micro test 3 study guide

8/4/2019 Micro Test 3 Study Guide

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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 you’re 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

○ Don’t have any way to get rid of H2O2

○ Don’t have catalase or peroxidase enzymes, so don’t 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 don’t 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: 10°C; Optimal: 30°C; Maximum: 45°C

Narrow temp range: Stenothermal; 30-38° (STD’s)

• 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 cells–shrink. Bacteria–stop 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

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

3/29

Viable count: know what it is

• Only way to find it is spread plate & dilution technique (?)

Batch cultures – closed systems

• No fresh media is provided to org nor wastes removed

• Exponential growth is reached fast (quickly)

Direct Counting: don’t distinguish dead from alive cells

• Petrof Hausser counting chamber: used to count prokaryotes

○ Don’t get viable count bc cant tell whether cell is alive or dead

○ Has low density cells- won’t 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 don’t 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

Turbidostat-



• 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 doesn’t 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 don’t 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 140°C for 3 sec.

• In Europe, served at room temp

Tyndalization

• 100°C for 30 min

• eventually free of microorganisms

Dry heat sterilization: put in oven. 170°C 2-3 hours.

• Incineration: you degrade everything to CO2 and H2O

Low temp: slow down growth or stop but doesn’t kill

• Liquid N2: 175°

• Dry ice: -70°

Freezing temp: fast (doesn’t 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”: doesn’t 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 C ontrol: (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

○ don’t use internally, doesn’t 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 don’t 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 don’t 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

Made by bacillus

Effective against G+ orgs, but restrictive on topical applications

• Isoniazid (INH)

○ Good against mycobacteria, which causes TB & leprosy



○ 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 & traveler’s 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 don’t 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



03/23/2011

micro test 3 study guide

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