Microbiology Lab Practicle 2

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Selective Media

used to isolate specific groups of bacteria

Phenylethyl alcohol agar, crystal violet agar, 7.5% sodium choloride agar

Selective media agars

Differential/selective media

can distinguish among morphology and biochemically related groups of bacteria. chemically altar bacteria to show a physical change in appearance of bacterial growth or medium surrounding the colonies, sometime differential and selective media characteristics can be combined.

Mannitol Salt agar, MacConkey agar, EMB agar

Differential Media agars

Enriched media

media that have been supplimented with high nutritious materials to cultivate fastidious organisms

Blood agar

Enriched media agar

Phenylethyl alcohol agar (PEA)

tests for gram positive organisms, and inhibitory to gram negative

Crystal violet agar

selective for gram negative, and inhibitory for gram positive

7.5% sodium chloride agar

inhibitory to most organisms, detects for salt loving organisms (halophilic) most useful in detection of staphylococcus

Mannitol salt agar

contains a high salt concentration, PH indicator for detecting acid. detects for staphlycocci-->yellow zone around bacteria if it ferments the mannitol salt

MacConkey agar

allows the isolation of gram negative two groups: coliform bacilli and (dysentery, typhoid and paratyphoid bacilli)

Coliform bacilli

test used for detection of e.coli (gram negative), if lactose positive (red)

Dysentery, typhoid and paratyphoid bacilli

non lactose fermenters, and therefore appear transparent (no lactose, no color)

EMB agar

selects for gram positive, and lactose positive. e.coli will have a blue-black with a metallic green shine

Enterobacter aerogenes

will produce thick pink colonies

Blood agar

gamma hemolysis, alpha hemolysis, beta hemolysis

Gamma hemolysis

no lysis, no change in appearance

Alpha hemolysis

incomplete hemolysis, green halo around the bacterial growth

Beta hemolysis

lysis of red blood cells with complete destruction and use of hemoglobin by the organism results in a clear zone surrounding the colonies (shiny glowing yellow green)

Triple sugar iron (TSI) agar test

designed to differentiate among the different groups of the enterobacteriaceae which are all gram negative bacilli capable of fermenting glucose with the production of acid AND to distingiush between other gram negative intestinal bacilli

Alkaline slant (red) and acid butt (yellow) with or without gas production (breaks in the agar butt)

only glucose

Acid slant (yellow) and acid butt (yellow) with or without gas production

eats sugars (lactose and sucrose)

Alkaline sland (red) and alkaline butt (red) or no change (orange-red) butt

both reactions the same, eat protein-->basic (non acidic)--> so therefore remains red

TSI COLOR CHANGE

the acid-base indicator phenol red is also incorporated to detect carbohydrate fermentation which turns the medium to yellow in the presence of acid

Enterobacteriaeae

short, gram negative, nonspore forming bacilli

Pathogens, occasional pathogens, normal intestinal flora

Types of Enterobacteriaeae `

Pathogens

salmonella and shingella only in humans

Occasional pathogens

proteus and klebsiella

Normal intestinal flora

Escherichia and enterobacter, which are saprophytic members of the intestinal tract

Indole test

tests for e.coli and turns cherry red if indole positive, however the have to be careful or red pigment vs red chunks (24-48 hrs) Kovac's reagent-->produces the cherry red layer

Citrate test

medium becomes alkaline, this medium turns from green to deep prussian blue (if blue it is citrate positive)

Methyl Red Test

separation of e.coli and e. aerogenes. turn yellow if a negative test, and red if a positive test (tests for presence of ACID)

SIM

forming a soluble black ferrous sulfide precipitate seen along the line of stab inoculation, H2S is present (positive) if a pipe cleaner thing is present. culture growth not restricted to line of inoculation. SALMONELLA WILL BE BLACK BUTT

Urease test

identifies proteus vulgares, if ammonia present, it causes the phenol red to turn a deep pink (super base)

Catalase test

by adding H2O2 (hydrogen peroxide) if it bubbles it is catalase positive,

Phenylalanine Deaminase Test (PPA agar)

identifies proteus, if color reaction turns green, its positive THEN dissapears. reageant--> 10-12% ferric chloride solution

Chemotherapeutic agents

chemical substances that destroy or inhibit the growth of microorganisms in living tissue (antibiotics/synthetic drugs)

Antibiotics

living bacteria, actinomycetes and fungi that destroy the growth of other microorganisms

Synthetic drugs

made in lab to destroy the growth of other microorganisms

Concentration

higher concentrations: more rapid death, toxicity must also be considered

Length of exposure

sensitive forms destroyed more rapidly than resistant ones (environment and consistency must also be considered)

Types of microbial population to be destroyed

spores--> resistant, acid-fast-->more resistant than non acid-fast, older less active cells more resistant than young cells

Environmental conditions

Temperature, and pH

Temperature

the increase in temp increases the rate of reaction in which cells are killed by the chemical reaction taking place

pH

pH conditions affect the microorganism AND the compound, extreme pH are harmful to microorganism but also increase the antimicrobial action of a chemical

Types of mediums

the microorganism may not stand a change against the combo of chemical and organic cellular molecules

Phenol Coefficient test

determined by dividing the highest dilution of chemical being tested that destroyed the microorganism in ten minutes but not five minutes

Skin normal flora

Staphylococci

Genitourinary tract

protozoa such as Trichomonas

strep and staph, hemolytic microorganisms

The Blood agar plate differentiates between

A chocholate agar plate detects

neisseria (ghonnorea): pink to purple black colonies

A mueller hinton tellurite or tinsdale agar plate detects

cornyebacterium (diptheroids), or colonies with a black appearance

Sabouraud agar plate detects

yeast; mold colonies will appear fuzzy, powdery growths arising from a mycelial mat

MSA plate will detect

staphlyococci causing a yellow concentration around the growth

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