Lab Exam 2 Flashcards
Starch hydrolysis
Does microbe have exoenxyme to hydrolyze starch. (Amylase). Negative-blue/brown colour, positive-colorless. Reagent-Grams iodine colour change in presence of starch.
Catalase Test
IDs hydrogen peroxide as a product of aerobic respiration. If there are bubbles this is oxygen gas produced in reaction with H2O2.
Carbohydrate fermentation.
Phenol red is pH indicator. Red if neutral/basic and yellow if acidic. Gas bubbles caught in Durham tube. Fermentation products-gas,acid, alcohol. Lactose, sucrose and maltose.
Reagent
Chemicals that produce a visible evidence that indicates a biochemical reaction has occurred
Citrate utilization
Medium-Simmons citrate (only carbon source). Bromthymol blue is pH ID, green neutral, blue +7.5. Citrate permeases transport citrate into cell>pyruvic acid and CO2>CO2 diffuses out of cell reacts w/ Na>NaCO3 which makes medium more basic. Positive is blue vv green.
Gelatin Hydrolysis
Exoenzyme, medium is nutrient gelatin. If gelatin digested medium liquefies. Liquid=positive. Issues low temp and long incubation. Gelatinase hydrolyzes gelatin protein into smaller peptides and AAs.
SIM
Sulfur Indole Motility. Tryptophan>indole produced through tryptophanase hydrolyze into pyruvate, ammonia (deamination) and indole. Add Kovacs reagent has compound which reacts with indole > red. Sodium thiosulfate. Cysteine desulfurase can can catalyze putrefaction of AA cysteine to pyruvic acid which produces H2S gas. Combines with iron in medium to create ferric sulfide a black precipitate. Motility- radiating growth appears fuzzy allowed by reduced agar content.
Urease
Products: CO2 and ammonia. Urea broth with phenol red. If positive then colour orange>pink. Rise in alkalinity of medium from ammonia.
Negative citrate test
Transfer nutrient agar, lack citrate permease, heat killed bacteria. If you transfer nutrient agar the bacteria can grow even if it doesn’t have citrate permeases.
Sim tests for
H2S (cysteine digestion). Indole (tryptophan digestion). Motility.
Test that require pH change
Citrate and urease
Digest AAs tested by
Cysteine>H2S, tryptophan >Indole.
Broad vs narrow spectrum
Broad many microbes and taxonomic groups, narrow 1tax group few
Disk diffusion test
Used to determine the sensitivity of microbe antimicrobial agent, agar covered with microbe filter paper with agents placed on look for area of inhibition. 1. Measure diameter, 2. Compare with chart, 3. If gram +- both susceptible=broad spectrum.
Bacterial transformation
Insertion of new DNA into bacteria. Change in genes, insert gene new traits
pGLO plasmid
GFP, bla (ampicillin resistance), araC (arabinose operon).
Role heat shock and CaCl2
Heat allows genes to enter cell. CaCl ionizes Ca2+ ion neutralizes charge on the phosphate backbone and phosoLs of PM allowing plasmid to enter.
GFP and arabinose relationship
Medium has arabinose sugar allows for gene expression. In plasmid some of arabinose genes replaced with GFP. Arabinose in medium+successful transformation> GFP activated and GF proteins expressed.
Serology
Study antiG and antiB interactions.
Immunofluorescene test.
Coat wells with antiG/org. Add patient serum. If antiB specific to antiG in serum binds to antiG. Wash to remove unbound antiBs. Fluorescent dye that is specific to the immune complex is added. Look for fluorescence under microscope. E.g. If HIV+ then HIV specific antiB binds to HIV antiG in plate and fluorescent.
ELISA
Enzyme-linked Immunoabsorbent Assay. Neither ELIsA of western blot are antiG linked.
Titer
Most diluted concent of serum antiBs that produce a detectable immune response. Indicator of relative concent of antiG specific antiB in patients serum.
Possible ELIsa results
1st exposure: 1 zero ABs, 7 low level, 14 big number, 21 low again. 2nd exposure: 1 some circulating ABs, 7 big amount, 14 big amount, 21 big amount. Vaccine no exposure- low level AB constant circulation. 0s every day no exposure no current case.
Differential medium
Allows microbiologists to distinguish between groups of microbes.
