Lab Quiz #6 – Flashcards
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| What does the Tryptophanase/Indole test testing for? |
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| The ability of an organism to hydrolyze tryptophan into indole |
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| What type of media is used in the Trypophanase/Indole test? What does it contain? |
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| SIM agar which contains tryptophan |
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| What reagent is used to detect indole in the Tryptophanase test? |
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| Kovak's reagent |
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| Is tryptophanase an endo or exo enzyme? |
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| Endo |
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| What are the 4 tests in the IMViC series? |
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| 1. Indole/Tryptophanase 2. Citrase 3. Methyl Red 4. Voges-Proskauer |
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| What does a positive Indole test look like? |
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| Red reagent layer after adding Kovak's |
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| What does a negative Indole test look like? |
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| No reaction after addition of Kovak's. No color. |
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| What does the Citrase test test for? |
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| Citrase converts citrate into oxaloacetic acid and acetate which is converted into pyruvic acid and CO2 (alkaline) |
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| What does a positive citrase test look like? |
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| blue media (from alkaline) |
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| What does a negative citrus test look like? |
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| green media (no change in pH) |
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| What does the Methyl Red test for? What is this detected by? |
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| Production of acidic end products from breakdown of glucose (detected by pH indicator Methyl Red) |
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| What class of organisms does the Methyl Red test target and why? |
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| enteric bacteria because glucose is a major substrate used by this class. |
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| What does a positive Methyl Red test look like? what pH range is this for? |
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| pH rang of 4 (red) |
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| What does a negative Methyl Red test look like? what pH range is this for? |
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| pH of 6 (yellow) |
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| What does Voges-Proskauer test for? (include broad trait and specific end-product) |
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| neutral end products of glucose breakdown- acetylmethylarbinol |
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| What reagent is used in the Voges-Proskauer test? |
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| Barritt's reagent A and B |
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| What does a positive Voges-Proskauer test look like? |
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| deep rose color after 15 minutes with Barritt's agent added |
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| What does a negative Voges-Proskauer test look like? |
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| No rose color? |
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| What additional traits is the Indole media used to test? |
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| H2S production and motility |
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| What does the Hydrogen Sulfide test test for? |
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| breakdown of Sulfur containing amino acids and other inorganic molecules to create H2S |
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| What media is used to detect H2S |
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| SIM |
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| What does a positive H2S test look like? |
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| black precipitate |
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| How do you detect motility in a bacteria? what media is used for this? |
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| when culture growth is not limited to the line of inoculation (SIM agar) |
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| What enzyme is being detected in the Urease test? What are the substrates and products in the reaction and what is the products pH? |
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| Enzyme is urease breaks N and C bonds in amides to produce ammonia (alkaline) |
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| What are the two reagents in the Urease test? |
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| Urea (amide compound) and phenol red (pH indicator) |
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| What pH does phenyl red test for in the Urease test? |
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| alkaline |
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| What does a positive urease test look like? |
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| hot pink media |
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| what does a negative urease test look like? |
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| orange |
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| What does the Catalase test test for? |
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| presence of catalase which breaks down hydrogen peroxide |
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| What is the function of catalase breaking down hydrogen peroxide? where does this hydrogen peroxide come from? |
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| Hydrogen peroxide is a byproduct of superoxide dismutase breaking down toxic superoxides |
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| What does a positive catalase test look like? |
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| bubble production when hydrogen peroxide is added to culture (O2 bubbles) |
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| What does the oxidase test test for? |
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| Cytochrome oxidase removal of electrons of final protein of the ETC by O2. |
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| what reagent is used in the oxidase test? |
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| oxidase strip with oxidase reagent |
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| what does a positive oxidase reaction look like? |
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| dark purple color in 10-30 seconds |
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| what does ELISA stand for? |
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| enzyme-linked immunosorbent assay |
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| What does a direct ELISA test for? |
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| detect antigen |
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| what does an indirect ELISA test for? |
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| detect antibodies |
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| What is the binding order for Direct ELISA? |
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| antigen--> known antibody with fluorescent label--> observe under light |
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| What is the binding order for Indirect ELISA? |
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| antigen bound to slide--> patient serum added (antibody)-->florescent anti-antibody (antibody sandwich) |
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| For carbohydrate fermentation, what media and pH indicator were used to identify fermentation? |
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| media was nutrient broth with phenol red |
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| what were the three sugars used to measure carbohydrate fermentation? |
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| lactose, glucose, sucrose |
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| Which bacteria had acid and gas productions (yellow with bubble)? |
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| E. coli |
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| Which bacteria had no fermentation (no acid or gas)? |
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| A. faecalis and Pseudomonas |
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| Selective Media |
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| selects among a specific groups of bacteria- usually share common trait (gram positive, gram negative, halophiles) |
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| 3 types of selective Media |
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| -Crystal Violet Agar -Phenylethyl Alcohol Agar -7.5% Sodium Chloride Agar |
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| Phenylethyl Alcohol Agar (what does it inhibit, what does it select for) |
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| Inhibits Gram negative (alcohol disrupts membrane) Selects for Gram Positive cocci |
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| Crystal Violet Agar (what does it inhibit, what does it select for) |
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| Inhibits Gram Positive Selects for Gram Negative |
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| 7.5% Sodium Chloride Agar (what does it inhibit, what class of bacteria does it select for, give an example of a bacteria in this class) |
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| inhibits anything non-halophilic Selects for halophiles Ex: Staphylococcus |
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| Differential Media |
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| Selects specific organism within a group of bacteria (identifies species) |
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| 3 types of Differential Media |
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| -Mannitol Salt Agar -MacConkey Agar -Eosin-Methylene Blue Agar |
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| Mannitol Salt Agar (What group does it select for, what does it differentiate and how, provide examples) |
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| salt selects for halophiles- Staphylococcus Mannitol fermentation (pathogenic trait) differentiates epidermidis from aureus Ex: S. aureus ferments mannitol and turns red media yellow |
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| MacConkey Agar (What group does it select for, what does it differentiate and how, provide examples) |
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| Selects for gram negative (crystal violet inhibits gram positive) Differentiates lactose fermenting bacteria (coliforms) Lactose fermentation produces acids which turn the pH indicator red Ex: E. coli, citrobacter, Enterobacter, Klebsiella |
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| Eosin-Methylene Blue Agar (What group does it select for, what does it differentiate and how, provide examples) |
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| Selects for gram negative lactose fermenters (coliforms) Differentiates between E. coli and all other coliforms- E. coli produces metallic green color -Enterobacter aerogenes (coliforms)- pink colonies w central dark purple spot |
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| Enriched Media- example? |
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| Contain nutrients to support a wide variety of bacterial growth as well as additional unique requirements of fastidious growers Ex: blood agar |
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| Blood Agar (what does it differentiate? what are the three types of growth?) |
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| Differentiates hemolytic Streptococcus species (enriched and differential media) 1. Alpha hemolysis 2. Beta hemolysis 3. Gamma hemolysis |
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| Alpha hemolysis (what does it look like and what organism shows this type of growth?) |
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| Partial breakdown of hemoglobin shows green/light brown sheen around colonies (biliverden) Ex. E. coli |
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| Betta hemolysis (what does it look like and what organism shows this type of growth?) |
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| Full breakdown of red blood cells leaves clear space- lyse cells using beta-hemolysin (streptolycin) Ex: S. pyogenes |
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| Gamma hemolysis (what does it look like and what organism shows this type of growth?) |
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| no hemolysis- whitish grey sheen on surface of media Ex: S. epidermidis |
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| Enteric Bacteria (2 types) |
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| Bacteria that colonize the intestinal tract- coliform and non-coliform -ferment glucose -facultative anaerobes -usually gram negative bacilli |
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| Coliforms |
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| lactose fermenting enterics Ex: E. coli, citrobacter, Enterobacter, Klebsiella |
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| [image] |
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| mannitol salt agar |
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| [image] |
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| Alpha Hemolysis (Blood Agar) |
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| [image] |
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| Betta Hemolysis (Blood Agar) |
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| [image] |
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| Gamma Hemolysis (Blood Agar) |
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| [image] |
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| MacConkey Agar |
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| [image] |
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| Eosin Methylene Blue Agar (E. coli) |
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| [image] |
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| Eosin Methylene Blue Agar (Enterobacter Aerogenes) |
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| Psychrophil |
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| 5-15 degrees C |
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| Mesophil |
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| 15-40 degrees C |
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| Thermophil |
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| 40-80 degrees C |
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| Hyperthermophil |
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| 80-110 degrees C |
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| Best temp for S. marcenscense |
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| 20 degrees C (mesophil)- red pigment |
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| 3 toxic Oxygen forms (superoxides) |
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| Superoxide Peroxide Hydroxyl |
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| 3 enzymes for superoxide breakdown. Where are they found? |
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| Found in aerobes - Superoxide dismutase -Catalase -Peroxidase |
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| What agar medium was used in the oxygen requirement experiments? |
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| Brain heart fusions in deep tubes |
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| What area in the agar do aerobes grow? |
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| at the surface of the tube |
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| what area in the agar do obligate anaerobes grow? |
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| bottom of the tube |
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| what area in the agar do facultative anaerobes grow? |
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| throughout the agar with more at the surface |
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| What is the final electron acceptor for aerobes? |
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| oxygen |
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| what is the final elector acceptor for obligate anaerobes? |
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| molecules other than oxygen |
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| In the catalase experiment, what solution is added to the tubes |
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| hydrogen peroxide (H2O2) |
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| What represents a positive reaction in the catalase test? |
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| formation of O2 bubbles |
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| in a normal cell, where would the hydrogen peroxide being neutralized by catalase come from? |
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| superoxide dismutase breaks down toxic superoxides and produces hydrogen peroxide. |
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| What agar and reagent is used in the amylase test? |
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| starch agar and Gram's iodine |
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| What reaction is being tested for in the amylase test? |
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| Starch hydrolysis into simple sugars (glucose) |
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| What does a positive Amylase test looks like (with Gram's iodine added)? |
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| cleared zone (halo) surrounding the growth |
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| What does a negative Amylase test looks like (with Gram's iodine added)? |
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| Blue to black color in the medium |
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| What agar is used in the lipase test? |
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| Tributyrin agar (with lipid)- blue color |
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| What reaction is being tested in the lipase test? |
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| hydrolysis of ester bonds by lipase |
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| What does a positive lipase test look like? |
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| clear halo surrounding growth. Loss of apacity |
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| What does a negative lipase test look like? |
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| Medium remains opaque blue color surrounding growth |
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| What media is used in the Casein test? |
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| milk agar |
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| What reaction is being tested in the Casein test? |
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| breakdown of casein, a protein in milk, by protease |
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| What does a positive results in the Casein test look like? |
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| loss of opacity surrounding the bacterial growth |
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| What does a negative result in the casein test look like? |
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| medium surrounding the bacterial growth remains opaque |
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| [image] |
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| Casein Test |
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| [image] |
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| Amylase Test |
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| [image] |
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| Lipase Test |
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| [image] |
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| Gelatinase Test |
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| What agar and broth was used for the temperature experiment? |
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| Nutrient broth and TSA platse |
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| What method was used to inoculate the oxygen requirement bacteria in brain hear fusion? |
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| shake tube inoculation |
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| What are the 4 exoenzymes? |
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| 1. lipase 2. amylase 3. gelatin 4. casein |
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| in the gelatin experiment, what enzyme is breaking down gelatin and what is it breaking gelatin down into? |
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| gelatinase breaks gelatin down into amino acids |
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| how many ml to one micro liter? |
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| 1ml = 1000ul |
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| In the DNA extraction experiment, what is done to the strawberry cells to expose the DNA? |
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| The cells are treated with detergent (SDS) in a buffer solution |
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| In the DNA extraction experiment, how is protein precipitated out of the solution? |
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| presence of salt in the DNA extraction buffer |
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| In the DNA extraction experiment, how is cellular waste and particles removed from solution? |
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| filtered out |
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| In the DNA extraction experiment, how is DNA precipitated from the filtered solution? |
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| Addition of cold ethanol |
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| Transformation- What two genes were present in the plasmid vector? |
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| amp resistance and GFP |
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| Transformation- how did we select for transformed bacteria? |
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| plated on amp agar |
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| Transformation- what was done to E.coli to induce competency? |
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| CaCl2 and heat shock |
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| Transformation- what were the growth areas surrounding transformed bacteria called? |
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| satellite colonies |
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| What is another name for the disk diffusion test (antibiotics)? |
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| Kirby-Bauer |
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| What is the area surrounding an antibiotic disk that restricts growth called? |
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| zone of inhibition |
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| What are two typical gram + bacteria? |
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| B. cereus and S. aureus |
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| What three sugars do phenol red tubes contain for carbohydrate fermentation? |
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| 1. glucose 2. lactose (galactose + glucose) 3. sucrose (fructose + glucose) |
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| In carbohydrate fermentation, which bacteria usually produces acid? |
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| S. aureus |
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| In carbohydrate fermentation, which bacteria doesn't undergo gas or acid production? |
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| Pseudomonas |
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| What type of agar is used to test for dental caries susceptibility? |
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| Snyder Agar |
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| What does a Snyder test test for? |
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| acid production from the breakdown of glucose by lactobacilli |
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| What pH does Snyder agar test for? What is the color change for a positive result? |
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| test for acidic environment. Changes from green to yellow in presence of pH 4.4 and below |
