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Path Micro 2
Lab Practical 2
| Question | Answer |
|---|---|
| What is the Urease test used for? | detects ammonia present from the break down of urea[so bacteria can use it as energy]. (urea-> 2ammonia + CO2) |
| What does a positive result in Urease test look like? | color change from light peach to bright pink due to increase in pH (more basic) |
| Cytochrome Oxidase Test | looks for the presence of the enzyme cytochrome oxidase (a terminal step in electron transport chain) - positive result is a purple color change. Positive for oxidase follow with oxi/ferm. Negative for oxidase follow up with endotube. |
| What is Triple Sugar Iron Agar test looking for? | sugar fermentation (glucose, sucrose, lactose), peptones, gas production, and H2S production (reduction of sodium thiosulfate interacting with ferric ammonium citrate) |
| What components are in TSI Agar? | glucose (0.1%), lactose (1%), sucrose (1%), peptones, ferric ammonium citrate, sodium thiosulfate, phenol red. |
| What does TSI result K/K look like? | Alkaline slant/alkaline butt: no sugar fermentation. peptones in medium are used resulting in NH3 production and alkaline pH. PINK all over! no gas, no h2s. |
| What does TSI result K/A, with or without G look like? | Alkaline slant/acid butt: with or without gas. Only glucose was fermented resulting in an acid butt, with a yellow color. Slant is pink. Uses the Emben-Meyerhof-Parnas pathway to ferment glucose |
| What does TSI result of black coloration mean? | H2S is produced. (from sodium thiosulfate and ferric ammonium citrate). There must have been acidic environment first. |
| What carbohydrates were tested to see if they could be fermented? | glucose, sucrose, and lactose. |
| If the organism was able to ferment the carbohydrate, what color did the indicator turn? | The phenol red turned yellow. pH is lower. There may have also been gas production in the Durham tube. |
| What did the motility test look for? | The ability of the organism to move, with its flagella, through the medium. Tetrazolium red dye was used. |
| What tests are found in the IMViC series? | Indole, Methyl Red, Voges-Proskauer and Citrate |
| What is the Indole test looking for? | Indole. One of the byproducts of tryptophan breakdown by tryptophanase. other byproducts are pyruvic acid and ammonia. |
| What reagent is used for the Indole test | Kovac's Reagent (a-napthol)reacts with Indole, producing a red dye called rosindole, a positive result. |
| What does the Methyl Red test look for? | looks for the ability of the organism to ferment pyruvate into acid. ex. lactic acid, acetic acid and formic acid. |
| Methyl Red positive result appearance? | Positive result = Red color = mixed acid fermentation. Negative Result = yellow color = no mixed acid fermentation. |
| Voges-Poskauer testing for what? | The ability of the organism to ferment pyruvic acid into 2,3-butanediol and acetoin by way of the butylene glycol pathway. |
| What reagents are used for the the Voges-Poskauer test? | Barritt's A (a-napthol) & B (40% KOH Potassium Hydroxide) |
| What does a positive result for VP look like? | Positive = Red = neutral end products Negative = dark urine color |
| What does the Simmons Citrate Test look for? | to see if organism can utilized citrate as the sole carbon source. Also ammonium dihydroxide phosphate is the sole nitrogen source. CO2 interacts, increases pH, bromothymol blue shows blue at higher pH, green at lower pH. |
| What are the selective components of Eosin Methylene Blue (EMB) agar? | aniline dyes (eosin and methylene blue) - they inhibit Gram + organisms and selects for Gram - enteric organisms. |
| What are the differential components of Eosin Methylene Blue (EMB) agar? | Lactose. Lactose fermenters = pH reduction = purple or green metallic colonies slight fermenters = pink colonies non-fermenters = colorless |
| What are the selective components of MacConkey (MAC) agar? | bile salts & crystal violet - inhibit growth of Gram +, selects for Gram- |
| What are the differential components of MacConkey (MAC) agar? | lactose and neutral red pH indicator. lactose fermentors = reddish colonies non-fermentors = clear to colorless colonies |
| What are the selective components of Hektoen (HEK) agar? | bile salts and acid fuschin - inhibits Gram+ growth, selects for Gram- |
| What are the differential components of Hektoen (HEK) agar? | three carbohydrates: lactose, sucrose, salicin (when fermented, lowers pH, acid fuschin reacts Bromothymol blue = color change) Sodium Thiosulfate (to be broken down by organism and to react with Ferric Ammonium Citrate to form H2S = black color change) |
| Decarboxylase tests looking for what? | to see if organism can decarboxylate specific amino acids. |
| Why did the decarboxylase tests use mineral oil? | To maintain a lowered oxygen anaerobic environment so the enzyme can be active. |
| Decarboxylation results in what products for each amino acid (ornithine, lysine, arginine) | Ornithine -> putrescine + CO2 Lysine-> Cadaverine + CO2 Arginine -> agmatine +CO2 |
| Break down of carbohydrates yield what 3 products? | acid (lowered pH), gas (CO2 or O2), alcohol |
| What does a positive test for decarboxylation look like? | When decarboxylation occurs, pH increases and bromcresol purple changes to purple. Without decarboxylation, color remains the same or changes to yellow (reduction in pH) |
| What does the gelatinase test look for? | the organism's ability to produce gelatinase, which breaks down gelatin. If the organism produces it, no solidification of the gel will occur after being placed on ice. No production of gelatinase = solid gelatin when placed on ice. |
| What is gelatin a component of? | joints and connective tissue |
| What is the Salmonella-Sigella Agar used for? | To isolate Salmonellla and Shigella from lactose fermenting enterics. |
| What are the selective components of Salmonella-Shigella (SS) Agar? | bile salts and brilliant green die. selects gaingst Gram+ and many Gram- |
| What are the differential components of Salmonella-Shigella (SS) Agar? | Contains lactose and neutral red pH indicator. Lactose fermenters = pink to red non-fementers (Salmonella/Shigella) will appear colorless. Also Sodium Thiosulfate as sulfur source, reacts with ferric ammonium citrate to form H2S, causing black color. |
| Which organism produces H2S on SS Agar? | Salmonella |
| What is Xylose Lysine Desoxycholate (XLD) Agar testing for? | Isolates and identifies enteric pathogens. |
| What are the selective components of Xylose Lysine Desoxycholate (XLD) Agar? | Sodium desoxycholate - inhibits Gram+ bacteria |
| What are the differential components of Xylose Lysine Desoxycholate (XLD) Agar? | Xylose (fermentation = lower pH = phenol red changing to yellow) L-lysine decarboxylation = cadaverine and CO2 = higher pH = red appearance Sodium thiosulfate when reduced + ferric ammonium citrate = H2S = black color |
| What is the purpose of the Phenylalanine deaminase test? | To see if the organism produces deaminase. This enzyme removes the amine group from the amino acid phenylalanine and releases the amine group as free ammonia. As a result of this reaction, phenylpyruvic acid is also produced. |
| What does Phenylalanine agar, or phenylalanine deaminase medium, contain? | nutrients DL-phenylalanine |
| How is the Phenylalanine deaminase test performed? | After incubation, 10% ferric chloride (FeCl3) is added to the media; if phenylpyruvic acid was produced, it will react with the ferric chloride and turn dark green. If the medium remains a straw color, the organism is negative for deaminase production. |
| Enterotube | To quickly perform identification tests on oxidase negative organisms. Must perform cytochrome oxidase test first. Utilizes an algorithm to identify potential organisms. |
| Oxi/Ferm | To quickly perform identification tests on oxidase positive organisms. Must perform cytochrome oxidase test first. Utilizes an algorithm to identify potential organisms. |
| What color is phenol red at basic pH? | Red - it is yellow at acidic pH yellow when pH<6.8 red when pH<8.2 |
| What color is methyl red at basic pH? | yellow - it is red at acidic pH |
| What is the reason for using 16s rRNA sequence for identification? | the 16s region is highly conserved portions of the rDNA sequence from distantly related organisms are remarkably similiar They are easily and precisely able to be aligned to determine differences |
| What was our PCR procedure? | 5-10 min initial denaturation//Denaturation, 30sec-1min(95*C), Anneling 30 sec(50*C), Extension 90 sec (72*C) - cycled 20-40 times// followed by 10 minute final extension (72*C) |
| What is required in a PCR reaction? | DNA template strand, dNTPs(nucleotides), buffer, Taq Polymerase (enzyme), Primers (16sF, 16sR), MgCl (salt), and Bovine Serum Albumin (BSA)to reduce colony components. |
| What is electrophoresis? | Applying an electrical current to separate DNA fragments by size through a matrix (agarose). Current runs from negative (well side) to positive so negatively charged DNA will move through the gel. Agarose acts as a "sieve" - smaller fragments move faster |
| What is the purpose of an agarose gel? | Agarose acts as a "sieve" - smaller fragments move faster. |
| How are we able to analyze our DNA sequence to determine the bacterium’s identification? | 16S rDNA PCR Assay |
| What are the differences between sequencing reactions and PCR? | sequencing tells you the exact order of the nucleotides in DNA while PCR just tells you the length |
| What kind of broth was used for the Indole test? | Tryptone Broth |
| What was found in the loading dye we used for electrophoresis? | Glycerol - for weight Xylene cyanol - blue green color runs at about 4000 bp Bromophenol Blue - violet blue color - 500bp SYBR green - colormetric way to visualize - intercalates |
| What are satellite colonies? | Wild-type bacteria colonies that grow in the clearing of antibiotic around colonies growing that contained the antibiotic resistant plasmid. |
| What color is neutral red pH indicator when the pH<6.8? | Red Colorless when pH>6.8 |
| How many base pairs are in the 16s sequence? | 1500 |
| What occurs during PCR? | takes one molecule of dsDNA and replicates a selected portion of that DNA which has been defined by primers. When the newly polymerized segment of DNA is denatured by the heat, it becomes the template for the next round of replication. |
| What does the broth contain for MR/VP? | peptone, buffers and glucose. Glucose can be utilized then by different pathways and these tests differentiate which pathway was followed. |
| What nutrients does Simmons citrate medium contain? | citrate - sole carbon source ammonium dihydrogen phosphate - sole nitrogen source only those bacteria who can utilize both will grow. |
| What color is Bromothymol blue at low pH (acidic)? | green (citrate is acidic so it will appear green in Simmons Citrate medium) |
| What is decarboxylation? | removal of the carboxyl group from an amino acid resulting in the release of CO2 and the alkaline amine substance. |
| CaCl2 transformation solution helps transformation how? | cell membrane is permeable to chloride ions, but is non-permeable to calcium ions. chloride ions enter the cell, water molecules accompany the charged particle. This influx of water causes the cells to swell and is necessary for the uptake of DNA. |
| Define: Minimum Inhibitory Concentration (MIC) | The minimum concentration of antibiotic required to inhibit bacterial growth. |
| How do you determine Minimum Inhibitory Concentration (MIC) | Serial dilutions of antibiotic are made and inoculated. Lowest concentration of antibiotic preventing appearance of turbidity (i.e. growth) is MIC |
| Define: Minimum Bactericidal Concentration (MBC) | the minimum concentration of antibiotic required to kill bacteria |
| How do you determine Minimum Bactericidal Concentration (MBC) | Following MIC, the dilutions can be plated on normal growth medium to see if organisms are recovered. The lowest concentration with no growth is the MBC. |
| What two important genes did the plasmid contain? | ampicillin resistance gene and gfp gene |
| What can yield a false positive on the Cytochrome Oxidase disk test? | Exposure to air will oxidase the substrate found on the disk after a certain amount of time. It is important to only inoculate 1/2 so when the other side changes color you know when it has been too long and your organism is for sure oxidase negative. |
| What concentration is typically used of an antibiotic after the MIC is determined? | 10x MIC |
| pGlo lab: what did the Ampicillin resistance gene do? | Allowed the organisms that picked up the plasmid to grow on a nutrient agar containing Ampicillin. The gene produces ampicillinase which breaks down ampicillin allowing E.coli to grow on LB + ampicillin plates |
| pGlo lab: how was the green fluorescent protein gene regulated? | Under the control of an arabinose inducible promoter (and AraC). Will only see fluorescence if arabinose is present. |
| What is agarose? | polysaccharide extracted from seaweed and is non-toxic - used at different concentrations to make gels for electrophoresis |
| pGlo lab: what are the components of the plasmid? | Ori - origin of replication bla - B-lactamase (ampacillinase) gfp - green fluorescent protein araC - Arabinose utilization |
| How was DNA extracted from the agarose gel? | dissolving gel, washing sample, binding DNA to column and collecting after eluting. |
| What is the Kirby-Bauer Antibiotic Sensitivity test? | organism plated on Mueller-Hinton Agar, disk diffusion test of 6 antibiotics at different concentrations. Zones of inhibition measured, compared to chart, susceptibility determined. |
| What 6 antibiotics were tested in the Kirby-Bauer Antibiotic Sensitivity test? | Penicillin, Ampicillin, Tetracycline, Chloramphenicol, Streptomycin, Erythromycin |
| Steps for transformation! | Add CaCl2, Plasmid and Culture Incubate on Ice Heat Shock Ice Nutrients Plate on appropriate media |
| Purpose for allowing incubation time during transformation with nutrient broth? | allows the bacteria time to generate the antibiotic resistance proteins encoded on the plasmid backbone so that they will be able to grow once plated on the antibiotic containing agar plate. |
| What is the first icing step in transformation for? | It makes the change in temperature very drastic which shocks the bacteria when placed in the heat - during this time it is changing the lipids found in the membrane to deal with the cold environment |
| What is the heat shock step in transformation for? | The cold adapted membrane becomes unstable when heated abruptly and increases plasmid uptake. |
| What is the second icing step in transformation for? | Restabilizes the membrane before nutrients are added. |
| What are peptones? | polypeptide products, formed in partial hydrolysis of proteins, that are soluble in water, diffusible, and not coagulable by heat; used in bacterial culture media. |
| What is the pH indicator in the urea test? | Phenol red |
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