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71 Cards in this Set
- Front
- Back
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What condenser setting is used for brightfield microscopy?
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Condenser should be at it's highest point
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What are four wyas to improve resolution when using brightfield microscopy?
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1. blue filter
2. condenser at highest point 3. oil immersion 4. the diaphragm should not be stopped down too much |
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How do you calculate total magnification?
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power of ocular by the power of the objective
ex: 10 X 40 = 400 |
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What is the procedure for focusing the microscope?
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Focus with the coarse adjustment know until you see the object (at 10X) the use fine focus knob. Once specimen is in focus, swith ocular to 20X and use fine knob, etc.
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Oil immersion theory
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(refreactive index oil = refractive index glass)
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When would you use darkfield microscopy?
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When looking at delicate trransparent living organisms, or when using fluorescence
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What condenser setting is used in darkfield microscopy?
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varies
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What human disease is often disgnosed using darkfield microscopy?
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Syphilis
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When would you use phase-contrast microscopy?
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When looking at living cells and their activities such as motility.
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What are the advantages of using phase-contrast over brightfield?
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you are able to differentiate the transparent protoplasmic structures and enhance the contrast between a cell and it's surroundings.
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What organisms are typically studied using the phase-contrast microscope?
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living cells, bacterium that are motile
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What is the procedure for calibration of ocular micrometer?
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determine how many of the ocular graduations coincide with one graduation on the scale of the stage micrometer. Ex: seven ocular divisions match up with one stage micrometer division of 0.01 mm to give an occular value of 0.01/7, or 0.00143 mm. since there are 1,000 micrometers in 1 millimeter, these division are 1.14um apart.
next, apply slide of organisms to be measured. count the graduations and multiply by the known distance between graduations. |
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What are the three basic shapes associated with bacteria?
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-rods (bacilli)
-cocci (spherical) -spirals (or curved rods) |
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What is the proper way to label a petri dish?
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Label the bottom of the perti dish with your initals, date, and specimens used. Use bottom because lids may be switched.
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Aseptic technique-what is the appropriate technique for flaming loops and needles?
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Flame the loop or needle until it is red hot to ensure that all the organisms are incinerated.
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What is the proper way to flame the mouth of culture tube?
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Shake tube, hole top of tube in the same hand as the flame or needle and flame the mouth of tube.
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How do you transfer organisms from one broth to another?
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Flame loop until red hot, gently shake tube, hold top of tube in same hand as loop, flame mouth of tube, remove a loopful of organisms from utbe, fmale mouth, recap tube, transfer organisms to sterile tube, flame mouth, insert loop, flame mouth, recap, flame loop til red hot.
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How do you transfer organisms from one slant to another?
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Flame loop, uncap slant, flame mouth, pick up organisms from slant with inoculating loop, flame mouth, recap. open sterile slant, flame mouth, streak organisms with loop, flame mouth, recap, flame loop.
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How do you transfer organisms from a petri plate to a slant?
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Heat loop until red hot, with free hand raise lid just enough to access a colony, pickup loopful of organisms, flame mouth of sterile slant, streak it's surface. Flame mouth of the tube, re-cap, flame loop.
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What is the correct procedure for pouring an agar plate?
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Liquefy a tube of nutrient agar, cool to 50°C, and pour the medium into the bottom of the plate. Be sure to flame the neck of the tube before pouring to destroy any bacteria around the end of the tube. Gently rotate the plate in a figure 8 motion to evenly distribute liquified agar. Failure to cool before pouring will result in condensation.
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Why is the lid of the petri plate vented after pouring?
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To reduce moisture/condensation.
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Streak plate method: theory
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Involve diltuing the bacterial cells in a smaple to an end point where a single cell divides giving rise to a single colony.
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Streak plate method: Procedure
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4 Methods:
Qudrant Streak: method A - Streak one loopful of organisms over Area 1 near edge of the plate. Flame loop, cool, streak 5-6 times from area 1 through 2. Flame, cool, make 6-7 streaks from 2 to 3. Flame loop, cool, make as many streaks as possible from area 3 through 4, using up the remainder of the plate surface. |
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Why should agarplates be incubated upside down?
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To ovoid condesation accumulating on agar surface.
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Subculturing Techniques
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Using loop, carefully pick up an isolated colony and aseptically transfer a colony to the broth tube or slant.
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What are the advantages and disadvantages of heat fixing?
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Cells adhere to slide, fast. Disadvantages: could shattern stain, kill organisms, cause shrikage.
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How do you prepare a smear from a liquid?
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Place two loopfuls of organisms in center of target circle. Disperse organisms over entire area of target circle. Air dry at room temp. Pass slide through flame several times to heat-kill and fix organisms to slide.
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How do you prepare a smear from a solid?
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Place two loopfuls of water in center of target circle. Disperse a small amount of organisms with loop in water over entire area of target circle. Air dry. Pass slide through flame several times.
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What are basic dyes?
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A dye that has a cationic chromophores. Ex: methylene blue
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What are acidic dyes?
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Dyes that have anionic chromophores. Ex: Eosin
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What three things can be determined from a simple stain?
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1. Pleomorphism-irregularity of form/demonstrating several different shapes.
2. Metachromatic granules - distinct reddish-purple granules within cells that show up when the organisms are stained with methylene blue. These granules are masses of VOLUTIN, a polymetaphosphate. 3. Palisade arrangement- parallel arrangement of rod-shaped cells. Picke fence arrangement, common to many corynebacteria. |
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How do you perform a simple stain? What dye is used?
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Prepare a smear, stain with methylene blue for one minute, briefly wash slide with water, carefully blot with bibulos paper
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Negative staining-procedure for preparing slides?
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Organisms are dipersed into a small drop of nigrosin or india ink, near end of slide. spreader slide is moved toward drop of suspension until it contacts the drop causing liquid to be spread along edge. spreader slide is pushed to the opposite side, dragging the suspension over the bottome slide. Air dry-examine under oil immersion
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What is the appearance of the organism and background using negative staining?
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Organisms appear transparent against dark background
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What are the differences between negative and simple staining?
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Negative stain - no heat fixing - no shrinkage. Allows for capsule around bacteria to be observed.
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What is the procedure for a gram stain?
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Prepare smear. Crystal violet for 1 minute. Wash 2 seconds. Gram's iodine 1 minute. Decolorize with alcohol - 10-20 seconds until solvent flows colorlessly. wash 5 seconds. Safranin - 30 seconds to 1 minute. Wash 2 seconds, blot dry.
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What color reactions occur at each step of the procedure?
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Gram + - Neat fixed - no reagent/clear. Crystal violet/purple. Gram's iodine/purple. Ethyl alcohol/pueple. Safranin/purple.
Gram - None/clear Crystal violet/purple Gram's iodine/purple Ethyl alcohol/clear Safranin/pink |
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What is the function of the idodine and othe reagents?
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Iodine is a mordant that complexes with the crystal violet and forms a
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What is the procedure for the Schaeffer-Fulton spore staining method?
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Cover smear with small piece of paper towel and saturate wil malachite green. Steam over boiling water for 5 minutes. After slide has cooled, remove paper, flush with water for 30 seconds. Counterstain with safranin for 20 seconds, rinse briefly with water to remove safranin. Blot dry and examine under oil immersion.
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What is the procedure for the Dorner spore staining method?
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Disperse several loopfuls of bacteria in 5 drops of sterile water, add 5 drops of carbolfuchsin. Heat suspension in beaker of bboiling water for 10 minutes. Mix several loopfuls of bacteria in a drop of nigrosin on the slide. Spread nigrosin mixture on slide. Allow to air dry, examine under oil immersion.
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What is the structural basis for staining results?
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Endospoer inside exosporium
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What would the spore and sporangium look like using the Schaeffer-Fulton method?
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Green endospore and pink sporangium
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What would the spore and sporangium look like using the Dorner method?
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Red spore with a colorless sporangium
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What are the two reagants used in the Shaffer-Fulton method?
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Malachite green and safranin
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What are the two reagants used in the Dorner method?
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Carbolfuchsin and nigrosin
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What is the chemical basis for staining results using the Acid-Fast staining method?
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Waxy material in cell walls of Mycobaterium and some Nocardia called Mycolic acid. This causes them to retain pink/red dyes when doing smears.
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What is the procedure for performing an Acid-Fast stain?
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Cover smear with carbolfuchsin, steam over boiling water for 5 minutes. After slide has cooled, decolorize with acid-alcohol for 15-20 seconds. Stop decolorization by rinsing briefly with water. Counterstain with methylene blue for 30 seconds. Rinse briefly with water, blot dry, examine directly under oil immersion.
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What are two human disease that can be diagnosed using the Acid-Fast staining technique?
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Tuberculosis and Leprosy
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What is Brownian Motion?
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Movement due to molecular bombardment of cells causing cells to shake or "jiggle about" but not move in any vectorial way.
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What is the full name of the medium we used to determine oxygen requirements?
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TGYA Shake - tryptone glucose yeast extract agar
FTM - fluid thioglycollate medium Brewer's Anaerobic Agar (contains tioglycollate and resazurin) |
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What are the components of the medium that act to lower the O/R potential?
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Thioglycollate
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What is the meaning of the pink color at the top of a tube of this medium?
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Indicates the presence of oxygen
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What is the name of the indicator dye in the medium?
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Resazurin
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What is the procedure for the Schaeffer-Fulton spore staining method?
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Cover smear with small piece of paper towel and saturate wil malachite green. Steam over boiling water for 5 minutes. After slide has cooled, remove paper, flush with water for 30 seconds. Counterstain with safranin for 20 seconds, rinse briefly with water to remove safranin. Blot dry and examine under oil immersion.
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What is the procedure for the Dorner spore staining method?
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Disperse several loopfuls of bacteria in 5 drops of sterile water, add 5 drops of carbolfuchsin. Heat suspension in beaker of bboiling water for 10 minutes. Mix several loopfuls of bacteria in a drop of nigrosin on the slide. Spread nigrosin mixture on slide. Allow to air dry, examine under oil immersion.
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What is the structural basis for staining results?
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Endospoer inside exosporium
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What would the spore and sporangium look like using the Schaeffer-Fulton method?
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Green endospore and pink sporangium
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What would the spore and sporangium look like using the Dorner method?
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Red spore with a colorless sporangium
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What are the two reagants used in the Shaffer-Fulton method?
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Malachite green and safranin
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What are the two reagants used in the Dorner method?
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Carbolfuchsin and nigrosin
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What is the chemical basis for staining results using the Acid-Fast staining method?
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Waxy material in cell walls of Mycobaterium and some Nocardia called Mycolic acid. This causes them to retain pink/red dyes when doing smears.
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What is the procedure for performing an Acid-Fast stain?
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Cover smear with carbolfuchsin, steam over boiling water for 5 minutes. After slide has cooled, decolorize with acid-alcohol for 15-20 seconds. Stop decolorization by rinsing briefly with water. Counterstain with methylene blue for 30 seconds. Rinse briefly with water, blot dry, examine directly under oil immersion.
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What are two human disease that can be diagnosed using the Acid-Fast staining technique?
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Tuberculosis and Leprosy
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What is Brownian Motion?
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Movement due to molecular bombardment of cells causing cells to shake or "jiggle about" but not move in any vectorial way.
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What is the full name of the medium we used to determine oxygen requirements?
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TGYA Shake - tryptone glucose yeast extract agar
FTM - fluid thioglycollate medium Brewer's Anaerobic Agar (contains tioglycollate and resazurin) |
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What are the components of the medium that act to lower the O/R potential?
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Thioglycollate
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What is the meaning of the pink color at the top of a tube of this medium?
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Indicates the presence of oxygen
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What is the name of the indicator dye in the medium?
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Resazurin
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Why is the medium boiled before innoculation?
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To ensure all oxygen has been drived off
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What is the name of the pigment produced by Serratia marcesans?
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prodigiosin
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What is the optimum temperature for Serratia marcesans?
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25
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