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Microbiology Test CH 6, 7, 8, & 34

Lipids, carbs, proteins, & nucleic acids: The nutrients they need in large quantities: CHONPS Hydrogen found all over the place main source is organic. (ex: CH4, H2O) Nitrogen Find it in proteins, nucleic acids, cell wall Source is atmosphere (which is 80% N) Phosphorous found in cell membrane (in phospholipids) & N.A major source of it is inorganic molecules The E source that gives P is ATP Sulfer Find it in A.A, some vitamins Major source is inorganic, especially sulfates Immunoglobulins; IGE is measured to find if youre allergic Also, K, Ca, Mg, Fe K= required for enzyme activity Ca= required for heat resistance (give it to spores) Mg= is a cofactor

Fe= (can be 2+ or 3+) Ex: hemeproteins, or cytochromes Fe is very insoluble, but required for e- carrier molecules, cytochromes Siderophores: bind to Fe & cross membrane in some bacteria, fungi (to deliver Fe) Co, Mo, Cu, Ni: Trace elements; use metabolic pathways

We can divide groups by Energy source: Phototrophs: use light Chemotrophs: use chemicals Litotrophes: if chemical source is of inorganic nature Autotroph: use CO2 as their sole source of Carbon. (plants) Heterotroph: use preformed organic molecules as their sole C source (Humans & most bacteria)

Does every org on the planet require ____ to survive? Why? O2- no O atom- yes Oxygen is required in organic compounds & inorganic salts like PO4, SO4 O mostly comes from the air

How would you define an organic compound? Ans: it has a Carbon.

Except: CO2, CO, & HCN: they are part of elemental gases Pseudomonas can utilize more than 100 organic compounds

Some organic molecules have to cross selective permeable membrane. In order to do this they can: 1)passive diffusion: high to low 2)facilitated diffusion: use permeases - carrier proteins imbedded in membrane 3)active transport: use E (from ATP); transport of solute molec to higher concentration 4)group translocation: actively transported & chemically altered when they go through the membrane

Endocytosis: engulfs nutrients and/or viruses; for uptake of nutrients into cell Pinocytosis: cell drinking

Different types of Media: Some will be liquid: Gets very fast growth bc you can shake it or add O2 to it We can monitor growth to determine how fast it grows Some will be solid: Main advantage is isolation to obtain pure culture & serial dilutions Some will be complex: With many nutrients & chemicals TSA

Some will be enriched: Special nutrients have been added

Some will be differential: Will help distinguish bw bacterial growths EMB: lactose +/-

Some will be selective: Differentiates groups of orgs; it favors the growth of a particular microorg. This may be accomplished by inhibiting growth of undesired microorg. EMB: select for Gram PEA: select for Gram +

pH= -log [H+] Measures # of H atoms released Org able to grow at: 1-5.5: Acidophile (ex: lactobacillus) 6-8: Neutrophile (ex: most bacteria & protists) 8.5-11:Alkalinophile / basophile (ex: bacillus)

According to O2 requirements, orgs can be divided into different groups: Aerobic: requires O2

TCA, e- transport system Anarobic: killed by O2 Dont have any way to get rid of H2O2 Dont have catalase or peroxidase enzymes, so dont have e- transport system Ex: clostridium Facultative: live with or without O2 Ex: E. Coli does better with O2 present If O2 present, it can produce 38 ATP If no O2, only 2 ATP Aerotolerant: metabolically resemble anerobes but not killed by O2 All these orgs are fermentative & grow very slowly Ex: streptococcus & enterococcus Microaerophilic: this group requires O2, BUT do it at reduced pressure So we use candle jar It does make CO2, but not completely anerobic Ex: Neisseria, lipids They need low pressure to make membrane lipids

3/25 Oxygen accepts e- & is reduced.

(Singlet Oxygen: O pulled to a higher state; it is a very reactive, powerful oxidizing agent that will quickly destroy a cell-p142)(it is probably the major agent employed by phagocytes to destroy engulfed bacteria) The result is reduction products: superoxide radical, H2O2, & hydroxyl radical. These are toxic because they oxidize & destroy cellular constituents so an org must protect itself or it will be killed. Phagocytic cells: use the toxic products to destroy invading pathogens Superoxide radical: O-; is formed during aerobic respiration (Use O2 as final e- acceptor) End product of enzyme is H2O2 detrimental to all cells The enzyme Super Oxide Dismutase (SOD) & catalase- catalyze the destruction of superoxide radical & H202 Peroxidase- can also be used to destroy/neutralize effects of H2O2 H2O2(w/ catalase)H2O + O2 If bubbles, catalase + Strep is catalase NEG H202(w/ peroxidase) H20 (no O2)

Aerobe: able to grow in presence of O2 Final e- acceptor: O2 SOD + catalase so able to get rid of toxic orgs Ex: pseudomonas, mycobacterium, corynebacterium

Anerobes: able to grow without O2 (O2 kills it?) No enzymes to get rid of toxic orgs

Facultative Anaerobes: Better with O2, but dont need it

SOD + catalase Ex: enterobacteria

Aerotolerant Anaerobes: ignore O2; grow same with or without it. Ignore glycolysis; fermentative SOD no catalase Ex: strep, lactobacillus

Microaerophiles- Damaged by normal levels of O2 & require 2-10% for growth SOD yes or no catalase Candle jar Ex: Neisseria

Temperature Cardinal temperature: Minimum: 10C; Optimal: 30C; Maximum: 45C Narrow temp range: Stenothermal; 30-38 (STDs) Ex: Neisseria

Wide temp range: eurythermal 0-44 Ex: strepfaecalis

Low temps: Psychrophiles 0-20 (optimum 15) Ex: Pseudomonas, bacillus

Mesophiles 15-45 (optimum 25-45) Most all bacteria: specially pathogens

High Temps: Thermophiles 45-80 (optimum 55-80) Ex: bacillus, stenothermophilus More than 100: hyperthermophiles

Osmosis In bacteria, regulated by cell wall

Physiological solution: same osmotic pressure as body fluid Isotonic solution: same as above Animal cells & bacteria: no change to cell

Hypotonic: soln w lower concentration Animal cell- swell, burst Bacteria- no action due to cell wall

Hypertonic: Animal cellsshrink. Bacteriastop growing Plasmolysis (in prok, fungi, & algae bc has cell wall) membrane shrinks away from the cell wall Osmophilic (hypertonic): like to grow in high temps, like yeast Halophilic: (hypertonic) like to grow at high NaCl concentration

Saccarophilic: like to grow at high sugar concentration

Water activity (WA): expresses the degree of H2O availability OAW spores! They have no H2O Most orgs need 80-95% humidity to survive Free H20 to vapor Staph 85% humidity to survive Molds 60% humidity to survive bc better at H20 extraction

Microbial Growth: growth increase in # cells or in mass Binary fission: Exponential growth: progressive doubling of # of cells or mass 1- LAG phase: the org adjusting to new environment. Not much growth. 2-log (aka exponential/ growth phase): org grows. Binary fission 3-transition phase: sporulation begins; stop making antibiotics 4-stationary phase: org is running out of media; cell division & death synchronized 5-death phase: too much toxic waste accumulated; die is also exponential

Microbial growth Viable cells you can directly count the number of dead/alive cells 2 methods to find them: (must use serial dilutions for both)

1) pour-plate, then count them. Colonies on inside surface of plate

2) spread-plate, with hockey stick 3/29 Viable count: know what it is Only way to find it is spread plate & dilution technique (?) dip in alcohol, flame hockey stick, spread org, incubate colonies on surface only advantages: very sensitive, viable counts disadvantages: clumps, we have to do serial dilutions

Batch cultures closed systems No fresh media is provided to org nor wastes removed Exponential growth is reached fast (quickly)

Direct Counting: dont distinguish dead from alive cells Petrof Hausser counting chamber: used to count prokaryotes Dont get viable count bc cant tell whether cell is alive or dead Has low density cells- wont give good, accurate count Coulter counter: used to count larger orgs like protists & yeasts Have optical eye that counts electrical resistance; counts the # of cells in a soln.

Only problem is dont know if count is bc live/dead cells. More expensive & No viable cells Indirect measurement of microbial growth Spectrophotometry: measures cell mass by how concentrated your soln is; light scattering Measures turbidity: higher concentration= less light transmitted=higher turbidity no way to tell if cell is alive/dead; so no viable cell count Standard curve Microbial dry weight: best for determining changes in cell mass Use a centrifuge Useful for growth of: filamentous fungi, large cultures Cant tell if cells live/dead

Continuous Culture Systems: Open systems continual provision of nutrients & removal of wastes keeps microbial population at exponential growth phase for longer

Chemostat Fresh media w/ limited qtys of essential nutrient being added Org grows at exponential rate always while you are feeding it fresh media. Can feed it more or less by how fast you want it to go


Use photo cell to measure cell density/turbidity of the culture Can regulate flow rate of media by measuring how fast/slow is growing

CH 7: Control of Microorganisms Sterilization: process all living cells, spores, & acellular entities are destroyed/removed; free of all orgs (spores, virus, prions ALL) physical agents mainly

Disinfection: killing or removal of incubation of pathogens Primary goal: destroy pathogens, but also reduces whole population Lister was 1st

Disinfectant: used on inanimate, nonliving objects Sanitation: microbial population reduced to safe levels for public Antiseptic: something used on living tissue to prevent infection by killing pathogen growth Not as toxic as disinfectants. Stronger (?)

___-static: inhibit the growth of microorgs but doesnt kill. Bacteriostatic

___-cidal: means it will kill bacteriocidal

Conditions that will affect use of antimicrobial agents: Kind of organism: Spore formers, older cells are harder to kill Surface: flat surface easier to keep clean (also, pop size, environment) Size: larger population takes longer to kill Amt: of your disinfectant; concentration if it works better at high or low of chemical Temp: if it works better at high or low T pH: orgs dont like acidic pH (except lactobacillus)

Autoclave: use moist heat to sterilize eqpt it will be 100% sterile (kills spores) 15-20 psi the high pressure increases the b.p of H2O to 120 leave it for 15-20 minutes

Pasteurization: used to kill the pathogen w/o harming the product Does not sterilize a beverage but reduces level of nonpathogenic spoilage microorgs. 63 C for 30 min TB decreases pathogenic viruses

High T short time flash pasteurization 72 for 15 sec lowers total microbial count milk refrigerator

Ultra high T

Heat milk under steam. Reach 140C for 3 sec. In Europe, served at room temp

Tyndalization 100C for 30 min eventually free of microorganisms

Dry heat sterilization: put in oven. 170C 2-3 hours. Incineration: you degrade everything to CO2 and H2O

Low temp: slow down growth or stop but doesnt kill Liquid N2: 175 Dry ice: -70

Freezing temp: fast (doesnt kill) Vs. slow (kill it bc H2O expands & will rupture membrane) Drying Most resistant to it: the more surface, the less resistant Cocci, rool, spiral

Alter osmotic pressure Make it hypertonic by adding salt or sugar Plasmolysis: shrinking. Used to preserve food

UV light: short wavelength & high E Noniodizing radiation Creates dimers (nitrogen bases bound together)

Like to form thymine dimers (in DNA)so org cant grow: the primary mechanism of UV damage

Ionizing radiation: very short wavelength & high E which can cause atoms to lose e- (ionize) Extremely dangerous (kills spores) Rays; free radicals cold sterilization: doesnt reach any kind of T whatsoever good for plastics, antibiotics, hormones, or anything heat sensitive

Filtration: removes bacteria; mostly for heat sensitive liquids o.45 Mfilters org w/o cell wall: microplasma (can get through this) 0.3 um 0.1 um

Positive pressure Negative pressure keeps heat inside room (for isolation) try to remove as much of org as possible, so are clean rooms

3/31 Use of Chemical Agents in Control: (mostly for disinfection & antisepsis) Phenolic compounds Burns tissues, denatures proteins, disrupts cell membrane Effective for organic material

Lysol contains phenol gives strong odor Hexachlorophene antiseptic; compound found on phisohex, staph, strep Org compounds can only be used as antiseptics, not ______ bc.? What org causes TB? Mycobacteria

Alcohols Disinfectant & fungicidal mostly. Occasionally a topical antiseptic. 70-80% isopropanol; good for lipid containing viruses will denature protein & dissolving membrane lipids good against org that causes TB/ HIV; not sporicidal if not organic material, alcohol is good for instrument disinfectant dont use internally, doesnt kill hepatitis virus, kills epithelial cells

Halogens: 7A; corrosive; denatures proteins; at high T, may kill some spores CL- used to disinfect H2O; very strong oxidation agent; can cause allergies F Br- H2O I- used for antiseptic (oldest) for minor abrasions; binds itself to tyrosine; denatures proteins; at high T, it denatures spores

Heavy Metals- denatures proteins, good for disinfect, not for use internally 1% AgNO3- on eyes of newborns to stop/kill gonorrhea silver sulfadiazine- burns to keep bacteria from infection; skin grafts

CuSO4- kill algae ZnCl2- mouthwashes Quaternary Ammonium Compounds (Quats) - disinfectant organic compounds added to detergents; serve as wetting agents or emulsifiers Will disrupt cell membrane permeability Anionic: some microbial activity Cationic: disinfectants, esp on Gram+ orgs; no effect on acid fast orgs; no endospores Ex: benzalkonium chloride Aldehyde functional group is ______. Sporicidal & chemical sterilants. Can be oxidized to an acid; reduced to an alcohol Glutaraldehyde: considered a liquid sterilizer; kills spores (kills us); extremely toxic, denature, etc. Cidex: 2% glutaraldehyde; 37% formalin (formaldehyde) disinfects. Sterilizing Gases: Cl- H2O disinfectant Ethelyne oxide: toxic; microbicidal & sporicidal; sterilizing agent Liquid: H2O2- most effective against orgs that dont produce catalase, so are anaerobic. Phenol coefficient: a disinfectant screening test in which the potency of a disinfectant is compared with that of phenol Test organisms: Staph aureus: G+

Salmonella typhi: G Chemotheraputic Agents: chemicals that can be used internally to kill/inhibit the growth of microbes within host tissues. They can be used internally bc they have selective toxicity. Antimicrobial hemotherapy Antimicrobial drugs: chemotherapeutic agents used to treat infectious agents Selective toxicity: they target the microbe & do relatively little harm to the host. (Understand well & how it relates to antibiotic) Most chemotherapeutic agents are antibiotics: chemicals synthesized by microbes that effectively control the growth of bacteria. 2 Groups: 1) antibiotics- naturally made. Made by fungi or bacteria 2) synthetic drugs lab made

CH 34- Antimicrobial Chemotherapy Paul Ehrlich father of ___ agents Found the dye trypan red could selectively destroy pathogens w/o harming human cells Discovered Salvarsan used to treat syphilis

Alex Flemming: discovered penicillin Good selective toxicity against G+ orgs

Selman Waksman: discovery of streptomycin filamentous bacteria found in soil. Treated TB. About 50% of all antibiotics are made by some species of Streptomyces

Few made from bacillus Penicillium, ecphalosporium

The Therapeutic Index: the ratio of the toxic dose to the therapeutic dose. The larger the T.I, the better the chemotherapeutic agent. A drug has a low T.I if it inhibits the same process in host cells or damages host in other ways

Narrow-spectrum drugs: they are effective only against a limited # of pathogens Penicillin G+ (Breaks down bacterial cell wall peptiglycan but has little effect on host cells bc they lack cell walls; therefore, its T.I is high)

Wide- affect large spectrum of bacteria; attack many different types of pathogens G+ or G neg Bad, bc normalflora is affected/destroyed. This is disadvantage Antibiotics dont destroy yeast. Why?

Super infections: Bc some orgs are resistant to?

Kirby-Bauer -Muller Hinton agar R. I. S (resistance- antibiotics have no effect) (Intermediate) (succeptibility-good ag particular orgs) M.I.C (minimum inhibitory concentration) this is the good one to use E Test combines k.b & MIC together What is good against viruses? Antiviral drugs, NOT antibiotics

4/5 Cell Wall Inhibitors Penicillin & Cephalosporins -both prevent final cross linking of peptidoglycan inhibit transpeptidation enzymes involved in cross-linking the polysaccharide chains of the bacterial cell wall peptidoglycan Penicillin: penicillin contains B lactam ring (glues molecule ____ characteristic) penicillium known as penicillin G narrow spectrum (G+)(-cidal) staph, strep, spirochetes injected, excreted/removed in 3-6 hrs by body orally, destroyed by stomach acid so came up with. Procaine penicillin 24 hour Penicillin V resistant to stomach acids Disadvantages succeptible to ____ & penicillinases Semisynthethic penicillin Methicillin no B lactam ring So now its been replaced by oxacillin

Amoxicillin Unfortunate patients can develop allergies to penicillin

Cephalosporins: cell wall inhibitors/ affect peptidoglycan Have similar B-lactam ring Given if patient is allergic to penicillin Broad spectrum(G +, some G NEG) (more____ for G NEG orgs)

Bacitracin & Vancomycin both: linear synthesis of peptidoglycan prevents transpeptidation of peptidoglycan subunits by binding to D-Ala-D-Ala amino acids at the end of peptide cross-bridges. Thus it has a different binding site than that of the penicillins. Vancomysin (MRSA) Narrow spectrum, (G+) Has to be given intravenously, extremely painful Toxic- blood levels must be monitored Alter cell membrane permeability

Bacitracin Isoniazid (INH) Good against mycobacteria, which causes TB & leprosy Made by bacillus Effective against G+ orgs, but restrictive on topical applications

Inhibits mycolic acid synthesis Narrow spectrum mycobacterial infections, principally TB Rifannpin Is a nucleic acid inhibitor

Cell Membrane Inhibitors Bind to phospholipid, so will alter cell membrane permeability. Only used for topical application so the therapudic index will not be as high Polymyxin B (-cidal) Produced by bacillus before they start sporulation Very good against G NEG orgs, esp pseudomonas binds to plasma membrane & disrupts its structure & permeability properties narrow spectrum G NEG only Bacitracin & Polymyxin B you can buy ointment w/o script. But if add neomycin: broad spectrum protein synthesis inhibitors Aminoglycosides (-cidal) bind to small 30S subunit & interfere w protein synthesis by directly inhibiting synthesis & causing misreading of mRNA Neomycin- Broad spectrum (G NEG, mycobacterium) Streptomycin- Narrow spectrum (aerobic G NEG) Nystatin & Amphotericin

Both antifungal Liver enzyme functions Primary choice for histoplasmosis bird droppings (farmers) & also coccioliomycosis (found on soil) Increase fungal membrane permeability Nystatin origin is NY state; extremely good for treatment of yeast

Protein Synthesis Inhibitors: happens on ribosome on euk ATP made on ribosomes important- if you use too high of a therapeutic index on a proteins synthesis inhibitor, you will damage the ribosome (?). be able to know if its index is too high, low, etc Nitroaromatics Orally, naturally occurring antibiotics w/ benzene ring

Chloramphenicol (-static) Streptomyces 50 S subunit to inhibit peptide chain elongation during protein synthesis blocks peptide bond formation extremely toxic broad spectrum antibiotic ( some G +, some G NEG, rickettsia & chlamydia) bc of its small size, it diffuses into the cell spinal fluid

meningitis, typhoid fever suppresses bone marrow activity a plastic anemia

Aminoglycosides (-cidal) Only account for about 3% of amino acids being used Also produce Streptomyces An example: streptomycin. (discovered 1944) Attached to it are: tobramycin, gentamycin, karamycin, neomycin Used as G NEG org, esp pseudomonas Neomycin- ointment used for triple antibiotic ointment Tobramycin- kids for cystic fibrosis 30S misreading of the genetic info carried by mRNA

Tetracycline (-static) Bind 30 S Prevent introduction of new amino acid Stop protein synthesis Streptomyces produces this Broad spectrum inhibits most G+/G NEG orgs Penetrates tissues extremely well so very good

Ag intracellular rickettsias or chlamyclias Good ag/ UTI & mycoplasma (no cell wall) pneumonia, syphillus, gonorrhea Candida- like to lead to super infections Sensitive to UV light avoid sun Can cause yellow teeth (esp in kids)

Macrolides (-static) Binds to 50 S subunit to inhibit peptide chain elongation during protein synthesis Broad (aerobic & anaerobic G+, some G NEG) Ex: erythromycin Drug of choice for patients that are allergic to penicillim Good ag/ staph, strep, ear infections, whooping cough, mycoplasma, G + orgs Cannot penetrate wall of G NEG outer wall

Eukaryotic Protein Inhibitor Cyclohexamide- antifungal Very dangerous goes for the whole ribosome 80S

Nucleic Acid Inhibitors Inhibit n.a synthesis Not as strong

Rifampin (-cidal)has a limited mode of action; blocks RNA synthesis so cell cannot divide Gets produced by streptomycin For patients that have TB or leprosy Used as a simple antibiotic Good- it can reach high levels of the cerebral spinal fluid. If too much, can give liver problems. Recommended as a profylaxis. Ex: Neisseria, meningitis carriers

Quinolones (-cidal) Broad spectrum like to inhibit DNA synthesis Block the enzyme DNA gyrase, therby blocking DNA replication & transcription Good for UTI treatment & travelers diarrhea

Cipro Synthetic antibiotic Braod spectrum Oral 40-50% gets secreted in urine Blocks DNA synthesis

Growth Factor Analogs Sulfa drugs The action of it (the mode of it): competitive inhibition

Able to synthesize its own folic acid (vit B), which is required for growth Humans dont synthesize their own, but e. coli can in order for it to grow.

E. coli paraamino benzoic acid (PAB) One of the precursers for folic acid synthesis The org competes with PAB. Sulfa drugs only work on orgs that make their own folic acid. Ex: silver sulfa dyazine Used for burns & eye infections