Lab Exam Ii Microbiology

Front 1

medium that is liquid at room temp

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Broth

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visible accumulation at bottom of tube.

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Sediment

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fine, cloudy appearance.

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Turbidity

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clumpy, floating growth.

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Flocculent

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growth around top rim.

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Ring

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heavy growth over surface.

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Pellicle

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to dry stained slides.

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Bibulous paper

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isolate colonies so solid lines of bacteria occur in first quadrants, and separate colonies in later ones.

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Spread plate

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to isolate a single colony subculture from a mixed culture.

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Slant tube

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move small volumes of material.

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Pipette

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clean slides.

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Kim wipes

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clean microscope lenses.

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Lens paper

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oxygen kills them.

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Obligate anaerobes -

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can use oxygen if present, but can change to anaerobic pathways if oxygen is absent, but grow faster if oxygen is present.

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Facultative anaerobes

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must have oxygen to live.

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Obligate aerobes

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require oxygen concentrations lower than in the atmosphere.

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Microaerophiles

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grow just as well with or without oxygen.

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Indifferents

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Describe change that occurs as agar cools from 100°C to room temperature.

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Agar solidifies.

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device that provides optimal growth conditions for bacteria.

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Incubator

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device that uses high temperature and pressure to sterilize heat-resistant materials.

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Autoclave

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is removal of an individual colony from a mixed culture using an inoculating loop.

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Fishing

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place a small amount of bacteria on a sterile medium for growth.

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Inoculation

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visible bacterial growth.

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Colony

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color.

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Pigmentation

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odor.

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Putrefactive

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contour of surface.

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Umbonate

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colony margin.

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Undulate

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shape of whole colony.

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Circular

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elevation.

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Raised

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undesirable organism growth in culture dishes.

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Contaminant

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What is the effect of flaming the inoculating loop?

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Blue part of Bunsen burner flame kills (sterilizes) all living cells on wire

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What should be our last step after all micro labs?

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Disinfect lab tops with 1% bleach solution

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What is the basis for biochemical identification of bacteria?

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Biochemical tests determine what enzymes bacteria produce. Can be used to identify species.

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Name some direct tests.

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Gram, endospore, and acid-fast staining, oxygen requirements, motility.

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What can be inferred from bacteria with the same morphology?

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Usually bacteria with the same physical features (morphology) will have same physiology.

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Amylase

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is an enzyme that catabolizes (hydrolyzes) starch by adding water to produce simple sugars.

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A negative result has what implication?

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A ________ result means that the tested bacterium cannot live on a strict starch diet. Iodine is added after incubation in a Petri dish.

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Describe a positive test.

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the starch in the medium will turn black/dark purple , and there will be a clear area around the growth where the bacteria have hydrolyzed the starch. A negative test will show black/purple up to the growth, no starch hydrolyzed.

Front 39

Explain the uses of negative acidic dye and positive dye in negative and simple stains. Contrast the images produced by a negative stain and a simple stain. What stain is used in each?

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4. Cytoplasm has a negative charge so a negative acidic stain (nigrosin) is repelled by the cytoplasm. Image is white, shiny against a dark background. In simple stain a positive dye (methylene blue) is attracted by the cell and is dark with a lighted background.

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What cell feature is responsible for the Gram stain reaction?

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Cell wall is determining feature.

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Describe the appearance of a Gram+ and Gram- reaction.

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Gram+ is purple;
Gram- is red/pink.

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What is the purpose of Gram's iodine?

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It is a mordant that helps set crystal violet stain.

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Which step in the procedure is most problematic?

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Decolorizing step is hardest to hit.

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Which microscope objective should be used to determine cell shape and color?

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Observe with 100X objective.

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What stain is responsible for the colors of each result?

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Purple is from crystal violet; red/pink, from counterstain safaranin.

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What is a positive test for catalase? What does this imply? What is the role of hydrogen peroxide?

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Oxygen bubbles appear because the bacterium produces the enzyme catalase that breaks down hydrogen peroxide (substrate) to water and oxygen. Bacteria that produce catalase are not strongly affected by hydrogen peroxide.

Front 47

Be able to outline the process for a smear preparation.

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1. wash a glass slide with powder cleanser, rinse it well, and dry thoroughly.
2. Obtain cultures of the bacteria to be stained and place them in a rack on the desk. Place a drop of water onto the center of the slide. For Broth cultures, no water is used. Light the Bunsen burner.3. Using the sterile technique, transfer a very small bacterial culture sample to the water drop on the slide. Place the loopful of bacteria into the drop of water on the slide and mix by swirling the liquid out to the area of a dime. Complete the procedure by reflaming the loop. For broth cultures, place a loopful directly on slide. 4. Air dry the slide until all the liquid evaporates by letting the slide sit on the desk top. 5. Heat fix smear.

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11. Be able to outline the steps in the endospore stain.
See p. 78, lab 12

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1. Prepare air-dried, heat fixed smears of the bacterial cultures. Label slide "Endospore" 2. Place slides on rack on pan and flood the smear with Malachite green. Steam the stain covered slides for at least 5 minutes. Do not let dye dry out during process! 3. Allow slides to cool for 2minutes. 4. Place slide on stain rack at sind and aply counterstain, Safranin, for 1 minute. 5. Rinse briefly. Blot Dry. 6. Examine stained slide with oil immersion microscopy. Look for green spores and red or pink vegetative cells. 7. Record observations (color, size, shape).

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Be able to outline the Gram stain procedure. For procedure, see p. 71.

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1.Heat fix a smear of bacteria 2. Applying primary stain (crystal violet) for 1 minute 3. Rinse with water 4. Apply iodine for 1 minute. Gram’s Iodine is the mordant. 5. Rinse with water 6. Apply decolorizer (Gram- decolorize faster). Most important step. Do not let decolorizer sit on slide pour decolorizer lightly over slide until it runs clear. 7. Rinse with water. 8.Add counter stain of Safranin for 1 minute – adds color to Gram- bacteria on smear 9.Rinse with water. Blot dry with bibulous paper 10. Add one drop of oil. View slide under 100x oil immersion lens
Gram + purple. Gram – orange/red/pink

Front 50

Describe heat fixing.

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by passing the slide through the Bunsen flame 3 times, allowing the heat to contact the underside. This will kill any bacteria that may still be alive, encourage stain penetration, and fix the cells to the slide so they do not wash off.

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Be able to outline the process for a smear preparation.

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1. Drop water, then inoculating loop with bactera
2. Spread out water-bacteria mixture
3. Air dry completely
4. Heat fix