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143 Cards in this Set
- Front
- Back
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What items go in the Sharps container?
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Slides, used and/or contaminated
Cotton tipped applicators Plastic inoculating loops Ampules and empty antibiotic disc containers used pipettes and micropipette tips |
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What goes in the red/orange biohazard bags?
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Anything heavily contaminated with bacteria (as long as is it not sharp)
Petri plates Plastic test tubes and micr-centrifuge tubes |
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What goes in orange disposal test tube racks?
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Used glass culture (test) tubes
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What goes in the broken glass container?
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All broken glass needs to be reported to instructor.
Disposal of broken glass will be done by your instructor. (glass box under instructor's bench) |
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What goes in regular trash containers?
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Gloves, paper towels, plastic, paper wrappers from swabs, paper items that cannot be recycled, and other trash items not contaminated with bacteria.
No food or beverage containers NO SHARPS!!! |
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When and how should you prepare your lab table before you work with bacteria?
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Before and after lab procedure with disinfecting spray and paper towels.
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The length of cells is estimated how?
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By dividing the field diameter by number of cells across the diameter.
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This magnifies specimen 10X
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Ocular lens
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This can be opened or closed to regulate the amount of light that hits the specimen
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Iris Diaphragm
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Ocular Lens: 10X
Objective Lens: 100X Cells fitting across field: 70 Determine the field size and estimated size of cells in mm and micrometers |
field size is 0.2mm
divide 0.2mm by 70 gives .003 mm which is the estimated cell size in mm multiply 0.003mm by 1000 to get micrometers which will give 3 micrometers |
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Why do you need to turn down the scope light before you turn it off?
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To prevent damage to the light
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As magnification size increases, field size ______________
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decreases
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The term that refers to the fact that once your specimen is in focus at low power it will remain in focus at a higher power is ________________
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parfocal
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Culture media whose chemical composition is known is called _____________ media.
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defined or synthetic
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In our lab, organisms are grown on _____________ media.
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complex or non-synthetic
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What tool is used to inoculate
liquid to liquid? environmental sample to plate? solid agar plate to deep? liquid to slant? |
liquid to liquid is a loop
env'l to plate is cotton swab solid agar to deep is needle liquid to slant is loop |
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What two types of information do you need to write on the media containers that you inoculate?
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your name and specimen name
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Remember to label the __________ of your petri plate and it must be ____________ before it is put in the rack for incubation.
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label the bottom of petri plate
close it before you place it in the rack |
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An example of a negative stain.
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India ink
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Beginning with a culture from a solid agar plate, outline the procedure for preparing a bacterial smear.
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1. Place one drop of distilled water on slide.
2. flame inoculating loop. 3. lift lid of Petri plate, do not completely remove lid. 4. gently run loop over agar to collect a small amount of bacteria. 5. place loop of bacteria on slide, smear/blend with the drop of water. 6. air dry 7. heat over bunsen burner briefly (2-3 secs) to heat fix. |
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Beginning with a bacterial smear, outline the procedure for a simple stain.
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1. place heat-fixed sample on tray in sink.
2. cover with methylene blue. 3. let sit for 3 mins. 4. rinse with distilled water to remove methylene blue. 5. blot with Kimwipe to remove excess water. 6. view under microscope with oil immersion. |
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Name one of the stains used for the gram stain.
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Crystal Violet
Gram's Iodine Safranin |
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How long do you leave on each stain/decolorizer in the Gram stain?
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Crystal violet: 2 mins
Gram's iodine: 2 mins Decolorizer: 3-5 secs Safranin: 3 mins |
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Gram positive organisms will stain ____________.
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purple
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Gram negative organisms will stain ____________.
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pink-red
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Name the Primary Stain, mordant, decolorizer, counterstain in gram stain process.
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Primary Stain: Crystal Violet
Mordant: Iodine Decolorizer: Acetone-alcohol Counterstain: Safranin |
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What color will gram (+) appear in each step of gram stain process?
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Crystal Violet: purple
Iodine: purple Decolorizer: purple Safranin: purple |
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What color will gram (-) appear in each step of gram stain process?
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Crystal Violet: purple
Iodine: purple Decolorizer: clear Safranin: pink-red |
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What is the function of the mordant?
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Adhere the color to the cells.
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Cultures used in gram staining should be younger than ____ old.
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24 hours
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What step in the gram stain is the most crucial and why?
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Decolorizer is the most crucial step. Gram (+) cells have already picked up their color once the decolorizer has been applied. Gram (-) cells need decolorizer to break down outer membrane and allow the safranin to bind to cells to give them color.
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What are two different types of environmental conditions that could trigger endospore formation?
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extremes in pH (high or low)
extremes in temp (high or low) |
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Many flagella covering the surface of a bacterial cell is termed ___________ while a single flagellum is termed ____________.
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many flagella is peritrichous
single flagellum is monotrichous |
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Name a bacterium that is acid-fast.
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Mycobacterium
Nocardia |
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What is the thick outer layer of special lipids in acid-fast bacteria called?
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Mycolic acid
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Organisms that can grow in the presence of oxygen but don't use it for energy production are termed ____________.
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aerotolerant
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Organisms that grow with or without oxygen are called _____________.
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facultative
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Why should you not use metal loops for the catalase and oxidase tests?
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Metal acts as a catalyst to give false positives.
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Write out the chemical reaction for the catalase test.
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H2O2 ----> H2O + O
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What are three characteristics of Enterobacteriaceae?
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Gram (-)
rods ferment |
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What pH indicators are used in the following media?
a. Citrate b. MR c. Carbohydrate tubes |
a. bromothymol blue
b. methyl red c. phenol red |
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Name two media that test for the presence of lactase in your bacteria?
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MacConkey
TSI Carbohydrate tubes |
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T/F
A red/orange color in a carbohydrate fermentation test indicates that the organism produces acid end products? |
False. A yellow color indicates acid end products
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T/F
Citrate agar contains citrate, glucose, and lactose as metabolic energy sources. |
False. Citrate agar contains citrate only as metabolic energy source.
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T/F
Basic T-Soy broth is used to grow organisms for the MR/VP test. |
True.
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T/F
The MR/VP test results can be -/-, -/+, +/-, but not +/+. |
True.
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T/F
Coliforms generally lack the ability to ferment lactose. |
False. Coliforms generally have the ability to ferment lactose.
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If you perform a SIM test and had a clear ring at the top of the tube and a black cloud around the stab, how would you interpret these results?
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Positive for S (H2S production and contains enzyme cysteine desulfurase)
Negative for Indole (no red ring at top) Positive for Motility (black cloud around stab) |
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If you performed a Phenylalanine test and the results indicated a green pool at the bottom of the slant, how would you interpret these results?
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This is a positive result for the enzyme deaminase.
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If you performed a Lysine/Ornithine test and the results indicated a purple color in the Lysine tube and a yellow color in the Ornithine tube, how would you interpret these results?
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The Lysine tube is positive for decarboxylase. The Ornithine tube is negative for decarboxylase as it had yellow color.
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If you performed a Urea slant test and the results indicated a yellow color in the slant, how would you interpret these results?
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This Urea test is negative for the enzyme urease. A positive result would be indicated by a fuchsia pink color in the slant.
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If you performed a SIM test and the results indicated a red ring at the top of the tube, with a clear stab line and no cloudiness, how would you interpret these results?
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Because the tube is clear with no cloudiness or precipitate, it is negative for H2S production. S negative.
It has a red ring which indicates the ability to metabolize the amino acid tryptophan. I Positive There is a clear stab line with no precipitate which indicates no motility. M negative. |
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What is the pH indicator used in Lysine/Ornithine broth?
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bromocresol purple
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What is the pH indicator used in the Urea agar?
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phenol red
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Name two sources of protein that can be used to prepare the media used for protein catabolism tests.
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Animal parts
Yeast Casein Soybeans Beef extract |
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T/F
If your unknown bacteria is negative for catalase, it is definitely a Streptococcal species. |
False.
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T/F
Bacitracin (A disc) is an antibiotic used to distinguish between organisms that produce alpha hemolysis. |
False.
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T/F
The bacteria that is used to streak down the middle of a CAMP test is Staphylococcus saprophyticus. |
False. The bacteria used to streak down the middle of a CAMP test is Staphylococcus aureus.
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T/F
An alpha hemolytic reaction indicates partial hemolysis of sheep red blood cells. |
True.
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The bile esculin test determines if an organism can digest ____________ in the presence of _________.
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digest esculin in the presence of bile.
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You have an unknown gram (+) bacteria that bubbles in the presence of catalase. Also, when this bacteria is grown on T-Soy with a Novobiocin disc, the bacteria grows to within 25mm of the disc. How would you interpret these results?
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Bacteria is sensitive to Novobiocin.
Bacteria contains catalase. |
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The selective ingredient in MacConkey is?
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Bile salts, analine dyes
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The differential ingredient in MacConkey is?
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lactose
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Name a media containing:
a. Methyl green b. Ferrous sulfate c. ferric citrate d. bromocresol purple |
a. DNAse
b. SIM c. bile esculin d. lysine/ornithine |
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Name the three physical forms of media.
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liquid/broth media
semisolid media solid media |
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Culture media whose exact chemical composition is known is called _________________.
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Chemically defined or synthetic media
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In routine microbiology culturing, most organisms are cultured on ____________________.
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complex or non-synthetic media
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In addition to growth being cloudy or turbid, there may be clumps of cells; this is called ____________________.
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flocculant
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There may be a membrane or _____________ across the surface of the broth.
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pellicle
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If the cells have settled to the bottom, they form a ____________.
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sediment
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Always label the ____________ of your Petri plate.
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bottom
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The KOH test will do what do Gram (+) and Gram (-) cells?
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Leaving the KOH emulsion on Gram (+) cells will do nothing.
Leaving KOH emulsion on Gram (-) cells will make the cells release chromosomal material and the suspension will become viscous, stringy, and adhesive. |
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Name characteristics of Gram (+) cells.
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Thick peptidoglycan layer, resist decolorization as alcohol makes the walls less porous and better able to retain purple stain, Gram (+) stain purple.
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Name characteristics of Gram (-) cells.
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Thin peptidoglycan layer, lipopolysaccharide layer, lipids and polysaccharides take up Crystal Violet- Iodine, then cells lose CV-I mixture and the walls are exposed after alcohol decolorizer applied, pick-up red Safranin dye. Gram (-) stain pink-red.
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What is the purpose of capsule formation in bacteria?
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Capsules are composed of mucous-like polysaccharides and peptides and surround the wall of certain bacteria. They may provide the bacteria with the ability to evade phagocytosis and they allow some species to adhere to each other or to other cells and surfaces.
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What is the purpose of flagella?
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Flagella allow bacteria to be motile, moving about in their environment in response to various stimuli.
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Complete hemolysis of red blood cells, leaving a clear zone with a clean edge around the colony of bacteria.
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Beta hemolysis
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Incomplete hemolysis, producing methemoglobulin, leaving a greenish-brown cloudy zone around the colony.
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Alpha hemolysis
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No hemolysis and no change in the blood agar around the colony
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Gamma hemolysis
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What is CNA selective for?
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Isolation of Gram (+) bacteria.
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What is CNA selective against?
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Gram (-) bacteria. CNA is the selective material.
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What is the differential material in CNA?
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Sheep red blood cells
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What is a differential positive in CNA?
What is differential negative in CNA? |
Alpha, beta or gamma hemolysis.
No halo, therefore no ability for hemolysis. (Gamma hemolysis) |
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What is MSA selective for?
What is MSA selective against? |
Isolation of salt tolerant Gram (+) bacteria.
Salt inhibits the growth of salt sensitive gram (+) and gram (-). |
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What is the selective material in MSA?
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salt
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What is the differential material in MSA?
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Mannitol with phenol red as pH indicator.
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What is the differential positive in MSA?
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Phenol red color change to yellow = mannitol fermentation
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What is the differential negative in MSA?
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Red coloration = no mannitol fermentation
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What is MacConkey selective for?
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Isolation of Gram (-) bacteria
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What is MacConkey selective against?
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Crystal violet inhibits the growth of Gram (+) by interfering with the function of the peptidoglycan layer.
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What is the selective material in MacConkey?
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Crystal Violet
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What is a differential positive in MacConkey?
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Purplish/red colonies = lactose fermentation (high) to pink (low)
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What is a differential negative in MacConkey?
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Colorless to translucent = non-lactose fermenter
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What is the differential material in MacConkey?
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Lactose with neutral red as the pH indicator
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What is EMB selective for?
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Isolation of Gram (-) bacteria
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What is EMB selective against?
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Aniline dyes inhibit the growth of Gram (+) by interfering with the function of the peptidoglycan layer
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What is the selective material in EMB?
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Aniline dyes
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What is a differential positive in EMB?
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Purple (low) to metallic green sheen (high) = lactose fermenter
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What is a differential negative in EMB?
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No color to pale lavender
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What is the differential material in EMB?
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Lactose with eosin dye and methylene blue used as color indicators.
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Organisms that must have oxygen to survive, such as soil bacteria Pseudomonas
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obligate aerobes
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Organisms that cannot grow in the presence of oxygen, such as methane gas producers and Clostridium botulinum
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Obligate anaerobes
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Organisms that usually prefer oxygen but can grow with or without oxygen such as E. Coli in intestines
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Facultative anaerobes/aerobes
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Organisms, such as Streptococcus species, that require only low levels of oxygen. Many times atmospheric oxygen is too much and will inhibit growth.
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Microaerophiles
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Organisms that do no use oxygen at all but are not inhibited from growing in its presence
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Aerotolerant
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The catalase test is used primarily to separate organisms from the genus _________________ and the genus __________________
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Staphylococcus
Streptococcus Staph are positive for catalase Strep are negative for catalase |
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Why do we not use samples from a blood agar to perform the catalase test?
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Blood cells contain catalase and will give a false positive result
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In a TSI or triple sugar iron test, what are the different interpretations of the test results?
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K/K = red slant, red butt no change, no sugars fermented
K/A = red slant, yellow butt glucose fermentation only (possible pathogen) A/A = yellow slant, yellow butt lactose and/or sucrose and glucose fermented yellow slant indicates lactose and/or sucrose yellow butt indicates glucose fermented K/A = red slant, black precipitate, yellow butt red slant no lactose or sucrose, black precipitate H2S production (possible pathogen), yellow butt often obscured by black means glucose fermentation. |
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Of what is the TSI agar composed?
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Glucose, lactose and sucrose. Lactose and sucrose are 10X the concentration of glucose.
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What is the pH indicator in TSI?
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phenol red
pH of less than 6.8 produces yellow color |
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What is the sulfur source in TSI?
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Thiosulfate
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What is the H2S indicator in TSI?
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Ferric ammonium sulfate
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For what is TSI used to screen?
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sugar metabolism
acid production CO2 gas and/or H2S gas production H2S production |
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Possible results of TSI?
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Yellow color change (A) indicates acid production from sugar metabolism (glucose and either lactose and/or sucrose)
Red color reversion (K) indicates production of alkaline wastes from protein digestion. Bubbles and/or cracking indicate gas production. Black color indicates H2S production. |
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What do the results of the TSI test tell you?
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Results of this test allow you to determine the presence of the enzymes sucrase and/or lactase.
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What is the composition of Citrate?
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Simmon's citrate agar with no glucose or lactose for fermentation.
Provides citrate (citric acid) as a metabolic energy source. Bromothymol blue as a pH indicator. NH4 as a nitrogen source. |
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For what does the Citrate test screen?
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This test screens for the presence of enzymes necessary for metabolism of citrate.
It determines if an organism can use citrate as its sole metabolic (carbon) energy source. |
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What are the possible results of the Citrate test?
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A color change from the original green to a bright blue.
Bromothymol blue will be a blue color at pH 7.6 or above due to the production of sodium-carbonate, an alkaline end product. |
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Interpretation of Citrate test results?
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The blue coloration (citrate +) indicates that the organism has used citrate as a nutrient source and produced alkaline end products (sodium carbonate).
A green coloration (citrate -) indicates that the organism could not use citrate. |
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For what enzymes is the Citrate test testing?
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Citrate permease and citrase.
This test requires the presence of oxygen, do not tighten the lids all the way down. |
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What is the composition of the carbohydrate fermentation with Durham tubes?
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Peptone broth w/single sugar to be tested. One each of dextrose, lactose, sucrose, maltose.
Phenol red is pH indicator. A pH below 6.8 produces lemon yellow. Inverted Durham tube for gas collection. |
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For what is the Carb Fermentation testing?
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The ability of an organism to use a particular sugar as a metabolic energy source.
The production of acid or alcohol end products. The production of gas during fermentation or aerobic respiration. |
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What are the possible results of Carb Fermentation?
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Lemon yellow color indicates that the organism produces acids as fermentation end products.
Red/orange color indicates alcohol production from fermentation or aerobic respiration w/no fermentation. Bubbles represent gas production, primarily CO2. |
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Interpretation of Carb Fermentation results?
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Lemon yellow tube with no bubbles = sugar fermentation w/acid production but no gas.
Lemon yellow tube with bubbles in Durham tube = sugar fermentation w/gas production. Red/orange tube w/bubbles = either sugar fermentation w/alcohol and gas or aerobic use and exhaustion of sugar with gas production. |
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What is considered a positive test in Carb Fermentation?
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Only a bright lemon yellow color is considered a (+) test. Any other color change is (-).
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What is the composition of MR/VP?
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Basic T-Soy broth plus
1. Methyl red pH indicator added post-incubation. 2. Barritt's reagent (VP-A and VP-B) added post-incubation. |
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For what is MR/VP screening?
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MRVP is screening for the end product of glucose metabolism.
1. Mixed acids; various organic acids are produced. 2. Acetoin production; a possible end product is usually 2,3 butanediol. |
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What are the possible results of MR/VP?
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Methyl red in the incubation tube will remain red at pH 4.0 or below and turn yellow at pH 6.5 or above.
Barritt's reagent (VP-A and VP-B) produces a red ring at top of tube if either neutral or non-acidic end products are produced. |
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Interpretation of MR/VP results?
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The presence of a positive MR test is an indication that the organism produced mixed acids as a result of glucose metabolism.
The presence of a red ring at the top of the tube after at least 15 min incubation indicates the production of acetoin from glucose metabolism. |
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What results can be expected from MR/VP test?
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-/-, -/+, +/- but not +/+
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T/F
Stab and streak bile esculin slant. |
true.
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What does a positive test for bile esculin look like?
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a black color in the tube.
the black color indicates esculin hydrolysis in the presence of bile. |
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For what is the bile esculin test presumptively looking?
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Enterococci and Streptococcus bovis group
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What is the indicator in the bile esculin test?
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ferric citrate
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What is the purpose of peptone and esculin in the bile esculin test?
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They provide nutrients.
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What is the purpose of the bile in the bile esculin test?
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Bile inhibits growth of gram + organisms (except for Group D streptococci and enterococci)
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What is the purpose of sodium azide in the bile esculin test?
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Sodium azide inhibits gram - organisms.
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If you have beta hemolysis, positive catalase test, positive coagulase test, mannitol fermentation, and positive DNAse test, at what organism are you looking?
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Staphylococcus aureus
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If you have gamma hemolysis, positive catalase test, negative coagulase test, mannitol fermentation and negative DNAse test, at what organism are you looking?
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Staphylococcus saprophyticus
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If you have gamma hemolysis, positive catalase test, negative coagulase test, no mannitol fermentation, and negative DNAse test, at what organism are you looking?
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Staphylococcus epidermidis
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What are the characteristics of Staph aureus tests?
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Beta hemolysis
+ catalase + coagulase mannitol fermentation positive DNAse |
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What are the characteristics of Staph saprophyticus tests?
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Gamma hemolysis
+ catalase - coagulase mannitol fermentation negative DNAse |
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What are the characteristics of Staphylococcus epidermidis tests?
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Gamma hemolysis
+ catalase - coagulase NO mannitol fermentation negative DNAse test |
