-solid(broth solidified with agar)
What are/is 'inoculate'?: to place bacteria onto a medium
What are/is 'subculture'?: -to transfer bacteria from one medium to another
What are/is the 3 shapes of bacteria?: -Bacillus(rod)
-Coccus(round)
-Spiral
What are/is the arrangements of bacteria?: -Singles
-pairs
-clusters
-chains
What organelles are typically found within a bacteria? (9 of them): -Capsule
-cytoplasm
-ribosomes
-cell wall
-plasma membrane
-nucleoid
-plasmid
-inclusion bodies
-fimbrae
What functions are required of a bacterial cell? (5): -enclose internal contents and separate from environment
-allow entry and exit of specific molecules
-make cellular components
-replicate genetic info and pass on to next gen
-evolve
What are some optional functions of a bacterial cell?: -movement
-attach to other cells or surfaces
-store nutrients for later use
-create survival structures(ie: spores)
-exchange gen info with other bacteria
-communicate with other bacteria
What are the organelles typically found within a procaryote?(5): -cytoplasm
-nucleoid
-ribosomes
-cell wall
-cytoplasmic membrane
What is cytoplasm? What 3 components is it made of?: -cytoplasm is a gel-like matrix of the cell and the site of most cellular metabolic reactions

-made up of: cytosol, nucleoid and inclusion bodies
What are/is 'cytosol'?: -a component of cytoplasm

-90% water; 10% proteins, lipids carbohydrates and ions
-ribosomes are for protein synthesis
What are/is the nucleoid?: -nuclear area NOT enclosed by a membrane
-80% DNA; 20% RNA and protein
-single, circular highly folded chromosome (DNA)
-may have accessory DNA molecules (plasmids)
What are/is 'inclusion bodies'? Give some examples.: -optional organelles in bacteria. NOT present in all bacteria

eg:
-Granules(act as reserve stores)
-Magnetosomes (magnetic FE3O4; orients bacteria in magnetic fields; required for magnetotaxis)
-Gas Vacuoles (permeable to oxygen; expands and contracts to adjust buoyancy)
What is the significance of the cell envelope? AKA cell wall.: -major structural component of cell
-determines gram staining reaction
-contains chemical subunits found nowhere else in nature
-target where some anti-microbial chemicals act
-interface between cell and it's environment
What's the difference b/w a gram +'ve and a gram -'ve membrane?: -Gram pos has one membrane (plasma membrane; peptidoglycan layer[thick Xlinked-traps dye easily]; no outer membrane)

-Gram neg has two membranes (plasma membrane; peptidoglycan layer[thin-dye easily washes out]; outer membrane; has periplasmic space; has lipoprotein; has an outer membrane)
What are/is the 'plasma membrane'?: -common to both Gram pos and neg
-consists of a phospholipid bilayer (hydrophilic alcohols and hydrophobic fatty acids) and proteins
What are the functions of the plasma membrane? (2 of them): 1) Permeability barrier; prevents leakage of components out of cytoplasm; gateway through which nutrients and small molecules enter the cell

2) Site where many types of metabolic proteins are located
What are/is the 2 ways that nutrients enter or leave the cell?: 1) Passive Transport: based on diffusion; doesn't require energy

2) Active Transport: moves molecules from lo to hi conc; requires energy; uses proteins to pump stuff
What is the function of Peptidoglycan layer?: -gives cell its characteristic shape
-gives strength, rigidity and protection to the cell (prevents rupture)
What is the outer membrane composed of? (3 things): 1) phospholipids
2) proteins: Braun's lipoprotein=anchors OM to PG; Porin protiens= hollow channels for entry or nutrients
3)Lipopolysaccharide
What is the Lipopolysaccharide composed of? (3 things): 1) O-antigen; 4-5 sugar units
2) Core polysaccharide
3) Lipid A: glucosamine & fatty acids; anchors LPS to OM

**1&2 project out from the cell surface, 3 is buried within the OM
What is the significance of Lipopolysaccarides?(4): -only found in procaryotic cells
-helps stabilize outer membrane
-gives surface of bacteria -'ve charge
-important in disease(eg: O-antigen stimulates immune system)
What is periplasmic space?: -a gel like area between OM and plasma membrane
-contains degradative enzymes which partially break down nutrients before transport into cytoplasm
For peptidoglycan, which side does the long chains come from? The Nam or the Nag side?: -chains hang from the Nam side
Give 2 examples of 'weird' cell envelopes.: 1) Mycobacteria species(eg:M.tuberculosis) has a mycolic acid layer along with PM and PG

2) Mycoplasma species(eg:M.phneumoniae) no OM, no PG... just a PM... very fragile and a bad stainer
What is a capsule/slime layer/glycocalyx?: -a loose polymer of polysaccharides and protein
-diff chem structures with diff spp
-highly viscous, seem slimy and wet
-found in most pathogens
-needs special stains to be seen(ie: -'ve stain)
What's the 2 functions of capsules?: 1) Attachment to solid, inanimate surfaces (forms "biofilms")

2) Protection against drying, starving, chemicals, antibiotics, immune system, etc.

*** enables bacteria to grow and survive in a natural environment
What is a biofilm?: -a collection of cells on a surface held together by a capsule
How do bacteria make a biofilm?: -through cell to cell communication called "Quorum sensing"
-cells attach and secrete chem sig called "homoserine lactone"
-HSL increas triggers cells to make capsule, aggregate and form biofilms
What are/is 'taxis'?: -a response to stimuli which results in movement toward or away from a certain environment

-eg: chemotaxis, phototaxis, magnetotaxis
What 3 components make up the flagella?: 1) basal body- rings buried in cell envelope
2) filament- appendage extending out from cell
3) hook- dido
What is pili/fimbriae? What are the 2 types?: -hair like filaments
-shorter and thinner than flagella
-fimbriae occur at the ends of the cell (for adhesion to surfaces)
-pili are generally longer than fimbriae (for adhesion to other cells)
-required for attachment, NOT for mobility

1) sex pili- attaches to other bac
2) common pili- attach bac to euc. cells
What are/is 'spores'?: -a dormant, metabolically inactive form of some bacteria
-a type of bacterial cellular diferentiation( converting one cell form to another)
Describe the process of sporulation.: 1) Multilayered wall forms around bacterial DNA
2) Spore loses water(70%-15%)
3) mater spore is released as the rest of the cell degrades (resists chem & radiation due to thicker walls; survives heat and cold due to lower water content)

*Spore can later germinate and resume growth as a vegetative cell
How is growth of bacteria observed?: -observed as a population, NOT at level of single cells
How do cell pop's 'grow'?: -by binary fission
-increases exponentially
What is generation time(G)?: -the time required for one cell division

Expressed as
1) G in min/generations
2) Growth rate (generations/time
What 2 factors influence G's maximum value?: 1) Species dependant
2) Environment dependant
What are the 3 methods used for counting bacteria?: 1) Direct microscopic count: can't distinguish b/w live and dead cells

2) Turbidity: amount of light blocked is proportional to # of cells present; can't distinguish b/w live and dead cells

3) Plate count: cell # expressed as CFU/ml (Colony Forming Units); counts only LIVE cells
What are the 4 Growth Stages of Populations?: 1)Lag Phase: metabolise but not dividing

2)Exponential(log) phase: active cell division at a constant rate

3)Stationary Phase: nutrients exhausted, division stops; may form spores to survive

4)Death Phase: cells starve, may begin to lyse
What are/is a 'chemoheterotroph'?: -uses organic material for carbon
-gets energy during chemical breakdown of organics

*most bacteria are chemoheterotrophs
What are/is 'chemically defined media'?: -the exact composition of media is known

eg: agar
What are/is 'complex media'?: -the exact chemical composition is not known

eg: peptone and beef extract
What is Selective Media?: -contains chemical additives which prevent growth of some bacteria but not others

eg: Bismuth Sulfite Agar
What is Differential Media?: -has no inhibitors therefore anything can grow
-differentiates species on basis of appearance on media

eg: blood agar- diffs b/w hemolytic & non-hemolytic
What is the purpose of using MacConkey Agar?: -it's both differential and selective
-bile salts and crystal v. inhibit
-lactose and pH indicator differentiates
What are the 3 physical growth requirements?: 1) Temperature: optimum temp favors fast growth

2) Oxygen Concentration: 5 diff prefs

3) Osmotic Strength
What 4 names are used to describe bacteria based on temp preferences?: 1)Psychrophiles-opt. 10C; food spoilers

2)Mesophiles-opt. 37C; most species

3)Thermophiles-opt. 70C; hot springs, compost

4)Hyperthermophiles-opt. >80C; mostly archaebacteria
What 5 names are used to describe bacteria based on oxygen concentration preferences?: 1)Obligate Aerobes- needs it

2)Obligate Anaerobes- gets killed by it

3)Facultative Anaerobes- grows best with oxygen but doesn't need it

4)Aerotolerant- same growth with or without oxygen

5)Microaerophiles- grows best at low oxygen levels
How does osmotic strength affect the physical growth of cells?: -internal cell conc must be highter than external so water can enter the cell and for cell division

-so... if external conc increases, then internal conc increases by increasing production of sugars and acids and by increasing the pumping of ions into the cell

-but, if it gets out of control, everything stops...(ie; food preservation)
What are osmotolerant bacteria and halophiles?: -bacteria that can survive in high NaCl conc. solutions.

-Osmo bac can live in 10% soln
-Halophiles can live in 30% soln
What is 'genotype'?: -sum total of genetic information in organism
What is 'phenotype'?: -outward appearance of organism, as directed by genotype
What topics are covered by Bacterial Genetics?: 1)Structure and replication of DNA

2)Turning genetic info into functional protein

3)Transfer of genetic info

4)Manipulation of genetic info at molecular level
What did Griffith discover?: -transformation

-mice and S/R experiments
-live S had caps and killed, live R had no caps and not lethal, you could take it from here.
What did Avery, Macleod, McCarthy discover?: -shows that DNA is transforming agent
What did Lederberg discover?: -bacteria exchange DNA by mating
What did Cairns discover?: -saw bacteria chromosome by EM
What did Venter discover?: -saw first DNA base sequence of a bact. chromosome
What is the structure of DNA in Bacteria?: -double stranded helix of phosphate, deoxyribose, nucleic acid bases
-single, circular chromosome
-highly folded(super coiled)
What is DNA gyrase?: -enzymes that creates supercoils
What is Topoismoerase I?: -an enzyme that removes supercoils
List 4 characteristics to chromosome replication in bacteria.: 1)Semi-conservative: each original strand acts as a template for one new strand

2)Begins at a single point on chromosome

3)Proceeds bi-directionally from origin

4)Begins the next cycle of replication before the previous cycle is completed
Compare the Genetic Information Flow between Euc.s and Proc.s: 1) overall biochemical events are similar

2) Euc's export mRNA from nucleus
Proc's don't export; mRNA is translated as soon as it's produced

3)Euc genes have non-coding introns
Procs don't have introns

4)Procs synthesize proteins way faster!

*Procs generally more simplistic and Euc's
How does diversity occur in euc's? Procs?: -in euc's; by sexual reproduction

-in proc's; by mutation+selection and by gene transfer+recombination
What are mutations?: -a physical change in DNA sequence= change in mRNA=change in protein

-mutations can have no effect on cell, lethal to cell ro favorable to cell

-environment provides selective pressure which determines whether mutation is retained or lost
What are spontaneous mutations?: -random errors introduced during chromosome replication (DNA polymerase enzyme makes mistakes)

-low freq events (1 in 1,000,000,000)

-DNA polymerase has 'proofreading' ability which minimizes number of errors
What are induced mutations?: -mutations via external agents (mutigens or carcinogens)

-higher frequency events than spontaneous mutations(1 in 10,000)

-eg: UV light cause Xlinking; chem's that bind DNA
How would you isolate a bacteria whose mutation caused a gain of function? What is this process called?: 1)Treat pop of bac with a mutagenic agent to increase mutation freq

2)Create environment where only desired mutants can grow

**Called Direct Selection
How would you isolate a bacteria whose mutation caused a loss of function? What do you call this process?: 1)Treat bac pop with mutagenic agent

2)put bacteria onto media containing proline(non mutants and proline autotroph mutants will grow)

3)transfer all colonies onto media without proline (with that 'stamping' method)

4)Compare colonies on +/- proline plates. Colonies on [+pro] but not on [-pro] are called 'proline auxotrophs'

***This is called Indirect Selection
What are the 3 uses of Mutagenesis?: 1)Basic Science- to create things

2)Industrial- select beneficial mutations which improve bacteria (ie: brewing)

3)ID'ing of chemical carcinogens- test chemicals on bacteria to see if mutations are induced
What is Ames test?: -used to ID chemical carcinogens

-inoculate nutrient diffiecient medium with culture that requires that certain nutrient. Mutagenic substance cause causes some organisms to mutate and frow on medium. Nonmutagenic substances does not case organisms to mutate, no none grown on medium
What are the 3 mechanisms to gene transfer and recombination?: 1)Transformation
2)Transduction
3)Conjugation
How does 'transformation' work?: 1)Dead bacteria lyse
-chromosomes breaks into short, linear fragments
-released as free, 'naked' DNA (=Donor DNA)

2)Donor DNA binds to surface of other living bacteria (=Recipient cells)

3)Recipient cells transport donor DNA into cytoplasm

4)Donor DNA joins with recipient cell chromosome (Recombination)

**Can be artificially induced by creating holes (by chem or elec) in cell wall so DNA can get in
What is Recombination?: -the joining of DNA from 2 seperate sources

-necessary because donor DNA is in short linear pieces and will not replicate unless it becomes part of recipient cell chromosome
How does Transduction work?: -DNA is transfered by Bacteriophage(virus)

-Virus=nucleic acid + protein coat
-No independant metabolism therefore depends on functions of host cell(ie: bacteria) for growth and reproduction
Describe the life cycle of a "lyctic" bacteriophage. (5 step process): 1)Phage attaches to bacteria and penetrates cell wall. Injects DNA into cell cytoplasm

2)Phage DNA is transcribed and replicated
-Bacterial chromosome degrades into small linear fragments

3)Phage mRNA translated and new phage proteins are made

4)Phage proteins assembled into new intact phage particles

5)Bacterial cell wall degrades; eventually ruptures and releases mature phage particles

**total time is: 20-40min
**Yield: 100-200 new phages per cell
Are they're any uses to Bacteriophage Lytic Proteins?: -low conc. of lysin will kill pure cultures of bacteria(degrades PG layer)

-effective against Streptococcus, Bacillus anthracis, other gram +'ves
What is a Transducing Particle?: -the result of transduction gone wrong

-Bacterial DNA put into new phage heads instead of phage DNA
-infects other cells but injects bacterial DNA... recombines into the chromosome
How does Conjugation(Bacterial Mating) work? (4 step process): -DNA is transferred by direct cell to cell contact
-requires presence of Plasmids

1)Sex pili of F+ contacts F- cell

2)Pili retracts, draws cells together

3)F+ plasmid replicates: one copy is passed to F- cell, the other one stays

4)Cells separate after plasmid transfer
-both cells are now F+ and are now able to transfer to other F- cells
What are plasmids?: -second form of DNA in some bacteria
-circular, supercoiled DNA, replicate independently of chromosomes
-smaller than chromosome (5-100genes)
-b/w 1 and 100 copies per cell
-usually carry accessory genes

-carry transfer genes: encodes proteins needed for conjugation(ie: sex pili)
What did Fracastoro suggest?: -that diseases are caused by invisible organisms
What did Leeuwenhoek do?: -saw first direct observation of microbes
What did Redi do?: -concluded that spontaneous generation is not true for higher life forms
What did Needham conclude?: -concluded that animalcules arise spontaneously from broth
What did Spallanzani do?: -found no animalcules inside a sealed flask... air is important
What did Pasteur do?: -made the swan neck flask-nothing came out
-disproves spontaneous generation theory
What are F+ plasmids?: -are plasmids carrying transfer genes
-genes code for sex pili and other proteins needed for conjugation
What's the diff b/w a simple stain and a differential stain?: -simple stains only use 1 dye
-differential stains use 2 dyes
Give 3 examples of special stains.: 1)negative stain
2)spore stain
3)flagella stain
What would happen if you treated bacteria with lysozyme?: -it degradates the PG layer, cells are suspect to lyses now
Who is Robert Koch?: -criteria for showing cause-effect relationship b/w microbe and disease

1) Microbe must be present in every case of the disease

2) Microbe must be isolated from host and grown in pure form

3) Disease must be reproduced when a pure form of microbe is given to a healthy host

4) Same microbe must by recovered from the infected host
What are the 4 arrangements of flagella present on bacteria?: 1)Monotrichous: single polar flagellum

2)Amphitrichous: single flagellum at each end of the cell

3)Lophotrichous: 2 or more flagella at one or both poles of the cell

4)Peritrichous: flagella distributed over the entire cell
What are Endospores?: -highly resistant bodies produced WITHIN the cells of certain bacteria like Closrtridium and Bacillus
-survival mechanism
-spores are resistant to the effects of heat, drying, chemical disinfection, and radiation
What are Cysts?: -an entire cell rounds off and develops a thick-walled, highly refractile cell
-this thick wall of the cell consists of an inner layer (INTINE) and an outer layer (EXINE)

eg:Azotobacter form cysts
What 7 characterisitics are used to describe colonies?: 1)Size
-large, pinhead, etc.

2)Elevation
-raised
-convex
-flat

3)Edge
-entire
-undulate
-lobate

4)Form
-circular
-irregular
-spindle-shaped

5)Surface
-smooth
-rough
-concentrically ringed

6)optical characters
-opaque
-translucent
-dull
-glistening

7)Color
How would you characterize Bacillus megaterium?: Shape:
-bacillus

Growth Form:
-chains

Gram Stain:
+'ve
How would you characterize Escherichia coli?: Shape:
-bacillus

Growth Form:
-pairs/singles

Gram Stain:
-'ve
How would you characterize Sarcina lutea?: Shape:
-coccus

Growth Form:
-4's
- 2D clumps

Gram Stain:
+'ve
How would you characterize Micrococcus roseus?: Shape:
-coccus

Growth Form:
- 3D clusters

Gram Stain:
+'ve

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