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[SCORE A]
Compound microscope - correct answers 1. Ocular-eyepiece lens (usually 10x)
- Objective-nosepiece lenses (commonly 4x, 10x, 45x, and 100x) At what magnification do you use the oil immersion technique? - correct answers 100x. Total Magnification = - correct answers Magnification (Objective) x Magnification (Ocular) Important Parts of the Microscope - correct answers Framework, Stage, and Lens System Framework - correct answers Arm and base are the structural parts of the microscope which support the basic frame. Stage - correct answers Holds the slide. The mechanical stage clamps the slide and moves the slide around the stage. Lens System - correct answers Oculars, Objective, and Condenser Oculars - correct answers Eyepiece lenses (usually 10x magnification)
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Objectives - correct answers Lenses attached to rotatable nosepiece, common magnification of 4x, 10x, 45x and 100x. Parfocalized microscope focusing adjustments are not to be made when... - correct answers changing the objective lenses. The oil has approximately the same refractive index as glass to prevent... - correct answers light loss due to diffraction (bending of light rays) which would occur if light traveled from one refractive index to another (eg. glass to air) As magnification of the objective lens increases... - correct answers the working distance decreases. Working distance - correct answers distance between the object on slide and the objective lens, when in focus. Condenser - correct answers directs light towards the objective lens in bright field microscopy. In dark field microscopy, the condenser... - correct answers directs light at oblique angles away from the objective lens in a manner that allows only objects in the field of view to redirect or scatter light into the objective lens. This causes objects to appear white on a dark field.
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Bacilli - correct answers Cylindrical organisms. Helicoidal - correct answers Spiral shaped organisms. Incompletely separated cocci may appear in a number of different patterns depending upon... - correct answers the plane in which they divide and how they remain attached. Diplococci - correct answers pairs. Divide in one plane. Streptococci - correct answers chains. Divide in one plane. Tetracocci - correct answers tetrads. Divide in two planes. Staphylococci - correct answers clusters. Divide in three planes irregularly. Sarcinae - correct answers cuboidal packets. Divide in three planes regularly. Coccobacillus - correct answers very short and almost appears spherical, but they are just slightly longer in one direction than the other. Fusiform Bacilli - correct answers tapered at the ends, appearing as football like in shape.
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Filamentous Bacillary Forms - correct answers grow in long threads. Most microorganisms are difficult to see using light microscopy due to... - correct answers their size and the lack of contrast between the cell and the environment. The contrast is improved with... - correct answers the help of dyes. Dyes are... - correct answers organic compounds containing a chromophore with affinity for cellular material. Types of dyes - correct answers 1. Cationic (basic dyes, positively charged chromophore) - Methylene Blue, Crystal Violet
- Anionic (acidic dyes, negatively charged chromophore) - Acid fuschin, Congo Red, Nigrosin
- Fat Soluble (no charge): Sudan Black stains granules of Poly-B-OH-butyric acid
- Insoluble Dyes (water insoluble): India Ink (colloid suspension of carbon particles) Negative Staining - correct answers Stains background, not the cell in brightfield microscopy (Not Dark field Microscopy)
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Why are gram negative cells easily decolorized? - correct answers because the ethanol dissolves the high lipid cell wall allowing the crystal violet-iodine complex to readily exit the cell. How do gram positive cells resist colorization? - correct answers due to the difference in cell wall consistency retaining the crystal violet-iodine complex. What color will the gram positive cells be after the primary stain (crystal violet)? - correct answers purple. What color will the gram negative cells be after the primary stain (crystal violet)? - correct answers purple. What color will the gram positive cells be after the mordant (iodine) is added? - correct answers purple. What color will the gram positive cells be after the decolorizer (ethanol) is added? - correct answers purple. What color will the gram positive cells be after the counterstain (saffranin)? - correct answers purple. What color will the gram negative cells be after the mordant (iodine) is added? - correct answers purple.
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What color will the gram negative cells be after the decolorizer (ethanol) is added? - correct answers colorless. What color will the gram negative cells be after the counterstain (saffranin)? - correct answers pink. Acid Fast Stain - correct answers a differential stain procedure that causes cells to stain differently based on characteristics of their cell wall. Cells are steamed in the presence of calbol fuschin and decolorized with acid alcohol. Cells which are "acid fast" (microorganisms have a high wax content in their walls) will not decolorize and remain red, while non acid fast organism will readily lose their stain and become colorless. These cells are then counterstained with Methylene blue. Acid Fast microorganisms have a high... - correct answers wax content in their walls, which requires the use of steam to allow dye to penetrate the cell. What color will the Acid Fast positive cells be after the primary stain (Carbol Fuschin)?
- correct answers red. What color will the Acid Fast positive cells be after the decolorizer (acid alcohol) is added? - correct answers red. What color will the Acid Fast positive cells be after the counterstain (Methylene Blue)? - correct answers red.
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Spore Staning - correct answers Some microorganisms produce heat and chemical resistant structured called endospores or free spores. To stain the spores the cells must be steamed to allow for the dye (malachite green) to enter the spores. Once the spores are stained, all other microorganisms and vegetative cells can easily be decolorized with water, while the free spores and endospores retain the malachite green. The other microorganisms and vegetative cells are then counterstained with Safranin. Endospores appear as... - correct answers a green center within a pink sporangium. Free Spores appear as... - correct answers small green oval bodies. Three genera of Spore forming organisms - correct answers Bacillus - Aerobic, gram
- rod Clostridium - Anerobic, gram + rod Sporsarcinae - Cocci Anerobic green - correct answers endospore/free spores Clostridium Aerobic green - correct answers endospore/free spores of Bacillus Anerobic pink - correct answers vegetative/sporangia of Clostridium
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Aerobic pink - correct answers vegetative/sporangia of Bacillus What color will a spore be after the primary stain (Malachite Green)? - correct answers green What color will a spore be after the steam? - correct answers green What color will a spore be after the decolorizer (water)? - correct answers green What color will a spore be after the counterstain (Safranin)? - correct answers green What color will a non spore be after the primary stain (Malachite Green)? - correct answers green. What color will a non spore be after the steam? - correct answers green. What color will a non spore be after the decolorizer (water)? - correct answers colorless. What color will a non spore be after the counterstain (Safranin)? - correct answers pink.
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Selective and/or Differential Media - correct answers Phenylethyl Alcohol Agar (PEA), Desoxycholate Agar (DES), Eosine Methylene Blue (EMB), and Blood Agar. Phenylethyl Alcohol Agar (PEA) - correct answers Selects for the growth of gram positive microorganisms, because Phenylethyl Alcohol is inhibitory to the growth of gram negative organisms Desoxycholate Agar (DES) - correct answers Selects for gram negative microorganism, because Desoxycholate Agar is inhibitor towards the growth of gram positive organisms. Differentiates for lactose fermentors (lactose + microorganisms from lactose negative). Lactose fermentors produce acid which precipitates the bile salts in the media and absorbs the neutral red dye, therefore appearing Red. Non- fermentors do not do this and do not appear red. Eosine Methylene Blue (EMB) - correct answers Selects for gram neg. organisms. Differentiates lactose +/- microorganisms. Lactose + show a color change, Lactose - do not show a color change Can further differentiate Lactose + fermentors based on the amounts of acid produced during lactose fermentation. Mixed Acid Fermentors produce more acid and produce colonies with dark blue-black centers (center is almost the size of the whole colony) and some microorganisms like Escherichia coli, produce a metallic green sheen Butanediol Fermentors produce less acid so that the colonies have pale pink to lavender centers. The centers are only a small part of the colony (ie bull's-eye colonies), and will not have a metallic green sheen like Enterobacter.
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Blood Aga - correct answers Differentiates microorganisms based on their reactions on blood. Gamma Hemolysis - correct answers No blood hemolysis, no zone of clearing around the colony. Beta Hemolysis - correct answers Complete blood hemolysis and complete clearing around the colony. Alpha Hemolysis - correct answers Partial blood hemolysis and partial clearing around colony. Partial clearing sometimes appears green due to partial reduction of hemoglobin in blood. Biochemical Tests - correct answers Tests used to determine physiological characteristics of microorganism, particularly in terms of bacterial enzymes and the chemistry of biooxidation. Starch Agar - correct answers Tests for the presence of Amylase, which hydrolyses starch to simple sugars. Iodine is added to starch plate and appears blue/black when interacting with starch. If amylase is present starch will be hydrolyzed and the blue/black color will not be observed around the amylase positive colonies.
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Tests for a Mixed Acid Fermentor Mixed Acid Fermentors produce drastic amounts of acid from the fermentation of sugars. This acid ultimately results in the lowing of the pH below 5.1, so when the indicator methyl red is added to the culture the methyl red remains red. Escherichia = MR + Voges-Proskauer (VP) - correct answers HCOOH ----> acetyl methyl carbinol ?2, butanediol Tests for 2,3 butanediol fermentor 2,3 butanediol fermentors produce less acid and more neutral products than Mixed Acid Fermentors. Because acetyl methyl carbinol (acetoin) is easier to detect than 2, butanediol, acetoin is tested for when determining if a microorganism is a 2, butanediol producer. Barrit's reagents, also known as VPI (alpha-naphthol) and VPII (KOH) is added to the test culture. When oxygen is present KOH will react with Acetoin to produce a brick red color and indicate that the microorganism is a 2,3 butanediol producer. Alpha-naphthol is used to intensify the red color. Enterobacter = VP + Catalase - correct answers 2H2O2----> 2H2O and O Hydrogen Peroxide is produced during oxygen utilization and must therefore be eliminated since hydrogen peroxide is toxic. Catalase is an enzyme which converts hydrogen peroxide to water and oxygen, and can be tested for by merely adding H2O to the culture of question and look for the production of oxygen bubbles being produced.
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Oxidase - correct answers Oxidase is an enzyme which can oxidize aromatic amines to form colored products. Aromatic amine used to test for oxidase is dimethyl-p-phenylenediamine hydrochloride which when in the presence of oxidase will turn a dark blue black color. Nitrate - correct answers NO3- + 2e +2H+ ----> NO2 + H2O NO2 ----> N2 + NH3 other products Tests for the ability of microorganisms to reduce Nitrate Organisms are grown in Nitrate broth which contains nitrates (NO3-), Reagents Nitrate I (Sulfanilic Acid) and Nitrate II (dimethyl-alpha-naphthylamine) are added to the broth, if NO2-, a product of nitrate reduction, is present the broth will appear red. If the broth is red at this point the microorganism is said to be Nitrate pos. If the broth is not red, Zinc is added to the tube. Zinc is a catalyst which will convert NO3- to NO2-. Since the nitrate reagents are still in the tube, if NO3- was still present the broth will turn red. If the broth turns red after addition of zinc, the microorganisms are said to be Nitrate neg. If the broth does not turn red, the NO3- of the Nitrate broth was reduced by the microorganism to products other than NO2-, and is therefore said to be Nitrate pos. Tryptophan (Indole) - correct answers Tryptophan ----> Pyruvic Acid and Indole Tests for the enzyme tryptophanase which converts trytophane to indole and pyruvic acid. Indole can easily be tested for by adding Kovac's Reagent (p- dimethylaminobenzaldehyde, amyl or butyl alcohol, and HCl) which will appear red in the presence of the product indole. Urea - correct answers Urea ----> 2NH3 + CO
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Phenylalanine (PPA) - correct answers Phenylalanine ® Phenylpyruvic Acid (PPA) + NH Tests for the presence of the enzyme phenylalanase which converts Phenylalanine to PPA and NH3. To test for the presence of PPA ferric chloride is added to the media. Ferric Chloride in the presence of PPA will appear a deep green color. This green color is indicative of a positive test for phenylalanase. Litmus Milk - correct answers Tests for Lactose fermentation, reduction of litmus, presence of caseinase, and the deamination of amino acids to produce NH Litmus Milk contains the pH indicator Litmus and powdered milk. From this mixture mutiple different Litmus Milk results can be obtained. Acid Reaction - correct answers Pink Liquid due to drop in pH from the fermentation of lactose Acid Curd Reaction - correct answers Pink Solid due to acid production and coagulation of proteins causing the solid formation Reduction - correct answers Litmus is reduced and is caused to be colorless and the tube appears white since only the Milk remains. Alkaline Reaction - correct answers Blue liquid which is usually caused when protein breakdown produces amino acids that are deaminated and release ammonia.
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Peptonization/Proteolysis - correct answers Clearing of medium (may be brown or amber) caused by enzyme caseinase which breaks down the white protein casein in milk. Acid Curd Reduction - correct answers Looks like Acid Curd but the tube turns white except for a small region at the top where oxygen reoxidizes the litmus to the colored form, which is red. IMViC - correct answers Set of four tests that are used to differentiate between Escherichia coli and Enterobacter aerogenes. Indol, Methyl Red, Voges-Proskauer, and Citrate What does E. coli test positive for in the IMViC? - correct answers Indol and Methyl Red. What does E. aerogenes test positive for in the IMViC? - correct answers Voges- Proskauer and Citrate. Motility Media - correct answers Tests if the bacteria are motile or not. Contains Tetrazolium chloride, a growth indicator which turn red in the presence of growing bacteria. Therefore Red color away from the inoculation line is an indicator of growth.
