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Microbio > Lab Test 1 > Flashcards

Lab Test 1 Flashcards

1
Q

When using the bunsen burner:

  • Flaming is done at the top of the smaller blue flame, which is the hottest part of the flame
  • you should adjust the height of the flame with the over on the lab bench
  • the flame should be uniformly blue, with no second internal flame
  • the inoculating loop or needle is held horizontally in the blue flame
  • the blue flame should be no more than 1 inch tall
A

A

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2
Q

Microscope slides are cleaned with powdered cleanser:

  • on the front of the slide
  • on the back of the slide
  • on both the front and the back of the slide
  • only if used before, new slides do not need cleaning before use
A

C

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3
Q

Preparing a smear from broth and preparing a smear from solid media (slant) differ in:

  • Which instrument is used to obtain the sample
  • how large the smear will be when complete
  • whether water is added to the culture sample
  • when heat fixing occurs
  • 2 of the above
A

E

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4
Q

While obtaining specimen from the culture tube, the cap should be:

  • held between the pointer and thumb of the dominant hand
  • held between the pointer and thumb of non-dominant hand
  • placed on the disinfected bench top
  • held in the pinkie of the dominant hand
  • held in the pinkie of the non dominant hand
A

D

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5
Q

Heat fixing should be done:

  • by gently passing the slide through the flame twice on each side
  • only when the smear is completely dried
  • while smear is drying
  • to the slide both before and after making the smear
  • two of the above
A

B

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6
Q

A “perfect” smear before staining should be:

  • visibly chalky to the naked eye
  • totally invisible to the naked eye
  • barley visible against a dark background
  • visible as a cream colored smudge against a white background
A

C

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7
Q

All bacteriological dyes should be applied for 1 minute

T/F

A

False

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8
Q

When bacteriological dyes are applied, the entire slide, not just the smear should be covered

A

True

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9
Q

If your stained, rinsed slide is not visibly colored to the naked eye while still wet, you should reapply the dye for an additional minute

A

False

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10
Q

Both the back of the slide and the front of the slide should be rinsed after staining

A

True

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11
Q

Rinsing the slide removes bacteriological dyes from the glass, but not the bacterial cells, due to their charge

A

True

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12
Q

Before viewing all stained slides must have a cover slip added

A

False

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13
Q

Why are two slides required for a single negative stain?

A

Because one slide is used to distribute the organism and dye on the other

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14
Q

Negative stains are NOT heat fixed

A

True

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15
Q

During the gram staining procedure, when should the slide be dried in the Bibulous paper pad?

  • After crystal violet dye
  • After the iodine step
  • After the safranin dye
  • Between each step
  • Only after all steps are complete
A

E

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16
Q

When should the slide be rinsed during the gram staining procedure

A

Between each step of the staining process

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17
Q

Match the reagent for each of the steps of the gram staining procedure

A

Step 1 - crystal violet (primary)
Step 2 - Mordant iodine
Step 3- Alcohol (95%) (decolorized)
-Step 4 - Safranin (secondary)

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18
Q

If you switched the primary and secondary dyes of the gram staining procedure which of the following would be true

  • Gram + cells would be colored correctly at the end
  • Gram - cells would be colored correctly at the end
  • Neither cell types would be colored correctly at the end
  • Both cell types would be colored correctly at the end
A

A

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19
Q

If you see a mixture of pink and purple cells after gram staining, the cell wall of the bacterial species viewed is probably composed of thick peptidoglycan only

A

True

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20
Q

In the gram staining procedure the alcohol dissolves lipids to remove the primary dye from the cells with a lipid rich cell wall

A

True

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21
Q

When using heat to force dye into a smear you must always

  • apply dye ONLY in the smear area of the slide
  • use the flame on the surface of the slide to gently heat the dye
  • use filter paper to absorb extra dye so that excess does not run off
  • re apply dye frequently during the steaming process
  • rinse the slide immediately once the timed steaming ends
A

D

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22
Q

With acid fast/capsule stains since heat is used during the staining process heat fixing is not necessary before beginning the staining process

A

False

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23
Q

The steaming step of both the acid fast and spore staining procedures is used to force dye into a structure or part of the cell that would not normally take dye. How do the two stains differ?

A

Heat melts the mycolic acid in acid fast to let the dye below, but loosens spore coat layers in spore to let dye into the spore

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24
Q

The term “acid fast” refers to a cell wall that:

A

Holds fast to dyes if under waxy layers, even with harsh chemicals

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25
Q

What do bacteria need to survive?

A
  • Moisture (#1 thing)
  • Carbon source (#2)
  • Correct temperature
  • Neutral pH
  • Correct oxygen level
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26
Q

What conditions do bacteria find hostile?

A
  • Metals because they shed ions that disrupt chemical reactions
  • Smooth surfaces - no nooks to store moisture/organic material
  • Toxic chemicals
  • Radiation
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27
Q

Extraneous bacteria

A

Bacteria which colonize the superficial layers of the skin and are easier to remove by hand washing
-These bacteria are more likely to be pathogenic as they are picked up throughout the day as things are touched

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28
Q

Normal flora

A

Bacteria which are attached to deeper layers of the skin and are resistant to removal via hand washing
-Hand washing flushes them up to the surface

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29
Q

On which side of the plate are you more likely to see extraneous bacteria growing? normal flora?

A
  • Unwashed = more likely to see extraneous

- Washed = more likely to see normal flora

30
Q

Are there more bacterial types present before or after washing?

A

More types before washing because prior to washing there are extraneous bacteria present as well as normal flora

31
Q

Does waterless hand sanitizer remove bacteria from hands or kill them?

A

Waterless hand sanitizer kills bacteria on hands but usually doesn’t kill them all (only those sensitive to alcohol)

32
Q

Does hand washing prevent the spread of disease?

A

Yes, because it removes the extraneous bacteria which are usually the pathogenic bacteria that could potentially cause disease

33
Q

Focus

what parts of the microscope adjust focus

A

Moving stage in large increments using coarse adjustment and then in small increments via the fine adjustment
-puts stage at the correct distance

34
Q

Magnification

A

Making an image LARGER

*ocular sense always magnifies by 10

35
Q

Total magnification

A

Ocular (10) times objective

36
Q

Resolution

A

Ability to see image more clearly instead of a blurry image (when it is just in focus).

37
Q

Parts of the microscope that improve resolution and how

A

Abbe condenser - focuses a big beam of light into a smaller one
Blue filter - Wavelength at which the eye sees best at
Immersion oil - Prevents DIFFRACTION of light
Iris diaphragm - controls light source

38
Q

Morphology

A
  • Coccus
  • Bacillus
  • Spirillum
39
Q

Arrangement

A
  • Diplo (pairs)
  • Staphylo (Clusters)
  • Strepto (chains)
  • Tetrad (Tetrads)
  • Single
40
Q

Microscopy steps

A
  1. Turn light on brightest setting, close iris diaphragm
  2. Move condenser all the way up, a 1/4 of the way down
  3. Put slide on the stage
  4. Raise stage to highest level using coarse adjustment
  5. Swivel nosepiece to put 10X in place
  6. Use coarse adjustment to see tiny cells
  7. Swivel to 40x and open iris diaphragm part way
  8. Use fine adjustment to see larger, but fuzzier cells
  9. Swivel between 40X and 100X objective
  10. Add 1 large drop of oil directly on top of the spot of light on the slide
  11. Swivel nosepiece to 100X objective, open iris diaphragm all the way
  12. Use fine adjustment to see large clear cells
41
Q

Most bacterial components have a slight overall _____ charge and will bind to ____ dyes

A

Negative; cationic (basic)

42
Q

Difference between a simple stain and a differential stain

A

Simple stains only use one dye, imparting the same color to all bacteria stained. Differential stains are more complicated and use two or more different colored dyes with a decolorizing step in between.

Simple stains allow you to view size, morphology, and arrangement of bacteria, differential stains reveal more specific info about cell wall construction or the presence of optional structures.

43
Q

Examples of differential stains

A

Gram stain, Acid fast stain, Spore and capsule stain

44
Q

You failed to flame your loop before you entered the tube to obtain a specimen to smear. How will this affect your results as you smear and stain the specimen? How will this affect the ability of later groups to use your same culture

A

This won’t affect the current lab since any bacteria that were on the loop won’t have enough time to reproduce prior to smearing and heat fixing. However, later lab groups will see the bacteria that were on the loop that had had the course of the day to reproduce.

45
Q

Gram stain

A

A stain used to separate all known bacteria into one of two groups: Gram positive or gram negative

Gram positive cells: Have a cell wall composed almost entirely of thick peptidoglycan. (Stain purple)

Gram negative: Have a thin layer of peptidoglycan and an outer membrane like layer composed of LPS (stain pink)

46
Q

When doing a Gram stain how does the decolorizing agent work

A
  • 95% alcohol
  • The gram negative cell wall, which is high in lipid content is deteriorated by alcohols lipid solvent action. The dehydrating action of alcohol also acts to increase the porosity of the cell wall, allowing the primary dye (crystal violet) to escape from the cell wall.
47
Q

Important thing to remember when choosing a secondary dye

A

You want your secondary dye to be lighter than the primary dye because it overlays the cells that are stained with the primary dye as well as the cells that had their colored removed from the decolorizing agent.

48
Q

What happens to gram positive cultures that are over the age of 24 hrs?

A

Many gram positives begin to lose their ability to retain the primary dye after the culture reaches 24 hours of age. This would cause them to stain as if they were gram negatives.

49
Q

How do older gram positive cultures tend to stain?

A

Gram variable - the older cells lose the primary dye while younger cells keep the primary dye as they should

50
Q

Gram stain procedure

A
  • Flood entire slide with crystal violet for 1 min
  • Drain dye, rinse with water
  • Flood slide with iodine for 1 minute
  • Drain, rinse with water
  • Decolorize with 95% alcohol (3 squirts - 8 seconds)
  • Immediately after rinse with water
  • Flood slide with safranin for 10 mins
  • Drain dye, rinse with water
  • Blot dry
51
Q

Acid fast organisms

A

Are gram positive, but are characterized by also having high amounts of a waxy molecule called mycolic acid incorporated into their cell walls.
“acid fast” refers to their ability to withstand harsh chemicals and retain the dye

52
Q

Genus that are primarily acid fast

A

Mycobacterium

53
Q

Why is the ability to detect Mycobacterium species advantagous

A

Because these bacteria are very slow to culture

54
Q

Acid-Fast staining process

A
  • Cover smear filter paper
  • Saturate filter paper with carbol fuschin
  • Insert bunsen burner into the sink trough to allow flame to heat bottom of slide until steam rises
  • Remover burner from trough and watch steam
  • Repeat steps 2,3 to keep steam rising for 5 minutes
  • Allow slide to cool
  • Rinse slide with water
  • Decolorize with acid alcohol until no more color drips from the slide
  • Cover smear with methylene blue
  • Rinse slide with water
55
Q

Three most common locations of spores

A
  • Terminal (at the end)
  • Central (in the middle)
  • Sub-terminal (just below the end by above the center)
56
Q

Spore staining procedure

A
  • Air dry
  • Heat fix
  • Cover smear with filter paper strip
  • Saturate filter paper with malachite green
  • Insert bunsen burner into the sink trough to allow flame to heat bottom of slide until heat rises
  • Remove bunsen burner from trough squatting to watch steam
  • Repeat steps 4,5 to keep steam rising for 5 mins
  • Allow slide to cool
  • Rinse slide with water (decolorizing step)
  • Cover smear with safranin
  • Rinse slide with water
  • Blot dry
57
Q

Reasons for using a Negative stain

A
  • Some cells have unusual cell walls that do not seem to bind to acidic or basic dyes
  • -Ex. Treponema species (includes syphillus) and Borrelia genus (includes lyme disease)
  • To observe the natural size, shape, and arrangement of bacterial cells for the purposes of identification
58
Q

What does the Negative stain avoid?

A

Both smearing and heat fixing and thus allows you to view organisms in a much more natural, undisturbed state

59
Q

Negative Stain procedure

A
  • At one extreme end of a slide, put a small drop of nigrosin, centered at the end
  • Aseptically add bacteria directly on top of the dye, using a dropper (or a loop). DO NOT SMEAR
  • Take the second clean slide and back up to the drop, allow it to spread along the edge of the angled slide, and then drag the dye/bacteria towards the opposite end of the slide
  • Allow the stained slide to dry
  • View the slide under low, high dry, and oil immersion
  • Once finished, discard all used slides in disinfectant
60
Q

The capsule stain

A

A modified negative stain, followed by a positive counter stain. The negative stain, using an acidic dye colors the background, leaving the cell and capsule unstained. The second part of the stain involves carefully adding a basic dye to the air-dryed slide, then draining the excess dye and air drying again..
The secondary dye stains the cell leaving the capsule visible as a clear “halo” between the stained cell and the stained background

61
Q

“Hand washing is useless in disease control, since it does not kill or remove bacteria from the hands.”

A

False
The population of bacteria that are removed by hand washing are the superficial extraneous bacteria, which are picked up from other people or inanimate objects. Extraneous bacteria are much more likely to be pathogenic than normal skin flora, which are much harder to remove from the skin. This is the reason that disease control is accomplished through hand washing, even though numbers of bacteria are often higher after washing

62
Q

What is a bacterial colony, and why are colony numbers used to extrapolate numbers of bacteria in an area sampled?

A

A colony is a visible mount of bacterial cells produced by the cloning of a single bacterial cell that was placed on the agar. Since each individual cell placed on the Agar forms a colony as the cells divide, colony numbers can be used to extrapolate numbers of bacteria in an area. (ex 4 colonies = 4 different types of bacteria)

63
Q

Differentiate between resolution and magnification, and describe the microscope parts and techniques which increase each.

A
  • magnification - the enlargement of an image through a lens system achieved by the ocular and objective lenses, which enlarge the image. Total magnification = ocular x objective
  • resolution - clarity of the image which is enhanced by:
  • –immersion oil - immersion oil has the same refractive index as glass, so using the oil prevents the diffraction, or the scattering of light as it passes from the specimen into the optical system of the objective lens
  • –Abbe condenser - focuses a big beam of light into a smaller one directly onto the specimen
  • –Blue filter - allows wavelengths of light that are best seen by the eye to reach us
64
Q

Why are basic dyes used in bacteriological stains?

A

Most bacterial cell components have a slight negative electrical charge, which influences which dyes will adhere to the cell wall. Basic dyes have a net positive charge which causes them to be attracted to and adhere to the negatively charged cell surface of most bacterial cells.

65
Q

What are the three different decolorizing agents used in bacteriological stains? for which stain is each used? How is their action different based on the purpose of the stain?

A
  • 95% alcohol - gram stain; gram negative cell walls have high lipid content, which is deteriorated by the alcohol. The alcohol also dehydrates the cell making the cell wall more porous, allowing the primary due to escape. Gr+ are not noticeably damaged by the alcohol
  • Water- spore stain; primary stain is only slightly basic and does not stick to cells well, so simple water removes the dye from all but the re-solidified spore coat
  • Acid alcohol - acid fast stain; harsh acid decolorizes even Gr+ with thick peptidoglycan leaving only waxy walled mycobacterium colored. Heat allows the dye under the waxy layer
66
Q

Why is heat necessary for the spore and acid fast stain but not for the gram stain?

A

-Spore and acid fast cells normally resist dyes, due to their thickness (spores) or waxy nature (acid fast cells). In order to force dye into the cells, heat softens and loosens the surface to allow entry of the dye into these structures.

67
Q

Why are negative stained slides not washed, but placed in a tumbler of disinfectant?

A

Negative stained slides are not heat fixed, which means the bacteria are still alive. Washing slide with hands runs risk of self contamination of live potentially pathogenic bacteria

68
Q

Why is the capsule stain referred to as a combination negative-positive stain?

A

A capsule stain is referred to as a combo neg-pos stain because a negative staining procedure is done first to stain the background and leave the capsule with the cell inside unstained. In order to see the morphology and arrangement of the cell producing the capsule, a basic dye is added to color the cell, leaving the capsule a colorless “halo” on a black background with a colored
cell.

69
Q

If a positive acid-fast stain is made from bacteria found in a skin lesion, what disease would you suspect? Why?

A

Organisms belonging to the genus Mycobacterium are the only ones which stain acid-fast. I would suspect M. leprae in a skin scraping, since this organisms is the
causative agent of leprosy.

70
Q

Why do you never heat-fix a smear to be used for a capsule stain?

A

Heat-fixing would fry or boil away the mucoid capsule, and therefore you would not be able to see the capsule on a stain.

71
Q

You are gram staining alone on a Saturday night, and realize you’ve run out of crystal violet. Making do with what you have, which stain would be the best one to use as the primary stain - carbol fuchsin or methylene blue? Why?

A

Probably the better stain for you to use would be the methylene blue, since the counterstain in the gram stain is safranin (pink), the pink dye carbol fuchsin would not provide sufficient contrast with the counterstain to offer an easy distinction between gram positives and gram negative.

Microbio (7 decks)

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