question

Disinfectant

answer

Chemical agent that oxidizes biological macromolecules (germicidal or microstatic). For inanimate objects. Ammonia and bleach.

question

Antiseptic

answer

Chemical agent applied to biological tissue. Alcohol is most commonly used.

question

Types of antiseptic

answer

Alcohol, iodine, crystal violet, heavy metal salts, detergents

question

How to prepare plates for disinfectant/antiseptic testing

answer

Spread plate the bacteria broth onto TSA plate. Add filter paper disks. Pipette volume of antiseptic/disinfectant onto disk. Add control disk. Examine for zone of inhibition (clear areas of lawn)

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Antibacterial activity

answer

Size of zone of inhibition

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Microflora/microbiota

answer

Microbial organisms comprising your intestines, on your skin, on other body surfaces

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Acne causing bacteria

answer

Gram + are better able to grow in skin conditions (dry, salty, low pH). Staphylococcus is most common

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Propionibacterium acnes (bacteria)

answer

grows in oil secreted by oil glands in skin where hair follicles grow.

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Corynebacterium diptheriae (bacteria)

answer

acne causing, opportunistic pathogen that can cause diptheria

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Pityrosporum ovalis (yeast)

answer

acne causing, main cause of dandruff; most yeast are opportunistic and responsible for serious skin infections

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Handwashing

answer

Removes transient microorganisms from skin and excess populations of indigenous microbiota

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UV radiation sterilization

answer

damages DNA (disrupts cellular processes)

question

factors in UV sterilization

answer

wavelength, intensity, exposure time

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Enzyme activity regulation

answer

temperature, pH, concentration of substrate/enzyme, affinity of enzyme for substrate

question

Catalase enzyme

answer

Aerobic bacteria oxidize H2O2 --> H2O + O2

question

Catalase test

answer

Drop H2O2 on inoculating loop to watch for evolution of bubbles. Distinguish Staphylococcus (+) from Streptococcus (-) when identifying gram + cocci. Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa are positive. Enterococcus faecalis is negative.

question

Factors affecting catalase test

answer

Young, viable culture (false negative if cells not actively dividing); corroded zinc on loop- test with loop only (false positive)

question

Cytochrome oxidase

answer

Terminal link in ETC during respiration- mediates transfer of electrons from reduced cytochrome c to molecular oxygen.

question

Cytochrome oxidase test

answer

false negative if cultures are not actively dividing; oxidase reagent turns purple in presence of air.

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Lysine decarboxylase

answer

identifies Enterobacteriaceae species and species capable of fermenting glucose VIA lysine decarboxylase. NOT appropriate for non fermenters. low pH produced by glucose fermentation activates decarboxylase (purple -> yellow)

question

Lysine decarboxylase media

answer

decarboxylase base Moeller broth w/ Lysine (test), w/o Lysine (control). layer of mineral oil protects culture from oxygen for fermentation to occur and avoids false alkalinization on surface

question

Lysine decarboxylase test control

answer

determine culture is viable and capable of growing in media as well as if the test is appropriate (capable of fermenting glucose)

question

Lysine decarboxylase- species can ferment glucose

answer

test: turn yellow due to usage of small amount of glucose in media at 24 hrs; if decarboxylation of lysine occurs the pH increases and medium reverts to original purple; control remains yellow. False negative (in test) possible for weak positive reactions (incubate for 4 days). If control is purple then there was no growth or bacteria does not ferment glucose.

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Phenylalanine deaminase

answer

catalyzes conversion of phenylalanine to phenylpyruvic acid

question

Phenylalanine deaminase test

answer

distinguish Proteus from other genera from Enterobacteriaceae. drop 10% ferric chloride on the slant. deep green is positive (E.coli). negative is Proteus vulgaris and Serratia marcescens.

question

Tryptophanase (Indole Production)

answer

catalyzes hydrolysis of tryptophan with production of indole, pyruvic acid, and water. differentiate Enterobacteriaceae members (Escherichia vs Klebsiella)

question

Tryptophanase (Indole Production) test

answer

drip Kovac's reagent. Presence of indole detected by formation of red layer on the top.

question

Gelatinase

answer

hydrolyzes gelatin from gel to liquid.

question

Gelatinase test

answer

. melts at 37 deg C so if that is the optimal temperature, it must be incubated with an uninoculated control tube, then prior to reading both refrigerated at 4 deg C until control medium resolidifies. keep up to a week if negative (in case of false negative)

question

Urease

answer

convert urea to ammonia and CO2. ex. Helicobacter pylori

question

Urease test

answer

Christensen urea agar slant. Fuchsia pink for positive result

question

Amylase test

answer

Bacteria incubated on nutrient agar + 0.2% soluble starch plates. Flood plate with iodine. Completely black = negative; Positive = clear zone where starch has been hydrolyzed

question

Nitrate reductase

answer

nitrate -; nitrite (NO2-)

question

Nitrate reductase test

answer

Detection for nitrite. Tests for dissimilation (nitrate as terminal electron acceptor of anaerobic respiration). nutrient agar with KNO3.

question

Nitrate reduction (nitrate reductase) POSITIVE RESULT

answer

added sulfanic acid and dimethyl alpha napthylamine. RED=clear positive.

question

Nitrate reduction NEGATIVE RESULT

answer

added sulfanic acid and dimethyl alpha napthylamine. CLEAR=could be false negative. potentially nitrate was dissimilated but then reduced to end products other than nitrite.

question

Zn for nitrate reduction

answer

Zn reduces nitrate to nitrite. It is added to detect if there is any nitrate that was unreduced. Red=neg nitrate reductase (any gas produced is from fermentation of sugars in medium); clear=pos nitrate reductase (nitrate was converted to other forms or released as gas)

question

Carbohydrate fermentation test

answer

Phenol red carbohydrate broth, pH indicator, and Durham tube gas capturing device. Yellow is positive for acid production. Bubbles is positive for gas

question

MR-VP test

answer

Methyl red, Voges Proskauer for two different glucose fermentation pathway. use same inoculated MR-VP broth but incubated for different lengths of time.

question

VP test

answer

identifies intermediate (acetoin) in butanediol fermentation pathway (pH;6.0) 24-48 hours after inoculation b/c acetoin will be used up, leading to false negatives. Preferentially only use with Enterobacteriaceae. Rich wine-red positive, bronzy brown negative. Color improves with time.

question

VP reagents

answer

MR-VP broth, alpha napthol then KOH + O2 (mix)

question

MR test

answer

identifies the multiple acidic end products after mixed acid fermentation with pH indicator methyl red (pH;5). let color develop on the surface, then shake (both results should be same). Longer time improves readability - minimum 3 days suggested. Deep pinkish red/organgish red = positive; yellow/pale tangerine orange = negative

question

Why is VP preferred over MR?

answer

Faster, easier to read = results more reliable

question

Citrate utilization

answer

carbon source for many Gram negative rods (Enterobacteriaceae).

question

Citrate test medium

answer

Simmons citrate agar with bromthymol blue pH indicator. organisms that can't use citrate grow poorly w/o the food source. green=negative (neutral pH), blue=positive (high pH)

question

Successful chemotherapeutic agent

answer

1. ability to kill or inhibit many species of pathogenic microbes 2. relatively low probability of development of resistant forms of pathogens 3. minimal side effects in host 4. minimal effect on normal microbial flora of host

question

Paper disk plate method

answer

Mueller Hinton media containing no chemicals that might affect normal functioning of antibiotics. Disks with known concentrations of antibiotics.

question

MIC method

answer

Minimum Inhibitory concentration (microg/mililiter). Broth microdilution technique to determine the smallest amount of chemotherapeutic agent required to inhibit the growth of microorganism (turbidity). Identity as well as dose!

question

Transformation

answer

extraneous DNA is taken up into a cell and incorporated into genetic make up of the cell

question

Competency

answer

Able to transform (add CaCl2)

question

Cloning

answer

transformation of bacteria with foreign DNA. DNA is extracted, gene of interest is amplified with PCR, PCR product is inserted into plasmid cloning vector, plasmids are transferred into cloning host (E.coli), clones are reisolated from colonies, DNA fragment is amplified via PCR, PCR products can be digested by restriction enz or sequenced

question

pGLO plasmid

answer

genes for beta-lactamase (enz provides antibiotic resistance to ampicillin). used as a selective agent, cells not transformed by pGLO will not grow on amp plate. when arabinose is present, araC protein promotes RNA pol binding to promoter region (Pbad). Transcription of genes in ara operon promotes breakdown of arabinose normally, but in pGLO plasmid they are replaced with gene for GFP

question

combinations for pGLO transformation

answer

+/-[pGLO LB/amp/ara]

question

Ascomycetes

answer

perform meiosis within sack shaped ascus, forming ascospores

question

basidiomycetes

answer

forcibly discharge basidiospores from end of basidium, specialized meiotic cell produced on fertile surface of mushroom

question

True fungi

answer

1. yeast-like growth via threadlike cells (hyphae) which form radially growing colony (myelium) 2. cell walls of chitin instead of cellulose or peptidoglycan 3. heterotrophic nutrition via absorption of nutrients from environment 4. reproduction via spores - sexual or asexual 5. formation of fruting structures which produce spores

question

basidiomycetes life cycle

answer

fruit body discharges basidiospores that were produced from meiosis in the basidium (2n). basidiospores germinate into primary mycelium (n). primary mycelium mate to make a secondary mycelium that grows into fruit body (n+n).

question

dikaryotic vs diploid cello

answer

The packaging of the genetic material is different; heterokaryotic cells contain two separate nuclei where each nuclei contains a different single set of alleles (for two sets total per cell) and diploid cells have only one nucleus containing both sets of alleles.

question

clamp connections

answer

characteristic of dikaryotic hyphae

question

"yeast"

answer

asexual reproduction by budding or fission

question

Schizosaccharomyces octosporus

answer

produces 8 ascospores

question

Saccharomyces cerevisiae

answer

produces 4 ascospores

question

ascospore stain

answer

heat not required to increase stain permeability. vegetative cells stain pink/red, ascospores stain green

question

mycelium

answer

vegetative, nutrient absorbing part of the mushroom; grows into and occupies the substrate the fungus digests

question

cultivation of Pleurotus mushroom species

answer

inoculate sterilized grain with mycelium of fungus. once colonized, the grain becomes knitted together by growing mycelium to form solid block the mushrooms will later develop on

question

nitrogen fixation

answer

nitrogen is the element most likely to limit crop productivity. the process is a major source of usable nitrogen. catalyzed by nitrogenase

question

Cyanobacteria family

answer

photosynthesize nitrogen fixers, free-living and non-symbiotic or commensal/mutualistic with plants. cells often grow in long chains. Mistaken as blue-green algae

question

anabaena azollae

answer

filamentous Cyanobacterium that forms mutualism with several species of Azolla (floating water fern). Housed in cavities under each Azolla leaf. Receives some nutrients from the plant. Higher rate of nitrogen fixation than Rhizobium and legumes.

question

heterocysts

answer

nitrogen fixing cells in Anabaena azollae. thick walled to prevent oxygen from interfering with nitrogenase. Round shape, relatively large size, thick walls, polar nodules at each end differentiate them from Anabaena vegetative cells

question

Rhizobium

answer

Gram negative. mutualism with legumes-bacteria attach to root hairs, enter plant via infection tubes, and invade plant's root tissue. Plant responds by forming root nodules (tumor-like growth containing bacterial cells). Rod shaped bacteria become irregularly shaped bacterioids in plant cells. Nitrogen gas fixed into ammonia. Plant provides nutrients.

question

Sym (symbiotic) plasmid

answer

contains genes enabling Rhizobium strains to produce N2 fixing nodules.

question

Leghemoglobin

answer

binds to oxygen to protect nitrogenase from oxygen. gives root nodules a pinkish color.

question

stain root nodule vs mannitol salt plate

answer

X, Y shaped bacteriods vs long chains of bacteriods

question

Agrobacterium

answer

plant pathogens that infect broad-leafed plants

question

Crown gall disease

answer

tumor-like growth caused by Agrobacterium tumefaciens

question

Agrobacterium tumefaciens

answer

infects wounded surfaces of susceptible dicotyledonous plants at the soil-plant stem interface. Once Ti (tumor induction plasmid) is transferred to the infected plant cell the tumor synthesis can occur. These transformed plant cells produce opines which most strains of bacteria use as carbon and nitrogen source. Ti can be mechanism for future genetic engineering. Carrot tumors!

question

yeast vs bacterium

answer

eukaryotic, unicellular fungus, visible budding, visible at 30x or lower and 5-6 micrometeter lengths long

question

verifying purity

answer

gram stain (24 hr) from the streak so entire plate is pure, verify with 3% KOH test

question

immediate ID tests

answer

catalase, oxidase, motility stab ; broth, Gas Pak and stab TSA deeps, endospore stain for Gram+

question

PCR

answer

POLYMERASE CHAIN REACTION: directed enzymatic replication of target sequence from DNA template using target specific olgionucleotide primer sequences

question

Stages of PCR

answer

1. denaturation: hydrogen bonds break but covalent bonds remain intact and DNA becomes single stranded 2. primer annealing: primer binds to target sequence 3. polymerization: DNA polymerase extends primer by adding dNTPs --> amplified 30x to greatly amplify target sequence

question

PCR cycle

answer

1. heat shock-7min-94 deg C 2. 35 cycle of denaturation (1 min at 94 deg C), annealing (15 sec at 55 deg C), and polymerization/extension (1 min at 72 deg C)

question

Agarose Gel Electrophoresis

answer

confirm target region has been amplified and determine concentration of product = single well-resolved band of expected length will be present under UV light; loading dye containing glycerol is mixed in to increase density of the sample before the voltage is applied and fragments migrate across the gel

question

DNA ladder

answer

series of DNA fragment of known concentration and size

question

PCR Purification

answer

unincorporated primers and dNTPs can interfere with analysis of amplified products. Exonuclease I (E.coli) digests single stranded DNA such as primers into dNTPs and Shrimp Alkaline Phosphatase (SAP) inactivates dNTPs by inactivating phosphate groups from them. After degradation, samples are heated to 80 deg C to inactivate the enzymes. ready for sequencing!

question

DNA Sequencing

answer

Frederick Sanger developed dideoxy chain termination method for DNA sequencing: single olgionucleotide primer is used, resulting in linear accumulation of a single stranded product. ddNTPs (lacking 3'hydroxyl groups for phosphodiester bond formation) added that truncate DNA fragments to different length. *differs from PCR

question

Enterobacteriaceae family

answer

Enterobacter aerogenes, Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris, Serratia marcescens

question

Entero Pluri Test

answer

rapid identification within Enterobacteriaceae family

question

Staphylococcus aureus latex agglutination test

answer

latex beads covered with fibrinogen and antibody to protein A bind to the proteins - clumping factor and protein A- that are found on the surface of S.aureus. S.aureus infections are issue in hospitals-less common, housed in nasal cavities. Colonies grown on blood agar plate to differentiate b/w Staph and Strep.

question

Staphylococcus vs Streptococcus

answer

on blood agar plate: S.epidermis: chalky white, non hemolytic colonies, beta or alpha-hemolytic S.aureus: white-yellow, 1-3 mm diameter, beta-hemolytic gram stain: Staph: grape like clusters of Gram + cocci Strep: pairs or chains of Gram + cocci catalase test: staph: + strep: - *blood agar contamination will be catalase +, >24 hr culture will give false negative

question

Stimulated HIV-ELISA testing

answer

ELISA plates are covered with HIV particles Blocking agent is added to fill spaces in the plate - prevents secondary antibody from binding to plastic base of well to give false positive. Diluted patient's blood serum is added to well. Any HIV specific antibodies present will bind to HIV antigen. Plate is washed with buffer to remove unbound serum. Secondary antibody is added (color producing enzyme bound to anti-human IgG antibody). Secondary antibody binds to IgG antibodies attached to HIV antigen Plate is washed with buffer to remove unbound secondary antibody. Enzyme substrate is added and colored product amount measured

question

False positive ELISA

answer

non-HIV proteins stick to the wells of ELISA plate in the first step patient with antibodies against foreign antigens may attach to the HIV antigen (not necessarily HIV antibody) --> secondary antibodies will bind to any human antibody --> color change false positive use Western blot to sort HIV proteins by molecular weight

question

Acid fast stain

answer

used to identify bacteria (Mycobacterium), stain yeast and ascospores (thick walled ascospores stain acid fast (red) yeast cells stain (green)

question

Kimchi

answer

Mixed acid fermentation initiated by Gram - coliform bacteria Enterobacter cloacae and Erwina herbicola. The bacteria are replaced with Leuconostoc mesenteroides (producing lactic and acetic acid). When 0.7% acidity is reached, Lactobacillus planatarum and L. brevis dominate and give the acid flavor. Salt used to draw out plant juices containing the sugars and inhibit growth of non lactic acid producing organism.

question

Yogurt

answer

Lactobacillus bulgaricus and Streptococcus thermophilus. Sharp acidic flavor is due to lactic acid and aldehyde product from fermentation of lactose. Acids also make proteins insoluble (coagulation/thickening).

question

Beer

answer

fermentation of complex carbohydrates present in grain, that must be broken down before they may be assimilated by yeast

question

Malting

answer

Grain is soaked in water, allowed to germinate, then dried in an oven. Temperature and duration of drying determine the kind of malt produced (higher temperature = carmelization of starch and sugar, producing non- fermentable dextrins (for body) and caramel (for darkness).

question

Wort

answer

malt + hot water. During mashing, enzymes released from malt are activated and convert starches into fermentable sugars

question

Hopping

answer

Wort is heated to inactivate enzymes and hops are added. Provides dryness, bitterness, aroma to beer; antimicrobial properties that discourage bacterial growth

question

Pitching the wort

answer

After wort is cooled, solid materials are separated and wort is pitched with yeast. For lager beer, bottom fermenting yeast is used. For ale, top fermenting yeast is used.

question

Primary fermentation

answer

vigorous growth as most sugars are converted to alcohol

question

Secondary fermentation

answer

air is excluded; beer clarifies as yeast settles to the bottom; after, carbon dioxide is added by gassing or priming the beer with small amount of sugar

question

CFU/ml

answer

(#Total Coliform Colonies)/(Dilution factor*100ml)

question

MPM method

answer

estimate coliform count (coliform=gram negative bacteria found in digestive tracts). uses multiple serial dilutions. check tubes for acid/gas production and compare to MPN index.

question

Membrane filter

answer

MI agar detects contamination by E.coli or total coliforms (mammalian intestinal bacteria)

question

Triple Sugar Iron agar

answer

differentiates Enterobacteriaceae on ability to ferment lactose, sucrose, and glucose. Phenol red pH indicator changes from red to yellow in presence of acid. Sodium thiosulfate forms black precipitate if bacteria can reduce sulfur to produce H2S.

question

TSI results

answer

neutral/alkaline slant = red ; acid butt = yelow --> only glucose fermentation occured acid slant = yellow & acid butt = yellow --> lactose/sucrose fermentation occured alkaline slant = red & alkaline butt = red/orange --> no carbohydrate fermentation black precipitate in butt = production of H2S

question

Eosin Methylene Blue (EMB) agar

answer

eosin = red stain; methylene blue = blue stain -- combined give burgundy color. inhibit gram + bacteria growth without affecting gram - growth. Lactose fermenters produce acid and are deep purple/pink with dark centers. Bacteria not fermenting lactose are colorless or pinkish without dark centers. E.coli produce high levels of acid and have dark colonies with metallic green sheen.

question

Blood agar plate

answer

Bacteria differentiated by ability to cause hemolysis of blood cells (Strep vs Staph). Beta-hemolysis: complete lysis, clear zone Alpha-hemolysis: green/brown discolored zone, not true lysis, more of bruising Gamma-hemolysis: no change in media, cells remain intact

question

Mannitol salt

answer

7.5% NaCl inhibits growth of most species; but not Staphylococcus (salt tolerant). Medium contains pH indicator that changes from red to yellow if mannitol is fermented to acid. Pathogenic strains do while nonpathogenic don't.

question

Capsule stain

answer

Capsule: thick polysaccharide material that surrounds bacteria and yeast. they attach bacteria to surfaces, tolerate dessication, and protect from predators. sign of pathogenicity. fragile, water soluble, damaged by heat, difficult to stain. can be visualized with pos-neg staining culture is grown in skim milk broth & mixed with india ink for counterstain. crystal violet used to stain bateria and dried media in background

question

Endospore stain

answer

gram positive bacteria. thick walls of endospores are resistant to staining but once stained they resist decolorization and counterstaining. malchite green --> safranin. plate must be streaked heavily and kept at appropriate conditions 5-7 days.

question

Gram stain

answer

1. primary stain: stains all cells 2. mordant: Gram's iodine, fixes primary stain to Gram + 3. decolorizing agent: alcohol, removes any unfixed primary stain 4. secondary/counter stain: safranin, restains decolorized cells in a different color

question

gram neg vs pos staining

answer

gram +: retain primary stain, purple gram -: lose primary stain, counter stained by safranin, pink

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