# Microbiology Exam I (Lab) - Flashcards

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 Class: BIOL 04140 - GENERAL MICROBIOLOGY Subject: Biological Sciences University: Northwest Missouri State University Term: Spring 2011
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 Why aren't the magnification of both ocular lenses of a binocular microscope used to calculate total magnification? Because the image only goes through one ocular to reach both eyes What is total magnification for each lens setting on microscope with 15x oculars and 4x, 10x, 45x, and 97x objectives? objective x ocular = magnification explain why light of shorter wavelength will produce a clearer image than light of longer wavelengths calculate for limit of resolution D=wavelength/(NAobject + NA condensor) visible light = 300-700 nm limited resolution decreased if wavelength decreased why is wavelength the main limiting factor on limit of resolution in light microscopy if wavelengths are so small = smaller limit. resolution
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 on a given microscope, the numerical apertures of the condensor and low power objective lenses are 1.25 and 0.25, respectively, you are supplied with a filter that selects a wavelength of 520 nm. What is the limit of resolution on this microscope? Will you be able to distinguish two points that are 330 nm apart as being separate, or will they blur? 1.25+0.25.. 520/(1.25+0.25) = 347 nm Wavelength/(NAo+NAc) they'll blur because it's a small limit of resolution Same microscope from following question--high dry objective lens has a numerical aperature of 0.85. What is the limit of resolution on this microscope? Will you be able to distinguish two points that are 250 nm apart as being separate or will they blur? limit of resolution = 246 nm you will be able to distinguish two points because it's a bigger limit of resolution if you were too look at the letter e under a microscope what would you see? the image is inverted because of the way the light waves cross With which objective was it easier to determine the sequence of colored threads? high-dry, oil lens would cause the colors to run
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 Why should closing the iris diaphragm improve your ability to determine thread order? by closing the iris diaphgram it closes the peripheral light giving it a better image What does the Colored Thread slide demonstrate about specimens you will be observing later in this class? Even though they are very small they still have depth You are told that viewing is best done with as little illumination as possible. Why will transparent cells be easier to view with less light? Essentially making the image clearer with better contrast. Would you expect Brownian motion to increase the longer you observe a hanging drop or wet mount preparation why? Longer on the microscope, longer heated. Brownian is result of kinetic energy things will wiggle in place more.
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 Considering the cultures used to inoculate each medium in this exercise, how many different microbial types should you expect to see in/on each medium? Should only have 1 if using good sterile technique You were asked to describe differences in appearance of growth in each culture, if present. In which medium was this the most difficult to determine? What made it difficult? More difficult to see in broth because you can only see if growth occured on the top or bottom etc. Better in slant because you can observe different growth patterns. Which medium was most difficult for you to transfer from? Which medium was most difficult for you to inoculate? Why? Most people don't like working with slants (solid) What was the purpose of incubating the unopened plates? What is an appropriate name for these plates? To be used as negative control--nothing should grow on these plates
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 If growth appears on both unopened plates, what are some likely explanations? What if growth appears on only one plate? How does growth on the unopened plates affect the reliability of the other plates? on both--most likely occured from contamination, or when the media was made on just one--most likely contaminated while making or using Why were the specific types of exposure (air, hair, tabletop, etc.) chosen for this exercise? Major sources of environmental contaminations The plates you are using for this lab will be autoclaved eventually to completely sterilize them. The measures taken to disinfect the tabletops (the source of the organisms on plates 2 and 3) are not as extreme why? Plates should have higher organism growth than bench tops, because the bench tops have no media that attracts them to make them grow there. What is the consequence of leaving a stain on the bacterial smear too long (overstaining?) this can make the cell appear larger than it really is, with a simple stain it might be okay but with gram staining it would ruin it
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 Whic is more likely to survive in a dry environment? Coccus or Rod of equal volume? Coccus--because it has a lower ration, it has an advantage because it can't lose moisture Which would be better adapted to a moist environment? Coccus or Rod of equal volume? Rods better in moist. Why doesn't a negative stain colorize the cells in the smear? Stain, chromophore and negative cells, the negativity of the cell repels the negativity of the ink staining only the background Eosin is a red stain and methylene blue is blue. What should be the result of staining a bacterial smear with a misture of eosin and methylene blue? Eosin--is acidic and acts as a negative stain Methylene blue--is basic The smears background would turn out red while the cells would turn out blue.
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 Compare the diameter of M. luteus cells as measured using a basic stain and an acidic stain what might account for any difference? Basic (simple stain) heat fixing--shrinks cells/smaller negative--cells are their actual size HOWEVER--there is very little difference!! Predict the effect of the following "mistakes" made when performing a Gram stain. Failure to add iodine, Failure to apply the decolorize, Failure to apply the safranin, Reversal of crystal violet and safranin stains Failure to apply iodine--everything will appear gram negative Failure to apply the decolorizer--everything will appear gram positive Failure to apply the safranin--gram (+) will be purple, gram (-) will be colorless Reversal of crystal violet and safranin--wouldn't be able to read and would have to toss it. Both crystal violet and safranin are basic stains and may be used to do simple stains on Gram-positive and Gram-negative cells. This being the case, explain how they stain different cell types in the Gram stain gram (+) can hold on to the crystal violet although it's decolorized If you saw large, eukaryotic cells in the preparation made from your gumline, they were most likely your own epithelial cells. Are you Gram (+) or Gram (-)? Gram-negative--although you usually don't do a gram stain on a eukaryotic
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 How does heating the bacterial smear during a ZN stain promote entry of carbolfuchsin into the acid-fast cell wall? Acid fast melt mycolic acid--that's what the heat is for? Are acid-fast negative cells stained by carbolfuchsin? If so, how can this be a differential stain? Get rid of carbalfuchsin in non-acidic fast by using acid alcohol Why do you suppose the acid-fast stain is not as widely used as the Gram stain? When is it more useful than the Gram stain? First do the gram stains acid-fast are a rare group of organisms unless it just happens to be or not be acid fast. Capsules are neutrally charged. This being the case, what is the purpose of emulsifying the sample in serum in this staining procedure? serum acts as glue to hold it to the slides
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 Some oral bacteria produce an extracellular "capsule" of what benefit is a capsule to these cells? capsules help stick to teeth internally capsules help prevent phagocytosis Eschericia coli gram negative rod shaped opportunistic pathogen UTI, diarrhea, meningitis motile Bacillus subtillis gram positive rod shaped Serratia marcesens gram negative rod shaped motile opportunistic: GI infection, gram negative shock septicemia found in H2O, and soil
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 Bacillus megaterium soil bacteria long rod shaped gram positive motile very concentrated Staphylococcus epidermidis gram positive circles-clusters found all over skin opportunistic if gets into wounds/implants Rhodospirillum rubrum subacute bacterial endocarditis (can cause heart infection) not motile helical gram negative Mycobacterium smegmatis normal human bacteria waxy/crumbly gram positive rods
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 Mycobacterium phlei found in timothy hay opportunistic gram positive acid-fast Bacillus mycoides gram positive rod shpaed chromogen is the colored molecule (often a benzene derivative), the portion that actually gives it its color is the chromophore Auxochrome is the charged portion of a chromogen and allows it to act as a dye through ionic or covalent bonds between chromogen and the cell
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 Basic stains (where auxochrome becomes positively charged as a result of picking up a hydrogen ion or losing a hydroxide ion) are attached to the negative charges on the surface of most bacterial cells common basic stains: methylene blue, crystal violet, safranin Heat-fixing kills the bacteria, makes them adhere to the slide, and coagulates cytoplasmic proteins to make them more visible. It also distorts the cells to some extent. Negative Stains Technique is uses a dye solution in which a chromogen is acidic and carries a negative charge. The negative charge on the bacterial surface repels the negatively charged chromogen, so the cell remains unstained against a colored background. This technique is used to determine morphology and cellular arrangement in bacteria that are too delicate to with-stand heat-fixing. Decolorization step occurs in gram staining between the application of two basic stains. This is the most critical step because Gram (-) cells are decolorized by the solution whereas Gram (+) cells are not. The Gram (-) cells are then colored by the counterstain safranin
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 Gram Stain Primary Stain is Crystal Violet. Mordant is Iodine Counterstain is Safranin Mordant using Iodine--enhances crystal violet staining by forming a crystal-violet-iodine complex. Acid-Fast Stains The presence of mycolic acids in the cell walls of acid-fast organisms is the cytological basis for the acid-fast differential stain. Mycolic acid is a waxy substance that gives acid-fast cells a higher affinity for the primary stain and resistance to decolorization by an acid alcohol solution.
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 Acid-Fast Stains: Ziehl-Neelsen Ziehl-Neelsen (ZN)--uses heat as part of the staining process. The phenolic compound carbolfuchsin is used as the primary stain because it is lipid-soluble and penetrates the waxy cell wall. Steam-heating further enhances by melting the wax and allowing movement of stain into cell. Acid-Alcohol is used to decolorize nonacid-fast cells; acid-fast cells resist this decolorization. Methylene blue is used as counterstain; acid-fast cells are red/purple, while the nonacid-fast are blue. Heat acts as the mordant. Acid-Fast Stains: Kinyoun Kinyoun method uses a slightly more lipid-soluble and concentrated carbolfuchsin as the primary stain allowing the stain to penetrate the acid-fast walls without the use of heat but makes this method slightly less sensitive than the ZN method. Decolorization with acid alcohol is followed by a contrasting counterstain such as brilliant green or methylene blue. Capsules are composed of mucoidpolysaccharides or polypeptides that repel most stains. Capsule Stain technique takes advantage of this characteristic by staining around the cells. Typically an acidic stain such as Congo red or nigrosin, which stains the background, and a basic stain that colorizes the cell proper are used. The capsule remains unstained and appears as a white halo between the cells and the colored background. It's a differential stain used to detect cells capable of producing an extracellular capsule.
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 Endospore is a dormant form of the bacterium that allows it to survive poor environmental conditions. They are resistant to heat and chemicals because of the tough outer covering made of protein keratin which also resists staining. Endospore Stain: Schaeffer-Fulton Method Malachite green is the primary stain and is forced into the spore by steaming the bacterial emulsion. Malachite green is water-soluble and has a low affinity for cellular material, so vegetative cells and spore mother cells can be decolorized with water and counterstained with safranin. Heat acts as the mordant. Spores stain green while the vegetative cells are red.
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## List View: Terms & Definitions

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Why aren't the magnification of both ocular lenses of a binocular microscope used to calculate total magnification?Because the image only goes through one ocular to reach both eyes
What is total magnification for each lens setting on microscope with 15x oculars and 4x, 10x, 45x, and 97x objectives?objective x ocular = magnification
explain why light of shorter wavelength will produce a clearer image than light of longer wavelengthscalculate for limit of resolution
D=wavelength/(NAobject + NA condensor)
visible light = 300-700 nm
limited resolution decreased if wavelength decreased
why is wavelength the main limiting factor on limit of resolution in light microscopyif wavelengths are so small = smaller limit. resolution
on a given microscope, the numerical apertures of the condensor and low power objective lenses are 1.25 and 0.25, respectively, you are supplied with a filter that selects a wavelength of 520 nm. What is the limit of resolution on this microscope? Will you be able to distinguish two points that are 330 nm apart as being separate, or will they blur?1.25+0.25..
520/(1.25+0.25) = 347 nm

Wavelength/(NAo+NAc)

they'll blur because it's a small limit of resolution
Same microscope from following question--high dry objective lens has a numerical aperature of 0.85. What is the limit of resolution on this microscope? Will you be able to distinguish two points that are 250 nm apart as being separate or will they blur?limit of resolution = 246 nm
you will be able to distinguish two points because it's a bigger limit of resolution
if you were too look at the letter e under a microscope what would you see?the image is inverted because of the way the light waves cross
With which objective was it easier to determine the sequence of colored threads?high-dry, oil lens would cause the colors to run
Why should closing the iris diaphragm improve your ability to determine thread order?by closing the iris diaphgram it closes the peripheral light giving it a better image
What does the Colored Thread slide demonstrate about specimens you will be observing later in this class?Even though they are very small they still have depth
You are told that viewing is best done with as little illumination as possible. Why will transparent cells be easier to view with less light?Essentially making the image clearer with better contrast.
Would you expect Brownian motion to increase the longer you observe a hanging drop or wet mount preparation why?Longer on the microscope, longer heated.
Brownian is result of kinetic energy things will wiggle in place more.
Considering the cultures used to inoculate each medium in this exercise, how many different microbial types should you expect to see in/on each medium?Should only have 1 if using good sterile technique
You were asked to describe differences in appearance of growth in each culture, if present. In which medium was this the most difficult to determine? What made it difficult?More difficult to see in broth because you can only see if growth occured on the top or bottom etc.
Better in slant because you can observe different growth patterns.
Which medium was most difficult for you to transfer from? Which medium was most difficult for you to inoculate? Why?Most people don't like working with slants (solid)
What was the purpose of incubating the unopened plates? What is an appropriate name for these plates?To be used as negative control--nothing should grow on these plates
If growth appears on both unopened plates, what are some likely explanations? What if growth appears on only one plate? How does growth on the unopened plates affect the reliability of the other plates?on both--most likely occured from contamination, or when the media was made
on just one--most likely contaminated while making or using
Why were the specific types of exposure (air, hair, tabletop, etc.) chosen for this exercise?Major sources of environmental contaminations
The plates you are using for this lab will be autoclaved eventually to completely sterilize them. The measures taken to disinfect the tabletops (the source of the organisms on plates 2 and 3) are not as extreme why?Plates should have higher organism growth than bench tops, because the bench tops have no media that attracts them to make them grow there.
What is the consequence of leaving a stain on the bacterial smear too long (overstaining?)this can make the cell appear larger than it really is, with a simple stain it might be okay but with gram staining it would ruin it
Whic is more likely to survive in a dry environment? Coccus or Rod of equal volume?Coccus--because it has a lower ration, it has an advantage because it can't lose moisture
Which would be better adapted to a moist environment? Coccus or Rod of equal volume?Rods better in moist.
Why doesn't a negative stain colorize the cells in the smear?Stain, chromophore and negative cells,
the negativity of the cell repels the negativity of the ink staining only the background
Eosin is a red stain and methylene blue is blue. What should be the result of staining a bacterial smear with a misture of eosin and methylene blue?Eosin--is acidic and acts as a negative stain
Methylene blue--is basic

The smears background would turn out red while the cells would turn out blue.
Compare the diameter of M. luteus cells as measured using a basic stain and an acidic stain what might account for any difference?Basic (simple stain)
heat fixing--shrinks cells/smaller
negative--cells are their actual size
HOWEVER--there is very little difference!!
Predict the effect of the following "mistakes" made when performing a Gram stain. Failure to add iodine, Failure to apply the decolorize, Failure to apply the safranin, Reversal of crystal violet and safranin stainsFailure to apply iodine--everything will appear gram negative
Failure to apply the decolorizer--everything will appear gram positive
Failure to apply the safranin--gram (+) will be purple, gram (-) will be colorless
Reversal of crystal violet and safranin--wouldn't be able to read and would have to toss it.
Both crystal violet and safranin are basic stains and may be used to do simple stains on Gram-positive and Gram-negative cells. This being the case, explain how they stain different cell types in the Gram staingram (+) can hold on to the crystal violet although it's decolorized
If you saw large, eukaryotic cells in the preparation made from your gumline, they were most likely your own epithelial cells. Are you Gram (+) or Gram (-)?Gram-negative--although you usually don't do a gram stain on a eukaryotic
How does heating the bacterial smear during a ZN stain promote entry of carbolfuchsin into the acid-fast cell wall?Acid fast melt mycolic acid--that's what the heat is for?
Are acid-fast negative cells stained by carbolfuchsin? If so, how can this be a differential stain?Get rid of carbalfuchsin in non-acidic fast by using acid alcohol
Why do you suppose the acid-fast stain is not as widely used as the Gram stain? When is it more useful than the Gram stain?First do the gram stains
acid-fast are a rare group of organisms unless it just happens to be or not be acid fast.
Capsules are neutrally charged. This being the case, what is the purpose of emulsifying the sample in serum in this staining procedure?serum acts as glue to hold it to the slides
Some oral bacteria produce an extracellular "capsule" of what benefit is a capsule to these cells?capsules help stick to teeth
internally capsules help prevent phagocytosis
Eschericia coligram negative
rod shaped
opportunistic pathogen
UTI, diarrhea, meningitis
motile
Bacillus subtillisgram positive
rod shaped
Serratia marcesensgram negative
rod shaped
motile
opportunistic: GI infection,
gram negative shock
septicemia
found in H2O, and soil
Bacillus megateriumsoil bacteria
long rod shaped
gram positive
motile
very concentrated
Staphylococcus epidermidisgram positive
circles-clusters
found all over skin
opportunistic if gets into wounds/implants
Rhodospirillum rubrumsubacute bacterial endocarditis (can cause heart infection)
not motile
helical
gram negative
Mycobacterium smegmatisnormal human bacteria
waxy/crumbly
gram positive
rods
Mycobacterium phleifound in timothy hay
opportunistic
gram positive
acid-fast
Bacillus mycoidesgram positive
rod shpaed
chromogenis the colored molecule (often a benzene derivative), the portion that actually gives it its color is the chromophore
Auxochromeis the charged portion of a chromogen and allows it to act as a dye through ionic or covalent bonds between chromogen and the cell
Basic stains(where auxochrome becomes positively charged as a result of picking up a hydrogen ion or losing a hydroxide ion) are attached to the negative charges on the surface of most bacterial cells

common basic stains: methylene blue, crystal violet, safranin
Heat-fixingkills the bacteria, makes them adhere to the slide, and coagulates cytoplasmic proteins to make them more visible. It also distorts the cells to some extent.
Negative StainsTechnique is uses a dye solution in which a chromogen is acidic and carries a negative charge. The negative charge on the bacterial surface repels the negatively charged chromogen, so the cell remains unstained against a colored background.
This technique is used to determine morphology and cellular arrangement in bacteria that are too delicate to with-stand heat-fixing.
Decolorizationstep occurs in gram staining between the application of two basic stains. This is the most critical step because Gram (-) cells are decolorized by the solution whereas Gram (+) cells are not. The Gram (-) cells are then colored by the counterstain safranin
Gram StainPrimary Stain is Crystal Violet.
Mordant is Iodine
Counterstain is Safranin
Mordantusing Iodine--enhances crystal violet staining by forming a crystal-violet-iodine complex.
Acid-Fast StainsThe presence of mycolic acids in the cell walls of acid-fast organisms is the cytological basis for the acid-fast differential stain.
Mycolic acidis a waxy substance that gives acid-fast cells a higher affinity for the primary stain and resistance to decolorization by an acid alcohol solution.
Acid-Fast Stains: Ziehl-NeelsenZiehl-Neelsen (ZN)--uses heat as part of the staining process.
The phenolic compound carbolfuchsin is used as the primary stain because it is lipid-soluble and penetrates the waxy cell wall. Steam-heating further enhances by melting the wax and allowing movement of stain into cell. Acid-Alcohol is used to decolorize nonacid-fast cells; acid-fast cells resist this decolorization. Methylene blue is used as counterstain; acid-fast cells are red/purple, while the nonacid-fast are blue.
Heat acts as the mordant.
Acid-Fast Stains: KinyounKinyoun method uses a slightly more lipid-soluble and concentrated carbolfuchsin as the primary stain allowing the stain to penetrate the acid-fast walls without the use of heat but makes this method slightly less sensitive than the ZN method. Decolorization with acid alcohol is followed by a contrasting counterstain such as brilliant green or methylene blue.
Capsulesare composed of mucoidpolysaccharides or polypeptides that repel most stains.
Capsule Staintechnique takes advantage of this characteristic by staining around the cells. Typically an acidic stain such as Congo red or nigrosin, which stains the background, and a basic stain that colorizes the cell proper are used. The capsule remains unstained and appears as a white halo between the cells and the colored background. It's a differential stain used to detect cells capable of producing an extracellular capsule.
Endosporeis a dormant form of the bacterium that allows it to survive poor environmental conditions. They are resistant to heat and chemicals because of the tough outer covering made of protein keratin which also resists staining.
Endospore Stain: Schaeffer-Fulton MethodMalachite green is the primary stain and is forced into the spore by steaming the bacterial emulsion. Malachite green is water-soluble and has a low affinity for cellular material, so vegetative cells and spore mother cells can be decolorized with water and counterstained with safranin. Heat acts as the mordant. Spores stain green while the vegetative cells are red.