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39 Cards in this Set
- Front
- Back
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For the identification of unknowns, why do we need to purify on a solid medium when we first obtain our species
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Because it will be provided in a mixed culture
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What are the stains we can use
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Gram
Capsule Endospore Flagella |
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Capsule and flagella stains can be strain specific what does that mean
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Some isolates of a given species are positive while other isolates of the same species are negative
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What is something else that is tricky about capsule and flagella stains
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Their appearance may be dependent upon growth conditions
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Should you rely on capsule and flagella stains when making distinctions among bacterial groups
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No
But use as confirmatory tests |
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What needs to be done for endospore tests
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Cells should be starved before staining them
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How to starve cells
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Transferring a loopful of cells onto a small patch on a plain agar or BG-11 plate (which lacks a carbon source)
After about a day the cells will be adequately starved and should form spores if they are spore formers |
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How can the list of candidate species be reduced considerably in the beginning
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With gram stain
Descriptions of cell Colony morphologies |
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What is the goal of this exercise
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To identify the species with a minimum number of tests
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How does the biological reduction of sulfure to hydrogen sulfde work
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Anaerobically
Two different ways, depending upon metabolism of organism Enzyme cysteine desulfurase catalyzes the hydrolysis of cysteine to pyruvic acid with production of H2S and annomia. The enzyme thiosulfate reductase catalyzes the reduction of sulfur as the terminal step in anaerobic respiration in a group of bacteria known as sulfidogens (sulfide producers) When H2S combines with ferrous ions, a black precipitate of ferric sulfide forms |
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How to test hydorgen sulfide production
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Using a microscopic needle stab a tube of sIM agar and insert 2/3 of depth just once. ncubate tube for up to 2 days
Blackening means H2S production |
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How do you detect indole production in a growth medium
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By the addition of dimethylamino-benzaldehyde which react with indole to produce a red-colored compound
Stab an SIM tube and incubate for up to 2 days After incubation add a few drops of Kovacs' reagent Appearance of a pink or red color in the reagent layer within several seconds indicates the preseance of indole |
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What are the products of prokaryote fermentation of one or more carbohydrates
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Acids and gases
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How to test for acid from lactose or mannitol
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Inoculate a tube of BP (bromcresol purple) plus carbohydrate broth with your culture, and incubate without shaking. Check for up to 7 days regularly
Acid production indicated by change of the purple pH indicator to yellow |
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What is gelatin
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Protein that can be hydorlyzed to amino acids by organisms that possess the extracellular enzyme gelatinase
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How to do gelatin hydrolysis
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A gelatin-solidified medium is liquefied by such bacteria. Using a microbiological needle, stab a nutrient gelatin tube once with a heavy incoulum of your organism
Incubate at room temp for up to one week examine daily for liquefaction of the gelatin For weak hydrolyzers, liquifaction may be apparent only at the surface of the medium |
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What is the voges proskauer reaction
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A number of acidic prods only developed from fermentation of carbs by various bacteria
Some convert their acids to dialcohol 2,3-butanediol Intermediate is acetoin, which can be detected with a simple test from a broth in which cells ferment glucose |
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What is the voges-proskauer reaction (how to do)
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Inoculate a tube of 5 ml VP broth, and incubate without shaking ofr at least 5 days
Transfer a 2 ml aliquot to an empty tube taking care not to disrupt any pellet or surface pellicle of cells Add 0.6 ml VP Reagent A and shake the tube gently for one minute to oxygenate the medium Add 0.2 ml VP Reagent B and gently shae the tube. Place the tube in a rack and do not move it for 20 minutes A positive result will be indicated by coloration of pink or red iwthin 10-20 minutes with the storngest color change at the surface Negative resutls are yellow to copper colored |
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What bacteria have electron carrier cytochrome oxidase
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Bacteria that perform aerobic respiration
Which catalyzes the temrinal reduction of O2 to H2O Many species of aerobic respirers have an alternative enzyme in the electron transport chain, and are thus cytochrome oxidase negative |
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What is decarboylase enzyme do
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Required for the breakdown of amino acids to amines and carbon dioxide
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How to test for presence of ornithine decarboxylase
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Lightly inoculate a tube of liquid ornithine, decarboxyylase medium
Using a sterile pasteur pipet, overlay the medium with 0.5 ml of sterile mineral oil Incubate without shaking for up to a week Psoitive result = purple Negative result = yellow to orange |
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Each enzyme-catalyzed step does what
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Results in the modification of an intermediate compound, the product of which serves as the reactant for the subsequent step in the pathway, or is the final product of that pathway
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EAch enzyme is encoded by ____ gene
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One
(Multi-subunit enzymes reuqire as many genes as there are unique subunitsP |
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An inactivating mutation in one gene of the pathway will prevent _______
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the syntehsis of the final product
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What is an auxotroph
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A mutant unable to make a factor required for growth
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How can a mutatnt be restored (wild-type phenotype restored)
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If suppleid with intermediate compound that is positioned downstream of the pathway's block
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A distruption of the pathway leads to a buildup of the precosor or preceeding intermediate
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Precursor
So it will leach out of the cell and onto the surrounding medium |
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What is crossfeeding
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Complementation analysis
Put two mutatnt strands close to each other so a neighboring mutatnt may be able to absorb the leached intermediate from another strand and if the defective step of the recipent is upstream of that of the donor the recipent may be able to convert the intermediate ultimately to the ifnal product of the biosynthetic pathway |
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What is the experimental outline for the prodigiosin biosynthesis
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1) Dilute culture, plate and expose to UV light
2) Patch several potential mutants to a new plate to confirm a genetic defecit 3) Streak confirmed pigment mutatnts for isolated colonies |
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How many colonies do we want per plate
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200-500
Sp color mutatnts should appear but can still identify and pick out individual colonies |
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How many PG plates inoculate
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6
2 of the dilution calculated 2 will a dilution higher 2 with dilution lower |
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Do you remove the lid when doing UV rays
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Yes
The lid will block the UV rays fro mreaching your cells |
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When you examine your plates after patching potential pigment mutations what are the three types of strains you are looking for
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*Strains look like WT, pigmentation defect was not due to mutations
*Strains maintain their pigmentation defect but are starting to darken around the edges near WT or mutatnt straints, defects in prodigiosin Strains maintin their pigmentation defect throughout and not variable among edges caused by mutation of enzyme late in prodigiosin pathway or other number of genes not in synthesis |
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What two mutations should you be choosing
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Ones with fringe and without any color change
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What plates are you swabing
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Streaking one strain (A, B, C, D,E, WT and two mutatnts_ on each plate with braod swath
Then do combinations A/B, etc(Streak one mutatnt strain onto each of the combination plates.) Should be only a few mm apart but not touching |
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Are capsules resistant to stains
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Yes
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What is a way to visualize capsules
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Negative stianing (stain the background)
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How to perform a capsule stain
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1) Place congo red stain at one end of a clean slide. Add a drop of serum to the congo red.
2) Add a small gob of cells and mix the suspension well on the slide 3) Take a second clean slide (a "spreader") place on surface of the first slide and drawit back into the drop. When the drop flows across the wsith of the spreader, push the spreader to the opposite end of the stained slide to draw out a thin layer of stain and cells 4) Flood in Maneval's stain for a minute 5) Rinse with water and blot dry and stain in red Capsules are clear halos around cells on pink background Use brightfield 100X |
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Why don't you heat fix a capsule stain
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Becaue causes shrinkage and leaves an aritfical halo around the cells that might be interpreted as a capsule
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