Micro Exam 2 Test Questions – Flashcards
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Unlock answersLab definitions: Peptone Fastidious Axenic |
p: short peptide units, fragments of amino acids f: picky, may not grow without an enrichment a: pure culture |
What is important to match when determining bacteriological media? |
Match physiology with medium thats appropriate in order to be cultured
- some species cannot be cultured because the medium needed is unknown |
Culture medium |
nutrients required by a particular group of microorganisms |
Photolithotrophic Chemolithotrophic Chemoorganotrophic |
p: photosynthetic, source of reducible carbon, energy of sunlight to ATP cl: inorganic environment, ferrous sulfate co: chemoheterotrophic, organic source of carbon, organic substrate for ATP yielding oxidation reactions |
Natural culture media |
- things that spoil (vegetables, fruit, milk) - human body, water, soil - Pasteur used a sliced potato |
3 artificial culture media |
1. Synthetic/chemically defined- ideal, but expensive, time consuming and difficult- exact amounts 2. Non-synthetic- micro lab, exact formulation unknown 3. Complex- include extra substances, fastidious substances will grow |
Agar |
convert liquid medium to solid seaweed/kelp extract, frequent solidifying agent |
Selective vs. differential media |
s: designed to allow the growth of the desire microorganism one is trying to isolate and suppress the growth of others (whats added or left out) d: distinguish between two types of growth on a plate; phyical appearence of colonies when cultured (pH indicator, fermentable carbohydrate) |
MacConkey's medium vs. blood agar |
m: selective medium, bile salts and crystal violet are incorporated to inhibit growth of some bacteria and permitting others b: differential medium, erythrocyte effects - gamma = no effect, alpha = destruction, beta = complete breakdown |
4 common non-synthetic media |
1. tryptic soy agar/broth- wide variety of bacteria (all purpose), chemooranotrophs 2. eosin methylene blue agar- dyes as indicators for differentiation that ferment lactose/produce acid 3. phenylethyl alcohol agar- select growth of a large group of bacteria (limits g-) 4. macconkeys agar- intestinal bacteria, ferment lactose (gray vs. red) |
Preparation of media |
1. proper amounts of each ingredient must be dissolved in water 2. check pH/adjust 3. medium dispense into containers 4. sterilized in autoclave or by filtration (121 degrees), can kill endospores |
T-streak technique |
use inoculating loop to distribute bacteria into 3 regions of petri dish - top to bottom, right to left, bottom to top - sterilize after each region - separate components of a mixture |
What to mark on petri dishes |
mark on bottom: medium, bacteria, group identifier, date, incubation temperature |
1. Serratia marcescens 2. Staphylococcus epidermidis |
1. g-, bacillus, broth, nosocomial infection (urinary or GI) 2. g+, coccal, broth, skin flora |
Gas pak system |
- experimental atmosphere - different atmospheric conditions can be established - used in labs that cannot afford glove boxes |
Acidity's effect on environmental growth (3) |
acidophiles- grow at pH of 0 or 1, below 5.5 alkalophiles- grow at pH of above 8.5 neutrophiles- grow at pH between 6.5 and 7.5 * majority of disease causing bacteria |
Helicobacter pylori |
Lining of the stomach- flagella to move grows in acidic condition helical shape g- |
3 ways a microbiologist can measure growth |
1. biomass measurements 2. direct cell counts 3. viable direct cell counts |
Turbidometric measurements |
- type of biomass measurement - taken by spectrophotometer or klett sommerson photometer |
Spectrophotometry |
- single wavelength of light (monochromatic) neturalizes yellow medium at 540 nm - registered on photo electric cell - read at 100% transmission - % transmission decreases as more light comes through - medium becomes darker color |
Advantage/disadvantage of spectrophotometry |
advantage: easy to do disadvantage: does not distinguish between living and dead cells |
Direct cell count (4) |
- Petroff-Hauser counting chamber - Hemocytometer- blood cell count - Coulter counter/FACS machine - DMC- direct microscopic count/breed smear ** all do not distinguish between viable and dead cells |
Coulter counter/FACS |
- AKA particle counter - thin capillary tube, electric current, electrical detector, automatic counter - 2/10 of a mL sample size - can set window/gate of what cells to be counted within a size range - FACS= fluorescence activated cell sorter |
Viable direct cell counts |
- standard plate count (SPC)- measure of formation of colony (most accurate method) - colony forming units/mL on plates- ableto divide and form a population of identical cells - growth takes time, not immediate results - plate count x dilution factor - absorbance graph/standard curve can show # of viable cells - MPN- most probable number, water quality |
Sign of food irradiation |
radura |
Physical vs. chemical control of microorganisms |
p: filtration, heat/temperature, irradiation c: anitbiotics, chemotherapeutic |
Sterilization |
- destruction or removal of all life forms, microbial or otherwise in or on a product referred to as sterile after - done by heat, irradiation, filtration, selected chemicals - physical means are usually more reliable or absolute than chemical measures |
Disinfection |
- the reduction of the number of organisms (with or without sterilization) to a level that is not harmful to the health of a living animal or the quality of perishable goods - destruction of vegetative cells but rarely spores - treatment of inani-mate surfaces and objects |
Sepsis |
- poisoning or breakdown of living tissue due to the growth of microorganisms or the presence of their products - usually manifests itself as the formation of pus |
Antiseptic |
- chemical agent used in the destruction or inhibition of microbial growth on living tissue - limiting or preventing the harmful effects of infection |
Sanitize |
- reduction of the number of microorganisms to an acceptable level - dictated by local public health agencies |
-static vs. -cidal |
- static means to inhibit - cidal means to kill |
Chemotherapetuic agent |
compound that is used for the treatment of infections/neoplasia in man or animals |
Chemoprophylactic agent |
compound that is used for the prevention of infection in man or animals - ex) the administration of quinones and quinone when entering areas indigenous with malaria |
Biohazardous material |
- food- Campylocacter & Salmonella spp. - blood/blood products |
Type of organism expected in antimicrobial procedures (6) |
- endospore- Bacillus/Clostridium spp. - Staphylococcus spp. (aureus) - Mycobacterium spp.- acid fast/mycolic acid makes more resistant - Pseudomonas spp. (aeruginosa) - fungal cells- dermatophytes - viruses- naked or enveloped |
Turbidity |
cloudiness of a fluid (broth) caused by individual particles - asses amount of microbial growth - why microbiologists need special instruments to measure growth * colorimeter or spectrophotometer at 500-700 nanometers |
Selection of an antimicrobial procedure (4) |
1. type of organism expected 2. microbial load 3. environment organisms are found 4. intended use of the material/situational considerations |
Microbial load |
- number of organisms (present/anticiapted) - logarithmic growth - best temperature, time length, and medium for 90% of population to become inactivated |
Decimal reduction value/time |
- time it takes for a population to become inactivated, very predictable, logarithmic - longer to inactivate larger population - decrease by 1 log at a time (10^8 to 10^7) |
D value varies with what 3 things? |
1. temperature (ex. E. coli at 70 degrees has smaller d value than 50 degrees) 2. genera of bacteria (heat sensitive is closest to axis, endospores farthest) 3. foodborne pathogens (Staph. aureus has largest d value) |
D values are important in what industry? |
food production - in order to prove sterilization |
Importance of the environment in which organisms are found in antimicrobial procedures |
- prescence of organic products (carbohydrates and fats)- more present, longer time to treat it - temperature, pH and extraneous environment |
Heat as a disinfectant/sterilant (2) |
1. dry heat- incineration or hot air oven 2. moist heat- boiling water, pasteurization, or heat with pressure |
Incineration vs. hot air oven |
- both dry heat i: place into flame (bunsen burner, benchtop incinerator) h: 160-170 degrees for 2 hours, will inactivate endospores |
Boiling water as moist heat |
- will kill vegetative cells in 5 minutes at 70 degrees C - inactivate enveloped viruses and fungi - not a sterilant because does not inactivate endospores and naked viruses - differential sensitivity- 30 minute exposure maximum |
Pasteurization as moist heat |
- developed by Pasteur for beer - used to prevent Mycobacterium bovis in milk - Coxiella burnetti is controlled by pasteurization- can cause respiratory disease |
Pasteurization types (3) |
1. Batch- LTLT- use vat at 62 degrees C for 15 minutes 2. Flash- HTST- tubes at 72 degrees C for 15 seconds **milk in grocery stores 3. ultrapasteurization- UHT- ultra high temperature, sterile, not refrigerated, 142 degrees C for 3-4 seconds |
Heat with pressure in moist heat |
- autoclave/pressure cooker - considered to be sterile - saves time - minimum of 15 minutes at 121 degrees C with 1 additional atm for 1 tube - more volume, more time- takes longer to penetrate the center (ex. molasses) |
Which media promotes/inhibits growth of gram + and gram - organisms? |
gram +: limited growth with MAC and EMB so PEA promotes growth gram -: limited growth with PEA so MAC and EMB promote growth |
Colony morphology |
- general all purpose growth medium - form, elevation and margin - elevation is 3D and seen with stereomicroscope - color and size - light passage |
What dyes inhibit peptidoglycan production? |
cationic- crystal violet, MAC, methylene blue |
Salmonella typhi |
typhoid |
MSA |
mannitol salts agar - 7.5% NaCl (high concentraion) - inhibitory to everything but Staphylococcus spp. - can tolerate salt, turns yellow - selective and differential |
Lactose fermenter colors depending on medium |
MAC- fermenter is red, non-fermenter is white EMB- fermenter is metallic green |
SBA |
type of blood agar with 5% blood cells - 3 types of hemolysins: * alpha- incomplete, viridans * beta- complete, strep throat * gamma- ahemolytic, without - classify Staphylococcus & Streptococcus spp. with agar - selective to differential medium |
Paul Ehrlich |
- important with antibiotics and chemotherapeutic drugs - first chemotherapetuic therapist - worked with Treponema pallidum - arsphenamine/compound 606/salvarsan- chemical containing arsenic that was effective in treating syphillis |
Sir Alexander Fleming |
- important with antibiotics and chemotherapeutic agents - second golden age of microbiology - work led to penicillin discovery - discovery of lysozymes - mold growing on Staphylococcus auerus helped penicillin discovery - zone of inhibition- cells died |
Antibiotic vs. chemotherapeutic agent |
a: chemical produced by certain molds and bacteria that kills or inhibits the growth of other microorganisms ca: chemical used as a therapeutic medication to treat a disease |
Types of antibiotics and chemotherapeutic agents |
a: Streptomyces spp., Penicillium notatum/chrysogenes, soil organisms c: compound 606 |
3 fundamental ways of "marketing" chemotherapeutic agents |
1. antibiotics- limit competition in environment with limited resources 2. Synthetic- man made in laboratory 3. Semi-synthetic- antibiotics produced by microorganisms then altered by organic chemist |
Antibiotic examples (3 species) |
1. streptomyces spp.- streptomycin, tetracycline 2. bacillus spp.- bacitracin, polymyxin 3. penicillium spp.- G and V
|
Synthetic and semi-synthetic examples |
synthetic: chloramphenicol, sulfonamides semi synthetic: penicillin derivatives- ampicillin and methicillin |
Classification based on effect on "target" (3) |
- cidal/static/removal - spectrum of activity - mode of action |
-cidal/-static-/removal |
classification base on effect on "target" - cidal = kill pathogen - static = inhibit pathogen - removal- depends on immune system of host |
Spectrum of activity |
classification based on effect on "target" - broad- comprehensive treatment when infectious agent is unknown (mycobacterium, g+ and g-) - narrow- select group; leaves normal bacteria flora intact (only mycobacterium, only g-) |
Narrow mycobacterium exmaples (2) Narrow spectrum g- explanation Broad examples (2) |
- mycobacterium: isoniazid- tuberculosis, dapsone- leprosy - G- is narrow because it effects treponema, penicillin and neisseria only - broad: tetracycline and chloramphenicol
|
Mode of action (5) |
classification based on effect on "target" - inhibit peptidoglycan synthesis - inhibit unique prokaryotic metabolic/enzymatic pathway - protein synthesis inhibitors - inhibit membrane function/destroy membrane activity/selectivity - interfere with nucleic acid synthesis |
Inhibitors of peptidoglycan synthesis (4) |
- penicillin - cephalosporin - vancomycin - bacitracin |
Inhibitors unique prokaryotic metabolic/enzymatic pathway |
sulfonamides -- sulfa drugs -- |
Protein synthesis inhibitors (3) |
- aminoglycosides - tetracycline - erythromycin ** act on ribosomes ** |
Inhibitors of membrane function/destroy membrane activity/selectivity |
- polymyxin - amphotericin B - nystatin |
Interference with nucleic acid synthesis (2) |
rifamycin- RNA transcription nalidxic acid- DNA replication |
Characteristics of an ideal chemotherapeutic agent (3) |
- does not induce drug allergy in host/patient - does not induce drug resistance in target organism/pathogen - exhibits high selective toxicity |
Specifics about characterisitcs of ideal chemotherapeutic agent |
- allergy: 10% of population allergic to penicllin - resistance: major problem today with control of disease (ex. MRSA- methicillin, vancomycin) - selective toxicity: high therapeutic ratio measured by chemotherapeutic index (ex. Pseudomonas- nosocomial/hospitcal acquired) |
Chemotherapeutic index |
- Paul Ehrlich - formula: chemotherapeutic index = lowest dose toxic to patient / minimal curative dose - minimal curative dose inhibits or kills pathogen |
Importance of biochemistry |
- comparative biochemistry - exploiting differences in basic biochemical processes between bacterial and mammalian cells - selectively exploit the difference in biochemistry between different microbial organisms |
Importance of studying bacterial morphology |
- comparative cytology - selective toxicity - exploit the differences in prokaryotic cell structure - knowledge of cellular staining characterisitics of different organisms is the first step to understanding toxicity |
Tug of war between microogranism and patient |
- buy time so the immune system can rid of the microbial invader - recovery if immune system beats organism due to innate resistance or acquire immunity - disease due to strong virulence mechanism |
Addition of a chemotherapeutic compound |
- unforeseen consequences - drug interactions- consider on individual basis - good outcome if compund is -cidal/-static - bad outcomes: metabolize, metabolizing inactivated, allergy, resistance |
Microbial control by cell wall synthesis inhibitors |
- major means of control - highly favorable chemotherapeutic index - not without problems (ex. hypersensitivity - allergy) |
Beta-lactam basics |
basic penicillin - all penicillins and cephalosporins are beta-lactams - characteristic beta-lactam ring in every beta-lactam organism - thiazolidine ring stabilizes beta-lactam ring but is substitued for different penicillins |
Semi-synthetic derivatives of penicillin |
- increase resistance to beta-lactamases and penicillinases - increase spectrum of activity (ampicillin carbenicillin) |
Cephalosporin structure |
- different class but still produced by fungi - after penicillin - beta lactam ring present - dihydrothiazine ring instead of thiazolidine |
Dihydrothiazine benefits to cephalosporin |
- relatively more resistant to beta-lactamases - exhibit wider spectrum of activity - not as likely to produce allergy - improved pharmacokinetics - more efficient in body |
Transpeptidases |
- penicillin binding proteins (PBP) - responsible for catalyzing reaction - prevent transpeptidation - many different ones in a cell - involved in cell division and growth |
Mode of action of beta-lactams |
- PBP important to interbridge and side chain - bactericidal in rapidly growing/dividing cells - structural analogy between penicillin and terminal d-alanine-d-alanine amino acids of the side chain |
PBP actions |
- similarities in structure can confuse PBP and it will bind and not reverse - typically catalyzes where interbridge connects to 1 d-alanine - beta lactams inhibit catalyzation - beta lactamases cause resistance |
Enhance beta-lactam activity |
- clavulanic acid has beta ring; AKA "suicide inhibitor" of beta-lactamases - beta-lactamase inhibitor works by forming a stable complex with beta-lactamases - extend spectra of some penicillins - amoxicillin, clavamox |
Peptidoglycan synthesis |
- complex, multistep process - large number of enzymes, transporters, structural and membranal proteins - intracellular and extracellular stages |
Other cell wall synthesis inhibitors |
- no ring, but are still cell wall inhibitors - prevent monomer release: bacitracin, d-cycloserine - prevent side chain from forming (block growth point): vancomycin, ristocetin |
INH |
isoniazid - similar to molecule that inhibits mycolic acid synthesis - inhibits mycobacterium/nocardia - used in combination with ethambutol or pyrazinamide for tuberculosis treatment to prevent resistance |
Sulfa drugs |
- class of drugs - sulfonamide is an example - prevent metabolic/enzymatic pathway - unique to prokaryotes - chemotherapeutic not antibiotic - common ring structure - enhance effectiveness of inhibtion & change pharmacokinetics |
Inhibition of an enzyme |
- enzymes fit in particular active site based on shape - inhibition by similar shaped organism taking up active site - less affinity - prevent enzyme from catalyzing appropriate reaction (enzymes fooled- form dysfunctional product) - leads to more discrimination by enzymes, increasing chance of resistance |
Enzymatic pathway |
-dihydropteridine, PABA, and glytamic acid synthesized by dihydropteroate synthetase *critical - dihydrofolate is formed, synthesized by dihydrofolate reductase - forms tetrahydroflorate (folic acid) |
PABA and sulfanomide |
similar structure (structural analogs) - competitive with each other to reach active site since similar shape - higher ratio of sulfanomide inhibits PABA attachment - PABA has SO2 & NH2 groups, sulf has COOH |
Intracytoplasmic granules |
- vary in size, shape, number, location and composition - composed of polymetaphosphate - AKA volutin granules |
Lipid deposit stain |
- Burdon's method - PHB: poly-b-hydroxybutyrate is revealed - heat fixed smear, sudan black B for 15 min, xylene for 10-20 sec, counterstain 5-10 sec with .5% safranin, first water wash - lipid deposits are black, rest of cell is red |
Endospore stain |
- Schaeffer fulton method - heat fixed smear, saturated malachite green for 5 min, water wash, safranin counterstain - endospores are green, vegetative cell is red |
Inhibition of the THF pathway |
- trimethoprim/pyrimethamine instead of dihydrofolate reductase - pathway between dihydrofolate to tetrahydrofolate is blocked - t/p is used in synergy - lower concentrations, not as competitive |
Does degree of selectivity increase or decrease going down groups? |
* decrease * - peptidoglycan synthesis - metabolic/enzymatic pathway - protein synthesis - membrane function/destroy membrane activity/selectivity - nucleic acid synthesis |
Protein synthesis inhibitors (3) |
- streptomycin - erythromycin - tetracycline |
Stages of protein synthesis |
- initiation- GTP (energy), initiation factors, subunits (70s ; 80s - E, A, P sites), start codon - elongation- time varies, peptidyl transferase enzyme - termination- stop codon |
Sites of protein synthesis interference |
- structural components of the ribosome - enzymes involved in the process - "reading" of mRNA |
Antimicrobials that inhibit protein synthesis |
- aminoglycosides - tetracyclines -chloramphenicol - macrolides |
Aminoglycoside examples, structure ; side effects |
- streptomycin, kanamycin, neomycin, gentamycin, tobramycin (-mycin) - structure: amino acids and sugars: glycosidic linkage, OH, NH2 groups - side effects: damage to 7th ; 8th cranial nerves (hearing), nephrotoxicity |
Aminoglycoside mode of action |
* concentration dependent - blocks initiation of translation - "locks up" the ribosome - misreading of mRNA - bactericidal (sulfa is static) - can attach to 50s instead of 30s - respiratory and electron transport system is a requirement - not effective against anaerobes ; fermenters |
Tetracycline |
-; 4 ring structure; different R groups - bind reversibly to 30s ribosome near codon; block tRNA attachment - broad spectrum antibiotic - super infection - shouldn't use with young children for long period due to teeth effects |
Chloramphenicol |
- binds at 50s ribosome - inhibits peptidyl transferase (elongation) - broad sprectrum chemotherapeutic agent - super infection - Clostridium difficile- g+, intestinal flora; can cause serious colitis - lincosamides- same action |
Macrolides |
- bind to 50s ribosome, blocks translocation - use instead of penicillin - outer membrane excludes the drug - effect g+ (pneumonia) - ex. clarithromycin, azithromycin, erythromycin |
Translation basics |
- first amino acid it formylmethionine - initiation complex of 30s, 50s, f-met, and initiation factors - p site- initiating tRNA carrying f-met binds here - a site- tRNA enter to donate their amino acid; acceptor site (second codon) - e site- tRNA exit after donating amino acid |
Translocation |
- ribsome advances one codon (ratcheting) - requires elongation factors - a site becomes p site - next codon fills a site |
Shine-dalgarno vs. Kozak sequence |
- sd: ribsomal binding site for mRNA, 8 pairs upstream from start codon, prokaryotes, align ribosome and start codon, anti-sd is at 16s ribosome - k: eukaryotic verison; 3 pairs upstream * both crucial to initiation of translation* |
Compounds that inhibit membrane functions/selectivity |
- due to absence of cholesterol - polymyxin B, amphotericin B, nystatin, daptomycin |
Polymyxin types and mode of action |
- 6 total (a-f); B & E/colistin most common; others are too toxic - used topically (skin surface) - hydrophobic tail, hydrophilic head - effective against g- |
Daptomycin |
- lipopeptide antibiotic - g+ bacteria only - cannot penetrate LPS/outer membrane of g- - causes ionic imbalance and depolarization of the membrane (membrane permeability altered) |
Amphotericin B & nystatin |
- anti-fungals - polyenes- double bonds in the structure - combines with ergosterol - decreased toxicity - no cholesterol |
Compounds that interfere with nucleic acid synthesis |
- sulfonamides - trimethoprim - rifampin - RNA polymerase/synthesis - metronidazole- DNA synthesis, protozoa and anaerobes, intestines - nalidixic acid- DNA synthesis, UTI's - quinolones |
Quinolones mode of action |
- fluoroquinolone - DNA is usually supercoiled for compactness, but unfolds for replication and transcription - quinolones prevent DNA topoisomerase II/DNA gyrase, so DNA cannot unfold - norfloxacin, ciprofloxacin, oxfloxacin (fluorine in all structures) |
Determining antimicrobial sensitivity (6) |
1. disc diffusion- zone of inhibition, kirby-bauer 2. broth dilution assays- what concentration will inhibit growth 3. antibiogram 4. broth dilution test- turbid/clear tubes in increasing concentration, MIC/MBC 5. E-strip- strip of concentrations 6. Vitek 2 system- sophisticated |
MIC vs. MBC |
MIC: minimum inhibitory concentration; want it to be low; utilize assays to find the lowest concentration of a specific antimicrobial drug needed to prevent growth; visible growth measured MBC: minimum bactericidal concentration; lowest concentration of a specific microbial drug that kills 99.9% of cells in a given bacterial strain; assaying for live organisms in tubes with no growth from MIC |
Dangers of broad spectrum antibiotics (3) |
- normal flora bacteria is equally susceptible - recall "balanced pathogenicity" - opportunistic bacteria are part of normal flora |