Microbiology Exam 2

• transformation
• transduction
• conjugation

because it helps them gain genetic variability and since they are so simple and don't reproduce sexually, their variability is limited

prokaryotic cells transfer DNA from Donors to recipients

• recombination: combining DNA from different sources
• recombinant: the resulting cell that has genes from different sources

• humans: vertical gene transfer to offspring only
• bacteria: lateral or horizontal gene transfer to another organism in the same generation

• causes disease
• virulence factor: makes it more dangerous ie a capsule

• Genetic material is taken up from bacteriums surroundings (if theres DNA in the environment)
• single stranded fragments are taken up by competent cells and inserted directly into the bacterial chromosome
• first discovered in smooth vs rough colonies of streptococcus pneumoniae

• Frederick Griffifth looked at "smooth" and "rough" colonies of strep pneumo
• smooth colonies contain a capsule and are virulent
• rough colonies dont have a capsule and dont cause disease.
• He injected killed smooth bacteria into mice and they did not die
• also injected live rough bacteria into the mouse and it lived
• but when live smooth bacteria injected to the mouse it died as expeced since this is virulent
• However, an injected combination of killed harmful smooth and live harmless rough killed the mouse.. why? The rough took up the DNA released when the smooth was killed and gained virulency

• no, only competant bacteria
• some have a "competence factor" that is released into the medium prior to transformation which allows them to open up to DNA fragments
• biologists can buy competent bacteria to transform

• bacteriophages introduce genetic material through transduction
• 2 types of phages

• Virulent Phage
• Temperate Phage

• Cause infection and eventual death (by lysis) of the bacterial cell
• uses the lytic cycle

• infect bacterial cells but go into lysogenic phase where they insert their DNA into the bacterial chromosome (now called prophage) and do not lyse the cell open immediatedly
• virus DNA exists in the bacterial chromosome
• temperate phage can later be induced to go into a lytic phase, make copies of themselves then lyse the cell open to infect neighboring cells
• during this process sometimes bacterial DNA is taken out during prophage excision and transferred to other bacterial cells during subsequent phage infections

• absorption: virus binds cell
• entry: virus injects DNA into cell
• replication: more virus are made in the cell, virus has overtaken cell machinery
• assembly
• release: viruses lyse the cell and go to infect other cells

• longer sneakier cycle
• Virus binds and injects DNA, which sneaks into bacterial chromosome, bacteria continues to replicate and therefore replicating this viral DNA as well, eventually they can undergo induction and enter the lytic cycle

switch from lysogenic to lytic cycle

viral dna that is existing within the bacterial chromosome

• generalized transduction
• specialized transduction

• lytic cycle
• we have a virulent phage, it injects the DNA, takes over the (donor) host cell machinery to make viral DNA and more protein coat
• the host chromosome is fragmented into pieces during replication and when new viruses are packaged some have viral DNA and some have host DNA
• the ones with host DNA (defective phages) will transfer bacterial DNA from one to another when it goes to infect a new cell
• viruses escaping to infect new cells have EITHER host (pure host DNA) or viral DNA

virulent phage that was packaged with host DNA

• Undergoes lysogenic cycle
• virus injects DNA into donor bacterial host cell. viral DNA becomes incorporated into the bacterial chromosome (a prophage)
• replication occurs and eventually induction, and when the cell lyses,the viruses escaping to infect new hosts have BOTH viral and bacterial DNA

• host bacterial cell has chromosome with genes A+ and B+.
• Virus infects host cell and Host DNA is hydrolyzed into pieces
• phage DNA and proteins are made in the host cell
• occasionally, a bacterial DNA fragment (in this example, a fragment containing the A+ gene) is packaged into a phage capsid
• that phage capsid infects another bacterial host cell (this one is A- B-) and inserts that A+ DNA, Crossing over occurs and now the new cell is A+ B-

• The bacteriophage infects the host A+B+ cell and its dna becomes incorporated into the host chromosomal dna between genes A and B
• the prophage DNA will usually exit the chromosomal to be packaged into the new virus capsids, but occasionally, prophage DNA exits incorrectly, taking adjoining bacterial DNA with it (in this case the A+ gene)
• The phage particals carry the bacterial DNA along with phage DNA
• When the phage infects a new A-B-cell, there is still phage DNA inserted, but that A+ gene is crossed over into the chromosome, transferring this gene

• uptake of genetic material from neighboring bacterial cells
• requires contact between donor and recipient cells
• can transfer larger pieces of DNA, even whole chromosomes

• passed through "F pilus" or "sex pilus" or "conjugation pilus"
• pilli are specific to prokaryotes, they form a bridge

• aka: fertility plasmids
• conjugative plasmids that contain genes for generation of a pillus, so when transferred it can make a cell that cant conjugate able to
• accessory DNA 
• F+ is the donor (male)
• F- is the recipient (female)
• allow the transfer of genes

• direct synthesis of conjugation pilli
• provide resistance to certain antibiotics
• induce the formation of tumors in plants

• The donor cell contains the F plasmid along with the chromosomal DNA
• the donor forms a conjugation bridge with pili and a single strand of the plasmid goes across
• replication then happens so that both plasmids are double stranded
• F- is now F+

eventually they will all become F+

• called Hfr cells
• come from F+ strains where the plasmid is incorporated into the bacterial chromosome rather than seperate
• can initiate some chromosomal DNA to be transferred along with the F Plasmid

• A cell where the f plasmid has integrated into the bacterial chromosome
• during conjugation a single strand of the plasmid and some chromosomal DNA moves to the F- cell, crossing over will occur and that piece will be integrated into the F- cell dna creating a new Hfr Cell

• sometimes an F plasmid that is incorporated into the chromosome can detach and once again become an F plasmid
• However, if it takes a long piece of chromosomal DNA with it, it is now called the F' plasmid because it differs genetically from the original F plasmid.
• the cell may not be able to function at all if the plasmid takes out an essential gene
• F- can get extra copies of genes if conjugation happens this way

• plasmid is integrated into chromosomal DNA
• becomes an F' plasmid when it comes out of chromosomal DNA and brings genes A and B with it. 
• conjugation occurs and a single strand of the f' plasmid containing these genes goes to the F- cell and they both replicate
• F- cell now has duplicate copies of genes A and B

• resistance genes often found on a resistance plasmid
• can move around from plasmid to plasmid and sometimes incorporate into the bacterial chromosome
• move in groups 
• contain transposable elements and another unrelated gene that is moved from place to place

the ability to move from one location to another with regards to transposons

• genes that can move around by transposition
• made up of: 1. genes that code for transposase 2. genes that are surrounded by inverted base repeats

enzyme that aids in transposition

• the purposeful manipulation of genetic material to alter the characteristics of an organism in a desired way
• transfer of genes between different species is possible in the lab
• altering genetic material to get certain characteristics

• transposition of DNA from one location on a chromosome to another
• if the control genes for one operon are moved in front of another operon, both sets of genes will be controlled together

• ie: Regulator gene---promoter--operator-5-4-3-2-1---regulator gene---promoter-operator-z-y
• second regulator gene promoter and operator transposed out and what remains, the first regulator gene controls production of it all
• regulator---promoter--operator-5-4-3-2-1-z-y

an organism where the cell wall has been removed

• two protoplasts can fuse and mix their genetic material, recombining two strains before the cell wall reforms
• useful in making genetically modified organisms (gmo)

• making lots of copies of a gene and therefore lots of product
• plasmids or bacteriophages produce rapidly within a bacterial cell
• used to make antibiotics, enzymes, amino acids

• DNA contains info from two different species
• if the DNA integrates into the sperm or egg cell DNA, all offspring will contain the gene as well. These offspring are termed "transgenic" or "recombinant"

• 1. invitro manipulation of DNA
• 2. recombination of another species DNA in a  bacterial phage or plasmid (vector)
• 3. cloning, or increased production of many genetically identical offspring of phage or plasmids (so we can harvest the protein)

bacteria or animals can be transgeneic

• named because they restrict bacteriophage division by chopping up phage DNA
• molecular scissors that let us cut a gene out of DNA and insert it elsewhere

• 1. DNA from one organism is cut into small segments using a restriction endonuclease
• 2. The same restriction endonuclease is used to cut the recipient vector
• 3. The DNA is incorporated into the vector using ligase
• 4. The finished vector is used to transform bacterial cells by heat shock or electroporation

a high voltage discharge is used to make bacteria temporarily permeable to take up DNA

• Make substances important to humans: interferon (interrupts virus infection), insulin (treats diabetes), blood products (for hemophiliacs- clotting factors), enzymes, antigens for vaccines
• Diagnosis of genetic defects in fetuses
• Gene Therapy: very successful, tailored to the individual. inserting functional genes into appropriate cells to cure a genetic disease
• Forensic Science

• 1. Fermentation of wine, beer
• 2. Degradation of normally useless plant material (cellulose)
• 3. Fuel manufacturing
• 4. Clean up of environmental pollutants (ie oil spills)
• 5. Extraction of metals from ore

• Control insects that destroy crops and mosquitoes
• high yeild seeds
• resistance to weed herbicides
• nitrogen fixation

• Used in medical application
• fusion of two cell types
• first was the fusion of a myeloma cell (bone marrow cancer cell) with a B cell that makes antibodies- allows rapid pure antibody production which you can inject to treat disease

• YES!
• 1. no lab workers have become ill because of known recombinant organisms
• 2. The lab strain of E.coli used in lab does not infect humans
• 3. Mammalian genes incorporated into E.coli make the bacteria less adaptable
• 4. Using a normal level of lab safety is considered sufficient to control mutant e.coli

• the science of classification
• taxonomists look at evolutionary relationships to see how related 2 things are

• criteria for identifying organisms
• arrange related organisms into groups
• provide information about evolution

• Members of higher level groups share fewer characteristics
• members of lower level groups are more alike

• the father of taxonomy
• created binomial nomenclature (it used to be 5 names)

• every organism has 2 names- genus and species
• genus is ALWAYS CAPITALIZED
• species is NEVER CAPITALIZED
• both are written in italics OR underlined

• within a species, there can be different strains of bacteria
• contain a genetic difference or mutation that distinguishes them from other members of the species
• ex: resistance to abx, presence of an antigen, need for particular nutrient
• E.coli K12- studied for plasmids
• E.coli 0157:h7- hemmorrhagic inflammation of colon

• Kingdom
• Phylum (and sometimes subphylum)
• Class
• Order
• Family
• Genus
• Spaghetti 
• KingPhillipCameOverForGoodSphaghetti

• used to identify organisms in the world
• asks yes or no and if then questions
• ie: is it gram positive? if yes go to #2 if no go to #3. 
• will only get you so far, gives an initial identification, good for nature hikes identifying plants and such

• they are a species if they are capable of DNA transfer during mating to fertile offspring
• ie- lyger is not a species because a lion and a tiger make it but it is not fertile

because bacteria reproduce by binary fission and the only DNA transfer is during recombination

• according to:
• 1. biochemical reactions (biproducts from metabolism)
• 2. chemical composition
• 3. cellular structures
• 4. genetic characteristics
• 5. immunological features that identify an organism to our immune system (how they affect us)

Bacteria, Archaea, Eukarya

• plantae
• animalia
• fungi
• protista
• monera

contains all prokaryotes

• Eubacteria
• Cyanobacteria
• Archaebacteria

• true bacteria
• includes pathogens

photosynthetic autotrophs (self feed)

• ancient bacteria that live in the harshest of environments (high temp, pressure, salt, etc)
• cell wall and RNA polymerase are different from eubacteria (different composition)

• mycoplasma
• cyanobacteria
• spirochetes
• gram negtive bacteria
• rickettsias
• archaeobacteria
• gram positive bacteria

• euglena
• diatom
• dinoflagellate
• ciliate (paramecium)
• sarcodine (amoeba)
• mastigophoran (trypanosoma- african sleeping sickness)
• apicomplexan (plasmodium- ie malaria)

• sac fungi- morchella
• club fungi- amanita muscaria
• algae like fungi- pilobolus
• molds- penicillium (produce pennicillin)

• A couple of drugs come from them ie quinine from the cinchona tree
• otherwise not very related to micro

• insects
• arthropods
• vertebrates
• nematodes
• platyhel minths (worms)

• Methanogens
• Extreme Halophiles
• Extreme Thermoacidophiles

• anaerobic
• make methane
• landfills have pipes that are venting methane out that the bacteria are producing

• obligate aerobes
• live in high salt conditions

• found in hotsprings, hydrothermal vents under the ocean
• contain extremozymes

• enzymes that allow high temp toleration, non denaturing
• Scientist took extremozymes from these bacteria to do PCR to amplify DNA

• acellular infectious agents
• contain nucleic acids (rna or dna) and protein coats
• not alive, therefore do not belong to any kingdom

• they evolve quickly because of short generation time
• similar morphology
• hardly any fossil record because theyre so tiny

• organisms are observed for as many characteristics as possible
• values of 1 are given if present 0 if not present
• computers analyze numerical data to detect patterns
• greater than 90% match means 2 organisms are in the same species
• harder than it seems to do

• Studies the similarities of DNA between organisms
• sequencing is improving knowledge of organisms, but is costly and time consuming

• Base composition
• DNA/RNA sequencing
• DNA hybridization
• Protein profiles

• A T C G bases: look at G and C content. 
• Varies from 23%-75% in different bacterial species
• same GC content does NOT mean they are related, but different G-C content means they are NOT RELATED

probe for unique DNA sequences

• taking DNA from 2 organisms and sticking it together to see if the strands stick together. 
• identical DNA sequences will anneal to eachother
• the more they stick the more similar they are
• if unrelated, very little annealing occurs

• PAGE
• measures size of proteins

• properites of ribosomes
• immunological reactions
• phage typing

• ribosomes evolve slowly bc we need the proteins to be made correctly so if the ribosomes change we risk changing the protein which could kill the cell
• compare 16s ribosomal rna between organisms

• use antibodies to stick to particular surface proteins
• if bacteria have similar antigens theyre related
• western blot

• determins which bacterial viaruses attack particular organisms
• viruses have to be able to stick to the cell before they can infect. they only bind to certain types of cells

• obligate intracellular parasites
• must get into a host cell and use its machinery

• Contains RNA or DNA but NOT both
• invades a host cell, takes over the machinery to replicate
• doesnt last long outside the cell

one virus particle

• nucleic acid core
• protein capsid
• some virions have envelopes

RNA or DNA in the virus

• a protein coat that protects the viral genome (genetic info)
• determines shape of the virus
• has subunits

• subunits of the capsid (look like nuts and bolts)
• make up the capsid

• some viruses have outer envelopes
• made of bilipid layers that are "stolen" from the host cell as the virion particles burst open the cell
• covers them and makes them look invisible to new hosts since they are made of the same material
• allow viruses to get in cells and hide from our immune systems
• help new virions to infect other host cells

• non enveloped, or naked viruses are more virulent
• the envelope makes the virus more susceptable to temperature and pH changes

• lipids, proteins and carbohydrates
• the same as our lipid bilayers
• contain spikes

• glycoproteins that project from the surface
• allow virions to bind to a host cell by binding receptors on the cell

by fusing with the host cell membrane

• helical: spiral of protein around core; ie tobacco mosaic virus
• polyhedral: many sides, looks geometric; ie adenovirus
• complex: some capsomeres are helical and others are polyhedral; ie tailed bacteriophage
• bullet: looks like a bullet; ie rabies
• thread: thread like; ie ebola

icosahedral has 20 sides

• bullet shaped
• has lipoprotein envelope
• knoblike spikes (glycoprotein S)
• genome unsegmented
• Linear negative sense RNA

depends on the host range and viral specificity

• types of hosts a virus can infect
• most viruses can only infect one type of animal, plant, fungus, protist or bacterium
• ie- the polio virus only causes disease in humans
• some can jump species but many are species specific

• the type of cell a virus can infect
• dependent on the specific enzymes and proteins made by the host cell to aid in viral replication
• also depends on the ability of the virions to escape the host cell and infect other cells

• by the ability of a virion to attach to the host cell
• depends on the proteins made by the host cell and how well the virus can escape that type of cell to infect more

attachment depends on the receptor sites on the host cell and attachement sites on the capsid or envelope of a virus

• papilloma virus can only infect skin cells (causes warts)
• in contrast, cytomegalovirus (CMV) can infect cells of the salivary glands, GI tract, liver, lungs, etc and can cause varied symptoms throughout the body depending on which cells are infected

• the virus is a key and the host cell is a lock, it is very specific
• although some (ie CMV) can infect many differernt types of cells

• by the number and arrangement of proteins in the capsid
• can also be categoriezed by whether its an RNA or DNA virus

• because they need host cells to replicate, some believe they probably came on the scene after primitive cells evolved
• some think viruses and cells evolved together
• some think they were once real cells
• some think they evolved from bacterial plasmids
• some think they were the precursers of cells

• originally based on the type of host a virus could infect (usually categorized this way) ie plant virus, animal virus
• then on which area of the organism was infected (pneumonic, neurotropic)

• more recently, viruses have been classified by their genetic structure
• RNA viruses vs DNA viruses

most are single stranded and have either a positive or negative sense strand

part of RNA viruses that has to do with how virus gets proteins

• acts as mRNA
• RNA can be used immediately to make proteins by host ribosomes
• HIV is an ex. + sense strand converted to DNA by reverse transcriptase then to RNA by the host cell polymerase

• a complimentary RNA strand must first be made in order to be used by the host ribosomes
• - sense RNA viruses must also carry their own enzyme for making the complimentary strand (RNA polymerase)
• - sense strand; complimentary mRNA made from this that can be translated into protein by the host cell

• Picornaviridae
• Retroviridae

these names only apply to bacterialphages, although there are viruses that infect other organisms that can hide in the cell

• icosahedral viruses with + sense strand, no envelope
• enteroviruses: polio virus
• rhinoviruses: nose cold, common cold
• hepatociruses: "liver" hepatitis A

• retroviruses
• ie: HIV
• 2 copies of + sense RNA 
• contain reverse transcriptase
• the viral DNA can get into the host cell nucleus and integrate or hide there

enzyme that converts RNA to DNA for a retrovirus inside the host cell

+ sense strand transcribed into DNA by reverse transcriptase which sneaks into the host DNA and stays there for years and will be transcribed to mRNA and protein in the cell

different viruses infect at different times

• Adenoviridae
• Herpesviridae
• Papovaviridae

• "gland"- affect glands
• extremely resistant to heat, pH or chemicals
• cause respiratory symptoms and severe diarrhea in humans

• "creeping" family
• they can hide and be latent then come out later
• different family members cause many diseases
• oral herpes, genital herpes, chicken pox, shingles, lymphoma, hodgkins disease, b cell lymphomas
• Kaposi's sarcoma

one member of the family is papilloma virus (HPV) which is responsible for most instances of cervical cancer

contains parts of the virus or an attenuated virus

• 1. Adsorption: attachement to host cell (sticks to it)
• 2. Penetration: entry into the host cell (DNA is inserted and now it can take over the host machinery)
• 3. Synthesis: generation of viral nucleic acid and capsid proteins using host cell machinery
• 4. Maturation: assemply of new virions from these pieces (capsids, proteins, etc)
• 5. Release: escape of new virions from cells to infect others, often destroys host cell by lysis

• 1. Adsorption
• 2. Penetration
• 3. Biosynthesis
• 4. Maturation
• 5. Release

phage is adsorbed onto bacterial cell wall

• phage penetrates bacterial cell wall and cell membrane
• phage DNA is injected
• bacterial DNA is disrupted/fragmented

• the phage DNA directs the cells metabolism to produce viral componenets- proteins and copies of phage DNA
• empty phage heads and pieces of phage DNA are synthesized

• collars, sheaths, and base plates have been attached to the heads.
• tail fibers are added last

bacterial cell wall lyses, releasing mature phages.

• a way to determine the numbers of bacteriophage
• serial dilutions are made from a suspension of phage
• each dilution is added to a bacterial lawn on a nutrient plate
• when phage gets into bacteria it will replicate and then lyse it and infect surrounding cells. bacterial cell lysis will form a clearing of bacterial cells whereever the original phage infected
• counting the plaques will tell you how many phage you started with when x by the DF
• # reprisented in PFU

plaque forming units

• environmental or chemical triggers
• ie- UV  radiation

• virus attaches to a specific host cell and injects DNA or RNA into the cell.
• the cell then takes that genetic material and makes so many copies that eventually cause lysis and death

• 1 &2- Adsorption and Penetration: phage is adsorbed to receptor site on bacterial cell wall, penetrates it and inserts its DNA; phage DNA inserts itsef as a prophage into bact. chromosome and is replicated along with the bacterial DNA; binary fission occurs and both daughter cells have phage DNA
• 3. Biosynthesis: the phage DNA directs the cells metabolism to produce viral components; empty phage heads and pieces of phage DNA 
• 4. Maturation: heads are packed with DNA; collars, sheaths, base plates attach to heads and then tail fibers added
• 5. Release: bacterial cell lyses relasing completed infective phages

• 1. Adsorption and fusion with the cell membrane (easier if the virus has an envelope)
• 2. Penetration- enters the cell and uncoating (Protein coat lost and its just the DNA/RNA)
• 3. DNA/RNA enters host cell nuclear membrane and the viral genes are transcribed by host cell machinery
• 4. Viral DNA is replicated and translation makes viral coat proteins (capsid)
• 5. Maturation- New virus particles are assembled
• 6. Release- some viruses bud out from the host cell and become covered by envelope protein stolen from the host cell; others acquire envelope as they emerge from the nucleus or while theyre in the cytoplasm

:)

• No envelope: a canyon (dip between capsomeres) on the virus will match up with host plasma membrane proteins like a key for binding; it must match in order to stick
• With envelope: viral glycoprotein (spikes) on the membrane will bind the membrane protein receptors on the host cell

cell culture- growing cells in a flask

• primary cell culture
• dipoid fibroblast strains
• continuous cell line

• cells come directly from an animal
• can last only a short time in culture because of limited cell division

• eventually, cells age out
• telomeres on the chromosome have repeating sequences that protect the ends of the chromosome
• with every division, small pieces of the telomere are lopped off until its gone, then what's left to be chopped is chromosomal DNA so the cell can't divide anymore

• cells that make connective tissue
• derived from fetal tissue so they divide longer in culture
• used for making vaccines

• cells will reproduce for an extended time
• HeLa cells
• Immortal cell line because they will divide indefinitly in culture
• continuous cell lines are not normal cell lines, usually cancer

Henrietta Lacks' cervical cancer cells were taken without her knowldege and John Hopkins sent to George Guy who did many studeis

she had an enzyme that regenerated her telomeres

• something that causes defects during embryonic development
• if you're pregnant avoid these and take precautions
• some viruses can be teratogens if they are transmitted across the placenta

• CMV: causes neurological defects
• Herpes Simplex 1 and 2: Acquired during birth process if infant touches an outbreak, can damage eyes and nervous system
• Rubella: can cause deafnes, heart defects, mental retardation

if there is a flare up near delivery they'll do a c section so baby doesnt touch it

• smaller than a virus, cannot replicate itself
• consists of single circular RNA
• no capsid or envelope
• do not require helper virus
• doesn't produce proteins
• RNA can be copied only in host cell nucleus
• only known to affect plants

• acellular misshapen proteins
• not viruses or caused by viruses, just misshapen incorrectly folded proteins
• proteinaceous infectious particle

• prion proteins
• tend to stick together inside cells which is fatal to the cell

• because they induce normal protiens to fold incorrectly as well
• they can cause the membrane proteins of cells near them to misfold and get in and cause a chain reaction

• Creutzfeldt Jakob Disease: fatal neurodegenerative disorder (occurred during surgery with equipment contamination)
• Kuru: natives of New Guinea, due to cannibalism?
• Scrapie: mad cow for sheep
• Mad Cow disease: bovine spongiform encephalopathy

• resistant to high temps that would kill a virus
• cannot be treated with radiation that would damage the nucleic acids of a virus
• not destroyed by nucleases (enzymes that break up RNA and DNA)
• have direct pairing of amino acids
• can cross species

• they are resistant to normal sterilization/heat
• need a very strong chemical that denatures proteins to remove

• cows were given feed containing "protein supplements" which came from brain matter of sheep
• some of these sheep had scrapie
• prions were passed up the food chain from sheep to cows to humans
• prions are NOT species specific

• unregulated invasive growth of abnormal cells
• chromosome # can be abnormal too
• cells that do not stop growing as they are supposed to can build up locally inot a neoplasm

• tumor
• can be benign (cysts) or malignant (invade surrounding normal tissue)

when malignant (cells that spread to surrounding normal tissue) spread to other tissues/organs of the body

first virus linked to cancer is RSV (rous sarcoma virus) which caused cancer in chickens

• 15% of human cancers are caused by known viruses
• Epstein-Barr Virus (EBV): Burkitt's lymphoms
• HPV: cervical cancer
• Hep B: 80% of liver cancer
• HSV-8: Kaposi's sarcoma

• 70
• senescence- deterioration with age

• can make them divide indefinietly
• make them not affected by contact inhibition (stopping growing once they touch)
• do not remain stuck to eachother or culture dish
• make them need less nutrients and growth factors in media
• make them exhibit abnormal chromosome numbers

• aka aneuploidy
• abnormal chromosome numbers

when a virus integrates or sneaks its DNA into the host genome, mutations are made in the host DNA

• the mutations can be in
• 1. tumor suppressors: usually DNA repair enzymes (BRCA1); when its time to stop cell division, tumor suppressors start normally
• 2. oncogenes: usually proliferative genes

• normal: proto-oncogene
• mutated by viral insertions/deletions: oncogene

• applied to non living objects
• decreases the number of microorganisms, but does not sterilize
• some slow growth while others kill

• applied to living tissue 
• ie neosporin

• killing or removal of all microorganisms in a material or on an object
• done by using an autoclave

NO living organisms or endospores are present

reducing the number of pathogenic organisms

prevents microorganisms from growing or slows their growth

kills bacteria

kills fungus

kills many microbes but usually not spores

kills endospores and fungal spores

incapacitates viruses

• 1. A definite proportion of organisms will die in a given time interval
• 2. The fewer organisms present, the shorter the time needed to achieve sterility
• 3. Microorganisms differ in their susceptibility to antimicrobial agents (some sterilization techniques kill quickly, others slower due to resistance, etc- this is true for dissinfectants too)

• effectiveness is increased by high temperatures
• effectiveness can be affected by concentration of chemical, pH and time left on microorganisms
• ethyl and isopropyl alcohols work best at 70% solutions because water is necessary to denauture bacterial proteins (and 100% can burn our lungs if inhaled)

• Phenol Test
• Filter Paper Test
• Use-Dilution Test

• phenol- a carboxylic acid
• a measure of the bactericidal activity of a chemical compound in relation to phenol
• phenol has a coefficient of 1.0
• The dissinfectant to be tested is tested along side phenol on a standard microbe (ie salmonella)
• Dissinfectants more effective than phenol have a coefficient greater than 1, less effective dissinfectants have a coefficient less than one, and equally effective have a coefficient of 1
• there are different phenol coefficients for each organism

• tiny filter paper disks soaked in chemical agent are put on agar plates with a bacterial lawn
• clear regions around the disks mean they killed local bacterial cells

• A metal tube is coated in bacteria and then dipped into different dilutions of chemical agents
• finally, the tube is put into growth media to see if bacteria from any of the tested dilutions grow

• 1.Fast acting  (you don't want something that has to sit a while)
• 2. Effective against all types of infectious agents without destroying human tissue or acting as a poison (so it will get viruses/bacteria etc)
• 3. Penetrate material easily without damaging it or us
• 4. Easy to prepare and stable in a number of environmental conditions (dont want it to degrade after a month)
• 5. Inexpensive and easy to use
• 6. Smell pretty

• 1. They denature proteins (3D tertiary structure is destroyed)- can be bacteriostatic or bactericidal
• 2. They can affect membranes- break down the lipids
• 3. They can affect nucleic acids (DNA/RNA and how they replicate) or energy production (if microbes can't metabolize they'll die)

• contain alkali and sodium
• kills streptococcus, micrococcus, Neisseria and the flu virus
• can be cationic + or anionic-

• anionic are less effective against bacteria because of the - charge of the bacterial cell wall
• some cationic detergents are quaternary ammonium compounds which contain four organic groups surrounding a nitrogen atom

• They trap particles so they can be washed away
• soaps have hydrophobic heads and hydrophillic tails
• soap surrounds particle (dirt, microbe, etc)by creating micelles
• micelles: the hydrophobic parts surround the particle and the hydrophillic tails face outward, this way when you wash with water it will wash the whole micelle including the particle away

• acids and bases
• heavy metals
• halogens
• alcohols
• phenols
• oxidizing agents
• alkylating agents
• dyes
• miscellaneous

• lactic acid and propionic acids are used as food preservatives- add to food to slow bacterial growth
• vinegar- low pH, can be used to clean

• silver, mercury, selenium
• not ideal because these are also harmful to us

• chlorine, iodine, bromine
• salts

• denature proteins, dissolve lipids, cell membranes
• do not sterilize

hydrogen peroxide- disrupts disulfide bonds in proteins, changing the conformation and therefore stopping function

• disrupt proteins and nucleic acids (but also can disrupt ours)
• can cause cancer
• formaldehyde, etc

• Acridine orange stops cell replication
• Methylene blue stops bacterial growth
• Crystal violet blocks cell wall synthesis and inhibits gram + growth

• plant oils
• nitrites
• tea tree oil, natural things can be effective
• antibiotics come from living organisms (ie fungus)

• Heat (most important one)
• Refrigeration
• Freezing
• Drying
• Radiation
• Filtration
• Osmotic pressure

the temperature that kills all bacteria in a 24 hour old broth culture at neutral pH in 10minutes

time required to kill all bacteria in a culture at a particular temperature

• time required to kill 90% of organisms at a particular temperature
• ex: D150C= 30 min

• Dry heat
• Moist heat
• Pasteurization

• big ovens
• slow to penetrate, used for metal instruments, glassware, oils and powders (powders would get wet with moist heat)
• used if you cant use moist heat due to ie rusting
• some things can't be dry heated or they will catch fire
• 171C for 1 hr, 160C for 2hrs, or 121C for 16hrs

• autoclave- heats under pressure
• Maintains temperature and pressure for a period of time
• Validation includes tapes and documentation
• 121C and 15lb/in^2

• Does not sterilize, but will reduce microbes
• 71.6C for 15 seconds (flash method)
• 62.9C for 30 minutes (holding method)
• ultra pasteurized gets rid of even more bugs, this is why heavy cream expires later than milk

• uses steam, temp, and pressure 
• needs to be calibrated and tested often

• buy ampules with a glass container inside that holds media, and on the bottom (not in the media) is a spore strip
• put this through an autoclave run, the pressure will break the inner glass tube and the media will come out and cover the spore strip
• after the run, incubate the tube, if the medium remains clear, it works, if it is cloudy, growth occurred meaning the autoclave did not do its job and sterilize

4-5C, slows microbial growth

• -20C preserves food for longer periods of time
• Microbes can be frozen at -80C or in liquid nitrogen at -180C

• Absence of water inhibits microbial enzymes
• Endospore-forming bacteria will survive drying

• lyophilization: dried after being frozen
• used for long term preservation of microbes

• Ultraviolet light
• Ionizing Radiation
• Microwave radiation
• Strong visable light

• 40-390nm but 200nm is most effective against microbes as it damages proteins
• UV light is absorbed by nucleic acids which damages them but they can use repair enzymes
• useful for destroying viruses
• does not penetrate well, but useful for decontaminating air

• Xrays (0.1-40nm) and gamma rays create ions in substances by dislodging electrons
• useful for killing bacteria and viruses in foods

• 1mm-1m
• only useful for sterilizing substances that contain water

• 400-700nm can oxidize certain bacterial enzymes
• certain dyes can increase the effect of strong light on microbes

• passing material through a strainer to rid it of microbes
• good for small quanitities, but can be expensive
• membrane filters have varying pore sizes that let organisms of different sizes pass throught
• used when heat sterilization would harm the substance
• 0.025um-25um
• 0.2micron filters are commonly used to sterilize cultures from bacteria
• HEPA filters have an average pore size of 0.3um and are used to filter air

• high concentrations of sugar and salt can cause plasmolysis (loss of water)
• this leads to bacterial cell death
• used in jellies, salted meats

generic term- any chemical agent used to treat any aspect of a disease

chemical agents used to treat diseases caused by microbes

• chemical substance produced by microorganisms which has the capacity to inhibit the growth of bacteria
• ie molds produce pennecillin

chemical agents produced in a laboratory

• chemical precursor is made in a laboratory and given to a microorganism to complete synthesis
• the microorganism finishes the job and then scientists harvest the product from the microbes

antimicrobial therapy and antibiotics

selective toxicity

• a substance that has selective toxicity will harm microbes without significantly damaging host cells
• depends on dose, exposure and metabolism
• If an antimicrobial agent is too toxic to take systematically, it can only be used topically, or on the skin
• anything taken internally must have selective toxicity

Drugs have variable ranges between the therapeutic dosage level (where the drug does what its supposed to do) and the toxic dosage level (where it starts to affect you)

• every drug has one
• it is the maximum tolerable dose per kg of body weight divided by the minimum dose per kg body weight that will cure the disease

• everything is toxic, it just depends on the dose, exposure and metabolism of the thing 
• just because something is natural doesn't mean it's non toxic

• ie alcohol
• Exposure: you can sit in a bathtub of vodka and be fine. You need to ingest it for the exposure
• Dose: 1 drink is not toxic wheras 15 drinks is
• Metabolism: 2 drinks in 45min overwhelms the metabolism and it cant keep up which is why you begin to feel those toxic effects

• a good antibiotic will target a broad spectrum of microbes
• ie: tetracycline targets gram + gram- clamydias and rickettsias while isoniazids only target mycobacteria and are specific
• use a broad spectrum of abx if you are not sure what is causing the infection to cover all your bases, bc different abx kill in different ways

• they target diff parts of the bacterial cell depending on which abx you use
• inhibit cell wall synthesis
• disrupts cell membrane function
• inhibition of protein synthesis
• inhibition of nucleic acid synthesis
• antimetabolites

• penicillin, cephalosporin, ammoxicillin
• contain beta-lactam rings that prevent bacterial enzymes from crosslinking peptidoglycan (gets in the way of the framework making peptidoglycan- fence like structure has holes in it)
• doesn't affect eukaryotic cells (no cell wall) or cells without peptidoglycan

• polymyxins: work on bacteria, especially gram -
• polyenes: work on fungal cells

• aminoglycoside antibiotics (streptomycin, chloramphenicol, erythromycin, tobramycin)
• target the translation part of protein synthesis
• work on 70s ribosomes

ie- rifamycin binds to bacterial RNA polymerase

• competitive inhibitors of enzymes (sulfa drugs and paraaminobenzoic acid (PABA) ie sulfonilmide, trimethoprim
• enzymes are very specific for substrates and these antimetabolites bind to the active site on the enzyme so it cant bind the substrate
• some, esp antiviral drugs, will act like molecules and mimic things- mimic receptor protein on the cell that virus binds to therefore tying the virus up so their bound to these drugs rather than the cell receptor
• some can "squeeze" into normal chemical structures (ie the mimicry)

• toxicity
• allergy
• disruption of microflora
• resistance of microbes

• can be really strong (ie TB drugs)
• can affect normal host tissues, particularly the liver (the detox station that breaks these things down)

• host immune system overreacts and treats the drug as foreign
• ie a penicillin allergy- recent study found many people thought they were allergic to penecillin but it was just the side effects

• antibiotics get rid of bad bacteria but also commensal bacteria, throwing off the balance
• other microbes can take over where good ones were killed off (called a superinfection) ie c. difficile

• a microbe that USED to be susceptable to an antibiotic is no longer affected by it- something has happened to make it resistant
• can be acquired through genetic changes (spontaneous mutation or uptake of plasmids during conjugation or transduction)
• some bacteria can change to "L form" and stop making a cell wall for a couple of generations which increases resistance against cell wall specific abx (won't affect them)
• can also evade abx by "hiding" in tissues (such as TB in the lung)- hides from the immune system

• Alteration of targets
• Alteration of membrane permeability
• Development of enzymes 
• Alteration of an enzyme
• Alteration of a metabolic pathway

• mutation in DNA changes a protein so that an antibiotic can no longer bind to it
• if we have an abx that targets a certain protein on the microbe, if theres a mutation that changes protein shape, the abx wont bind

mutation in DNA changes membrane proteins so that an antibiotic can no longer penetrate into the cell

• A new enzyme can break down an antibiotic
• ie- B-lactamase breaks down B-lactam rings of some antibiotics therefore maintaining the cell wall structure and the bacteria will live

• causes competitive inhibitors not to work
• an enzyme can change in some way to function even in the presence of a competitive inhibitor

• bacteria can bypass a metabolic pathway or a step in a metabolic pathway inhibited by an antibiotic
• ie: a bacteria converts substrate A to B to C to D and an antibiotic inhibits the conversion from A to B, the bacteria can find a way to convert A to C directly therefore bypassing that step and the antibiotic will have no effect

• 1. Use high enough dose for long enough period of time to kill ALL pathogens and their mutants
• 2. Use 2 antibiotics- synergism (additive effect)- ie penicillin inhibits cell walls and therefore allows more streptomycin to penetrate the cell
• 3. Restrict antibiotics only for essential use-not prescribed for viruses, can knock out good bacteria for no reason if not needed

• an example of an additive effect by synergism
• ammoxicillin with clavulanic acid
• clavulanic acid that binds b-lactamase and keeps it from inactivating amoxicillin

• When there are no antibiotics, you have a small population of highly susceptable organisms, a small population of highly resistant organisms, and the largest population of intermediate organisms (bacterial curve)
• When you start taking abx by day 3 you have knocked down the highly sensitive population, making a dent in the intermediate population, but you still have the highly resistant group
• By day 6, you are begining to feel better, your own immune system is stronger and beginning to kick in, the highly sup. pop is basically wiped out, as is most of the intermediate population, and you begin to make a dent in the highly resistant population
• by day 10, all are wiped out with the abx in combo with your immune system
• IF YOU STOP AT DAY 6 WHEN YOU FEEL BETTER: the resistant population is not yet wiped out and will take off replicating to replace the niche of wiped out organisms, making you ill again with more resistant bacteria, this type can also spread to other hosts

• Disk diffusion
• Dilution method
• Serum killing power
• Automated methods

• Kirby Bauer Method
• very similar to filter paper disinfectant test, except instead its coated with different concentrations of abx
• filter paper disks with different concentrations of abx put on a lawn of a known microbe
• clear areas around the disk indicate susceptability

• organism inoculated into broth in culture dish with many wells
• decreasing concentrations of antimicrobial agent added to wells

• helps to find a therapeutic dose
• uses blood from a patient recieving antimicrobial therapy and inthe lab microbe is added and theyll see if it grows
• is it concentrated enough in the blood to kill this bacteria

• trays with tiny wells support growth of a variety of microbes
• antimicrobial agents can be added and growth of the microbes is tested by a machine and analyzed by a computer

• the distance of cleared area around the filter paper in the disk diffusion method
• can be measured and a chart can tell you if a microbe is susceptable, intermediate or resistant

• 1. Soluble 
• 2. Selective toxicity
• 3. Toxicity not altered by food, other drugs, diseases in host (ie you don't have to take it on an empty stomach)
• 4. Doesnt cause allergies
• 5. Stable over many hours
• 6. Few strains are resistant to it
• 7. Long shelf life
• 8. Cost-effective
• 9. Patient compliance (patient needs to do what they're supposed to (ie kids taking medicine- wont bc it tastes bad, not complying)