Microbiology Test 2

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Transverse Binary Fission

Back 1

How bacterial cells divide; cells grow to 2x their original length then divide into 2 daughter cells

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Formula for # of cells after a certain length of time

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B = Bo x 2^n
logB = logBo + nlog2
n = # of cell divisions

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Factors needed by all cells to grow

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1- carbon, energy source
2-nitrogen- amino acids, proteins, ammonia
3-vitamins- coenzymes
4-metals- cofactors for enzymes
5-water

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Coliform

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indicator organism; E.Coli; indicates fecal contamination; suggests sewage, feces contaminating sample

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Pasteurization

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Done to kill pathogens
(raw milk: 50-100 coliforms/ml, pasteurized: <5 coliforms/ml)

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E.Coli as an indicator

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if present, means other harmful bacteria may also be present; ferments lactose to acid and gas (test pH and gas formation in Durham tube)

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Coliforms in Drinking water

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1-4/100 ml H2O
Urinary tract infections

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Coliforms in water w/ shell fish

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70/ 100 ml
<10,000 bacteria/ml --> no infection

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Coliforms in rec. waters

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1000/100 ml
> 10,000/ml --> infection

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Microscopic counts

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Petroff-Hauser chamber, count # bacteria in each square of grid, account for dilution factor- must kill cells to count (therefore counts living and dead cells)

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Electronic Counts

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- charged cell passes thru + charged pore --> measure voltage w/ galvanometer when cell passes thru pore; counts living and dead cells; blood counts done this way

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Standard Plate count

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counts only living cells; assumes each colony = 1 cell (problem for streptococci); 1 g or 1 ml placed in 9 ml H2O and then diluted by factor of 1:10,000 ; place one ml of solution on plate and count # of colonies, take avg. # colonies and x by 10^4 (or dilution factor); bias due to pH, carbon source, temp., oxygen presence; need to have an idea of what type of bacteria are present in order to use this method

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Most Probable Number counts (MPN)

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based on some growth parameter like fermentation of lactose for ex.; used to determine # of coliforms in a sample; add sample to lactose broth, determine # of positive tuves (acid + gas), look at statistical table which tells the # of coliforms based on the # of positive tubes

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Indirect Method

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test for CO2, RNA, DNA, protein to determine if bacteria are growing; technique used in probe to Mars (gave false positive though due to radioactive CO2)

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Factors affecting bacterial growth

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Temp, O2, pH, water activity

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Bacterial growth curve- phase I

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lag phase: period of metabolic adjustment, cells synthesizing enzymes necessary for growth

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phase II

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log phase: cells actively growing, "balanced growth" meaning constant composition/increase of DNA, RNA, protein; time when antibiotics most effective

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phase III

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Maximum Stationary Phase- cells growth begins to level off b/c cells produce toxic wastes that inhibit growth ("race suicide") and b/c they exhaust nutrients; no net increase in cells (no cells dividing, or # dividing = # dying), cells living off internal reserves, basal metabolism

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Phase IV

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Dath phase- all internal reserves exhausted, cells die- some resistant cells can survive on materials from cells that have lysed (kind of bacterial canabalism= autophagy)- death part of curve and resistant cells show why you must take full course of antibiotics even though you feel better, must kill all of the cells including the resistant ones

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D-value

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Decimal reduction time- time it takes to kills 90% of a population at a given temperature (canning companies either increase time or temp to kill all cells including resistant ones and endospores)

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Temperature range for bacterial growth

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-5 to 113+ C

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psychrophiles

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cold loving; -5 to 15 C; found in super-cooled waters of arctic and antarctic; bacillus, pseudomonas

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Mesophiles

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25-45 C; most bacteria fall into this group including ALL pathogens

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Thermophiles

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45-100+ C; found in volcanic hot springs, compost piles, hot water heaters, cooling towers for nuclear power plants

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Hyperthermophiles

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> 100 C

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relationship b/w treponema pallidum and plasmodium vivax

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treponema pallidum causes syphilis, tries to invade CNS, grows just below body temp.; plasmodium vivax is a protozoan that causes malaria, attacks red blood cells and when cell bursts parasite is released, causes fevers of >104; these high temps will kill treponema pallidum and will cure syphilis if patient has syphilis and malaria

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psychrotrophs

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mesophilic organisms that will grow at 4 C (not optimal growth though), fridge temp, causes food to spoil

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proteus vulgaris

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causes eggs to spoil, produces hydrogen sulfide which gives rotten egg smell

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campylobacter

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from chicken, causes gastroenteritis

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Pseudomonas

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vegetables, not pathogenic

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Leuconostoc

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grows on processed meats, breaks down meat, not a pathogen though

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osmophiles

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live in high sugar (low Aw); xeromyces (yeast), can spoil jams and jellies

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halophiles

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live in high salt conc., low Aw; halobacterium grows on salted fish, hides (non-pathogenic)

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Relationship of (low) Aw to osmotic pressure

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high sugar/salt, high osmotic pressure outside cell, low osmotic pressure inside cell, water trying to leave cell --> membrane shrinks, cytoplasm dehydrates; called plasmolysis

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sterilization

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complete destruction of all microbes incl. endospores, viruses and cells; can be done by steam pressure, incineration, gas such as ethylene oxide

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Disinfection

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destruction of pathogenic microbes and toxins; applies to INANIMATE OBJECTS; ex. bleach (1/10 dilution), lysol

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Antisepsis

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destruction of microbes ON THE SKIN; alcohol, betadine; more gentle than disinfection

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Bacteriocide (or virocide or fungocide)

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agent that kills vegetative cells but not endospores

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bacteriostatic (fungalstatic)

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prevents multiplication but does not kill cells, if removed organism can resume growth; ex. sodium benzoate, calcium propionate added to bread to retard spoilage

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factors involved in killing of microorganisms

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1. time of application
2. conc. of agent (ethyl acohol must dilute, isopropanol 100%)
3. environmental conditions
-temp. (higher temp., faster rxn)
- nature of surface, pourous, etc.
-presence of extraneous materials such as blood, feces, urine, meat juices, will reduce effectiveness of agent

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physical agents- heat

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1. dry heat (like oven)- slow process, oxidizes proteins
veg. cells- 1 hr at 170 C
endospores- 3 hr at 170 C
2. moist heat (like boiling water)- faster process, denatures proteins (can't be reversed)
veg. cells- 10 min. at 70 C
endospores- 15 min. at 121 C, 15 psi steam pressure--> autoclave

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pasteurization

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milk- flash pasteurized, 72 C for 15 sec.
milk is sterile in udders, streptococcus, lactobacillus occur on udder, harmless but cause milk to spoil
bacteria can also come from human handlers

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single serving cream or juice container

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treated with H2O2 at 140-150 C for several seconds

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Pathogens that occur in milk

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1. mycobacterium tb- M. Bovis in cows, bone TB
2. Coxiella burnetii- a rickettsia, grows in a host cell, causes Q fever in man, forms endospore-like structures and becomes more resistant to high temps.
3. salmonella- causes gastroenteritis, typhoid fever; usually from fecal contamination
4. Brucella abortus- malta or relapsing fever, causes spontaneous abortion in cows, can pass thru cracks in skin
5. streptococcus enterrococcus- sore throats, found in intestines of man and animals
6. listeria monocytogenes- gastroenteritis, causes meningitis if it enters CNS- raw milk, soft cheeses, cole slaw, hot dogs

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Radiation

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UV light, gamma (X) rays
cause mutations in DNA
causes DNA to fragment
used to treat potatoes, poultry, pork, fruit, spices

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Why is control of bacteria in food so important?

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76 million cases of food borne illness each year; 300,000 hospitalized, 5000 deaths

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Culprits of food borne illnesses

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salmonella, e.coli, campylobacter (poultry), staph, viruses

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light spectrum

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x-rays and UV: short range, high intensity and penetrating power
IR: longer wavelength, low intensity w/ little penetrating power
Microwaves: long wavelength- only kill microbes if they generate sufficient heat to do so (microwave ovens do not)

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Radiation resistant bacterium

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Dienococcus radiodurans- can withstand 1000 x dose that kills e.coli on food and can also repair breaks in DNA; isolated by the army

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Filtration

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beverages- beer and wine
filters out yeast and contaminants, does not remove viruses (needed a new method to clean up optical solutions)

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affect of cold/freezing on microorganisms

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- slows down metabolism
- affects membrane transport

-some cells die during freezing b/c micro ice crystals form in cytoplasm and puncture membrane

-some bacteria can increase during freezing if micropockets of water and nutrients are present, occurs when food is thawed

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chemical agents- phenols, alcohols

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-denature proteins
-ex. lysol, phenol, hexachlorophene
-ethyl acohol (want 50-80% conc.)
-isopropyl (rubbing alcohol)- want 100% conc.
-do not always kill viruses, e.g. hepatitis is volatile

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hexachlorophene

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biphenyl group, less toxic to skin then phenol, very effective at controlling staph aureus in hospitals (in green soap drs wash with)- staph causes post-surgical infections and infections of umbilical cords (babies given hex. sponge bath- don't put baby in bath b/c hex. can cause birth defects if it enters CNS)

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Soaps and detergents

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disrupt membranes
effective against many bacteria, some viruses (esp. flu which has an envelope which is destroyed by soap)

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oxidizing agents

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destroy biological molecules, esp. proteins and nucleic acids
ex. H2O2, Cl, I, Br, MnO4-, betadine (organic I)
-Cl used to treat H2O supply; requires 10 x to kill polio virus than coliforms
-bleach (NaClO), 1/10 dilution, good disinfectant

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Semmelweis

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1846, dr. in ob-gyn ward, made physicians wash up with bleach before delivery b/c women were dying of child birth fever (puerple sepsis) caused by streptococcus pyogenes- dries out hands and painful, but worked

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heavy metals

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combine with sulfur groups in proteins to denature them
-AgNO3- applied to eyes of newborns to prevent blindness caused by neisseria gonorrhoeae
-Hg used to treat syphilis in 1496; Hg is a major concern in food supply, in fish esp. tuna
-Pb- lead paint causes mental retardation in children
-organic forms of Hg, merthiolate, mercurochrome used as antiseptics

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alkylating agents

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formaldehyde- used to preserve tissue, bodies
ethylene oxide- gas, kills endospores, used to sterilize plastic, requires 8 hr. exposure
glutaraldehyde

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antibiotics

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produced by microorganisms, 1st antibiotic = penicillin (Fleming), used to treat staph

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antimicrobial

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chemical synthesis to produce
ex. sulfa drugs, used to treat bladder infections
ex. salvarsan- aresenic compound used to treat syphilis

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mode of action of antibiotics/antimicrobials

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1. inhibit cell wall (peptidoglycan) synthesis
2. disrupt membranes- can combine w/ sterols
3. inhibit protein synthesis on 70s ribosomes- ex. streptomycin, chloramphenicol
4. inhibit nucleic acid synthesis
ex. nalidixic acid- cipro- DNA, rifamprin- RNA (TB)
5. antimetabolites- sulfa druges, inhibit E. Coli from producing its own folic acid

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Folic acid

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coenzyme- assists an enzyme in metabolism
e. coli can synthesize its own folic acid
sulfa drug looks like PABA which is part of folic acid, but instead prevents e.coli from synthesizing folic acid and carrying out metabolism

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Side effects of antimicrobial therapy- hypersensitivity

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1. hypersensitivites to drugs (sulfa, penicillin)- drug binds to protein and body thinks protein is foreign and responds against the protein --> mild sensitiviy = rash, serious sensitivity = anyphylactic shocks

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side effects- drug toxicity

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chloramphenicol- aplastic anemia
streptomycin- dammage to 8th cranial nerve which is involved in hearing and can cause deafness

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side effects- super infections

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usually happens w/ chronic antibiotic use- antibiotics kill normal flora and opportunistic pathogens can grow- ex. candida albicans (yeast infection)

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side effects- drug resistance

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-efflux mechanisms- drug transported into cell but back out immediately, antibiotic doesn't have time to act
-mutations/changes on receptors where antibiotics bind on 70S ribosomes
- multiple drug resistance on plasmids
-B-lactamase destroys penicillin - staph aureus

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Virus- defn.

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from latin- poison
1st defined as infectious agents that could pass thru a fliter
- smallest agents capable of causing disease!

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Virion

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single virus particle

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Are viruses living?

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composed of proteins surrounding nucleic acid (like living), but cannot live independently (non-living)

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obligate intracellular parasites

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need to invade a host cell in order to survive and replicate (viruses)

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size of a virus

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20-250 nm; can only see under e- microscope

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specificity of viruses

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specific for a host and type of tissue (organotropism = tissue specificity)
-animal viruses only infect animals
-plant viruses only infect plants

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nucleic acids and proteins in viruses

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only one kind of nucleic acid; DNA or RNA, NOT BOTH!
protein coat called CAPSID surrounds genetic material

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envelopes

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viruses- membrane derived from host

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Classification of viruses

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no accepted scheme:
a- type of diseases (pheumotropic- lungs, dematotropic- skin, viserotropic- blood and internal organs, neurotropic- nerves, CNS)
b- organized into families base on structure, size, properties (ex. DNA plant, DNA animal, RNA plant, RNA animal, etc.)

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Helical virus

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ebola, rabies, tobacco mosaic virus
composed of nucleic acid inside capsid; capsomere (protein) protects nucleic acid

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polyhedral virus

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polio
icosahedron (20 sides)- capsid
nucleic acid
capsomere- globular protein subunits

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enveloped viruses

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capsid of virus enclosed by envelope (membrane) w/ viral spikes

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influenza spikes

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1. hemagglutin- causes RBC's to clump
2. enzyme that destroys a chemical on respiratory cells

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Bacteriophage

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bacterial virus
made of capsid on sheath, base plate w/ tail fibers (help degrade cell wall of bacterium) and spikes (have lysozymes)
binds to chemicals on surface of cell

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2 possible outcomes to a viral infection

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1. lytic cycle- cell death
2. lysogenic cycle- alteration of cell genetics

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1st stage of lytic cycle- binding

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attachment of virus- capsid, spikes, tail fibers bind to chemicals on the host cell

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2nd stage lytic cycle- penetration

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animal cells- endocytosis- virion surrounded by host cell membrane; virion enters cell in a membrane vesicle; capsid removed by host organism (host enzyme in vesicle)

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3rd stage lytic cycle- degradation of host cell DNA

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virus prevents host cell from dividing, making protein, etc. virus uses host cell systems to make viral components

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4th stage lytic cycle- biosynthesis of viral components

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copies of viral nucleic acid, viral components (capsides, tail fibers, sheath, base plates, etc); viral nucleic acid inserted into capsid; assembly of virions by self-assembly

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5th stage lytic cycle- release of virus from host cell

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a-lysis of cell
b- exocytosis- vesicle carries virus to cell membrane, virus released w/o vesicle
c- budding- virus released with host cell membrane forming an envelope, spikes

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lysogenic cycle- 1- attachment

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same as lytic

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lysogenic- 2- penetration of host cell

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same as lytic

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lysogenic- 3- viral DNA incorporates into host DNA to become a pro virus

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-provirus is a stable genetic element in host DNA
-provirus is copied each time host dna is copied and thus all progeny are infected
-no new virions are made
-host cell is immune to further infections by the same virus
-provirus can alter normal host cell gene function = genetic change
-virus can confer new genetic info on cell = lysogenic conversion
-if nothing perturbs virus (chemicals, UV light, etc.), virun will remain in cell for duration of cell lifetime

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lysogenic conversion of corynebacterium diphtheriase

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non-pathogenic, then bacterial DNA infected by virus, virus encodes for diptheriae toxin gene --> pathogenic, toxin inhibits protein synthesis on 80s ribosomes in your cells- diphtheria causes sore throat, membrane over throat

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end of lysogenic cycle

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-virus may enter lytic cycle
-provirus excised from DNA, viral DNA encodes for functions of lytic cycle, cell lyses and mature virions will infect new cells

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latent

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lysogenic

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viral DNA as stable genetic elements in human chromosomes

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may or may not be expressed, can be expressed several times over the lifetime of an individual; copied when chromosomes are copied

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Herpes type I

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causes cold sores, fever blisters, affects nerve cells in face

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Herpes type II

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genital herpes; Valtrex causes virus to go back into provirus state, does not get rid of herpes

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herpes zoster

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children- chicken pox
adults- shingles
nerves in trunk affected

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factors that cause expression of latent/lysogenic viruses

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- hormonal changes, e.g. menstrual cycle in women
-UV light
-stress (hormones)
-chemicals in diet

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transforming viruses

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cause normal cells to behave like cancer cells
-surface of the host cell becomes like a cancer cell b/c new chemical groups are expressed
-infected cells stop dividing in a controlled fashion and behave like tumor cells

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Ex. of transforming viruses

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1. HPV- herpes virus, cervical cancer
2. Epstein-Barr virus, mono in US, lymphoma in African populations (Burkitt's lymphoma)

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Retro viruses

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RNA used as template to make DNA, retrovirus used reverse transcriptase, works "backwards"

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Ex. of retro viruses

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-HTLV- human T-cell lymphotrophic virus
-AIDS- infects T- helper cells

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How do viruses cause cancer

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16% of human cancers caused by viruses
How:
1. virus integrates into human DNA, can inactivate a gene or cause the gene to be altered in its expression
2. some viruses carry oncogenes- normal genes picked up by virus, mutation occurs, results in oncogenes in virus- oncogenes take over functions in cells such as cell division causing cell to become abnormal

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Human cancer viruses

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-papilloma
-herpes C- liver cancer
-herpes virus 8- Kaposi's sarcoma- AIDS patients
-HTLV

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control of viruses- immune system

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-antibodies- aggregate virus cells, then WBC's kill viruses
-interferon- protein made by cells infected w/ a virus, inhibits viral replications, released by infected cells and taken up by uninfected cells to protect them

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control of viruses- vaccines

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stimulate the immune system- polio, mumps, measles, small pox

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control of viruses- chemical control

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-nucleotide base analogues- look like normal DNA bases, inhibit viral DNA replication- ex. AZT- AIDS; acyclovir (valtrex)- herpes type II
-agents that inhibit binding of viruses to host cells or inhibit the release of virions from infected cells- ex. amantadine- prevents attachment of flu virus; tamiflu- prevents release of flu virus
-reverse transcriptase inhibitors- prevent retroviruses from working
-protease inhibitors- some viral proteins must be cleaved by protease before the protein is used to make viral components (AIDS)
-chemical/physical agents- UV, heat, Chlorine, phenol, soaps, etc.

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Cultvation of viruses

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-fertilized chicken eggs- embryonic cells
-tissue culture cells- can also be used in detection; virus causes the cells to become abnormal = cytopathic effect

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detection of viruses

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1- some viruses (flu) cause RBC's to clump
2-viral pathology
3- plaques- clear areas in bacterial lawns where bacteriophage have caused lysis
4-observation w/ e- microscope

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viral pathology

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negri bodies- rabies
Lipschultz bodies- herpes viruses
Downey cells- abnormal WBC's in mono

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colds- why we get so many- flu

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rhino viruses (colds), >100 different viruses
flu- also different viruses and variations of viruses

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antigenic DRIFT

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type A virus --> mutation to type A1 (ex. mutated spike)

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antigenic SHIFT

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2 different influenza viruses can infect a cell at the same time- ex. human and avian flus- when virus is assembled a mosaic virus is made (ex. human capsid and avian spike)- immune system thinks it has not seen this virus and immune response must start from scratch

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viral like agents- viroids

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small RNA molecules, infect plants, mechanism for causing disease is unknown

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viral like agents- prions

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infectious protein particles
-degeneration of neurons in brain, loss of motor function, paralysis, death
-prions accumulate in brain to cause destruction of brain tissue- spongioform encephalitis
-prions are resistant to chemicals, autoclaving, UV, radiation- only destroyed by high temps. such as w/ incineration
-can "reproduc"- ex. converts neuron protein a-helix into prion

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diseases caused by prions

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Kuru- humans, new guinea
mad cow- cattle, humans?
scrapie- sheep
Jacob-creutzfeldt disease - humans

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origin of viruses

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run away genes --> acquired a capsid --> virus

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transposons

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genes that jump from one DNA molecule to another- ex. maize