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BIOL 270 > LAB EXAM #1 experiments deck > Flashcards

LAB EXAM #1 experiments deck Flashcards

1
Q

Aseptic technique:

A

Insures that there is no contamination of microorganisms

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2
Q

Loops are used to:

A

Transfer from liquid media to liquid media or petri plates

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3
Q

Needles are used to:

A

Transfer from solid media to solid media or petri plates

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4
Q

Purpose of smear prep:

A

To prepare the organism for staining

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5
Q

Smear preps are used to determine:

A
  • Cell shape
  • Arrangement of cells
  • Internal structures
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6
Q

Procedure of aseptic technique:

A
  1. Transfer org to slant
  2. Transfer org from slant to broth
  3. Transfer org from broth to plate
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7
Q

Procedure of smear prep from a broth: (3)

A
  1. Using AT, transfer 3 loops of org to the middle of the slide
  2. Air dry
  3. Heat-fix slide by passing slide through the bunsen burner flame 5 times
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8
Q

Procedure of smear prep from a solid media: (4)

A
  1. Using AT, place 3 loops of WATER onto center of slide
  2. Transfer org to the water
  3. Air dry
  4. Heat-fix the slide by passing through the bunsen burner 5 times
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9
Q

Purpose of heat-fixation of smear preps?

A

Kills the organism and adheres them to the slide so they aren’t washed off during staining

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10
Q

Purpose of simple staining procedure: (2)

A
  • Used to make bacterial cells easier to visualize on the slide
  • Used to determine bacterial cell morphology (cell shape)
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11
Q

2 types of dyes for staining:

A

Basic dye

Acidic dye

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12
Q

Basic dye:

A
  • Positively charged chromophore
  • Works well with bacterial cells because they have a negative charge
  • Stains inside of cell
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13
Q

Acidic dye:

A
  • Negatively charged chromophore
  • Repelled by the negative charge of the bacterial cell
  • Stains outside of cell
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14
Q

3 main shapes of bacteria:

A
  1. Rods (bacilli)
  2. Cocci (spherical)
  3. Spiral or curved
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15
Q

Dye use in simple staining experiment:

A

Methylene blue

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16
Q

Procedure for simple staining: (4)

A
  1. Using AT, make a smear prep by adding org to middle of slide
  2. Air dry and then heat-fix slide
  3. Add methylene blue
  4. Rinse slide and view under oil
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17
Q

Purpose of negative stain:

A

To demonstrate the size and shape of bacteria

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18
Q

Appearance of bacterial cell in a negative stain:

A

Negative stain stains the background of bacterial cell and bacterial cell will appear transparent against a dark background

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19
Q

Dye used in negative stain experiment?

A

Nigrosin or india ink

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20
Q

Procedure for negative stain experiment: (4)

A
  1. Add 1 drop of india ink near end of slide.
  2. Transfer org into india ink drop
  3. With another slide, place the edge of the new slide against the drop of india ink and push the spreader slide across the slide
  4. Air dry then view under oil
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21
Q

2 types of gel-like layer on the outside of the cell wall:

A
  1. Capsule layer

2. Slime layer

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22
Q

Capsule layer: (3)

A
  • Gelatinous layer
  • Attached tightly to bacteria
  • Made of polysaccharides known as glycocalyx
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23
Q

Slime layer:

A
  • Layer made of ECM

- Loosely attached to bacteria

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24
Q

Function of capsule and slime layer:

A
  • Help cells from being engulfed or destroyed

- Attachment to solid surfaces

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25
Q

In the capsule staining experiment, what is the first step?

A

Negative staining procedure

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26
Q

Under oil, how do capsule appear?

A

Halos around pink/red cells with a dark background

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27
Q

Dyes used in capsule staining experiment:

Include charges

A
  • Congo red: acidic dye, stains background

- Maneval’s: basic dye, stains inside of cell

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28
Q

Procedure of capsule staining experiment: (7)

A
  1. Add 1 drop of congo red to end of slide
  2. Transfer org into congo red
  3. With another slide, place the edge of the new slide against the drop of congo red and push the spreader slide across the slide
  4. Air dry
  5. Add Maneval’s, as the counterstain, to the congo red
  6. Gently rinse
  7. Air dry and view under oIL
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29
Q

Hans Christian Gram:

A

Danish physician that developed a staining technique to differentiate bacterial cells from eukaryotic nuclei

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30
Q

Purpose of gram stain:

A

Provides identification of unknown bacteria

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31
Q

Gram positive cells:

A
  • Appear purple

- 90% peptidoglycan 10% teichoic acids

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32
Q

Gram negative cells:

A
  • Appear pink or red

- 10% peptidoglycan 90% outer membrane

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33
Q

Purpose of crystal violet in gram stain?

A

Primary stain that gives it the purple color

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34
Q

Purpose of gram’s iodine in gram stain?

A

Mordant- combines insoluble complex with crystal violet in gram positive cells

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35
Q

Purpose of 95% ethanol in gram stain?

A

Decolorizing agent- removes purple from gram negative cells

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36
Q

Purpose of safranin in gram stain?

A

Counterstain for gram negative cells so they may be seen as pink or red

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37
Q

Dyes used in gram stain experiment: (4)

A
  • crystal violet
  • gram’s iodine
  • 95% ethanol
  • safranin
38
Q

Procedure for gram stain experiment? (6)

A
  1. Make a smear prep by adding org to middle of slide
  2. Heat-fix
  3. Cover with crystal violet then rinse
  4. Cover with gram’s iodine then pour off
  5. Decolorize with 95% ethanol then rinse
  6. Counterstain with safranin then rinse and view under oil
39
Q

Purpose of acid-fast staining:

A

Used to identify Mycobacterium and Nocardia that have mycolic acid in cell wall

40
Q

Acid-fast bacteria are stained:

A

Pink to red by the dye basic fuchsin

41
Q

Non-acid fast bacteria are stained:

A

Blue with the counterstain methylene blue

42
Q

Dyes used in the acid-fast staining experiment: (3)

A
  • Carbolfuchsin
  • Acid alcohol
  • Methylene blue (counterstain)
43
Q

Procedure for acid-fast staining experiment: (5)

A
  1. Make a smear prep by adding org to middle of slide
  2. Cover with carbolfuchsin for 5 min
  3. Gently rinse
  4. Decolorize with acid alcohol then rinse
  5. Counterstain with methylene blue then rinse and view under oil
44
Q

2 species of bacteria that form endospores are:

A

Bacillus and Clostridia

45
Q

How to enter an endospore in endospore staining?

A

Heat needs to be applied to help the stain enter the endospore- mordant

46
Q

In the endospore staining experiment, what color will the endospore and vegetative cell be?

A

Endospore: green due to malachite green

Vegetative cell: red due to safranin

47
Q

Dyes used in the endospore staining experiment:

A

Malachite green

Safranin-counter stain

48
Q

Procedure for endospore staining experiment:

A
  1. Make a smear prep by adding org to middle of slide
  2. Cover with malachite green
  3. Heat-fix and replenish malachite green as needed
  4. Rinse
  5. Counterstain with safranin then rinse and view under oil
49
Q

Motility is found in:

A

Spirochetes and rod shaped bacteria

50
Q

Purpose of motility experiment?

A

To identify the presence of flagella

51
Q

Procedure for wet mount in motility experiment:

A
  1. Make a smear prep by adding org to middle of slide
  2. Cover slide with cover slip
  3. View under 400x immediately
52
Q

Procedure for motility agar in motility experiment:

A
  1. Take one tube with TTC in it and stab needle into agar for org
  2. Incubate media at 25C
53
Q

What is TTC?

A

A colorless dye that when bacteria reduce it, it becomes red

54
Q

Motile organisms:

A

Move away from stab line and have a fuzzy appearance

55
Q

Motile organisms that can reduce the TTC:

A

Move away from stab line and have red fuzzy appearance

56
Q

Non-motile organisms:

A

Will not move away from stab line and the rest of the media will be clear

57
Q

Non-motile organisms that can reduce the TTC:

A

Red color in the stab line only and the rest will b e clear

58
Q

Who was the first person to utilize the pure culture technique?

A

Robert Koch

59
Q

Pure culture technique:

A

Helps obtain a single kind of organism from a mixed culture

60
Q

2 commonly use methods of the pure culture technique:

A
  1. Pour plate

2. Streak plate

61
Q

Streak plate method for the pure culture technique experiment:

A

Streak TSA plate with org using a quadrant streak

62
Q

Pour plate method for pure culture technique experiment: (6)

A

Day 1:

  1. Obtain 3 petri plates
  2. Obtain 3 TSA tubes from the water bath and transfer the mixed culture into tube #1, mix
  3. Before pouring into petri plate, transfer 1 loop from tube #1 into tube #2
  4. Pour tube #1 into petri plate
  5. Transfer 1 loop from tube #2 into tube #3, then pour tube #2 into petri plate
  6. Pour tube #3 into petri plate
  7. Gently rotate plate to mix content then flip upside down when agar has solidified

Day 2:

  1. Obtain 3 TSA slants and transfer 1 colony of each color onto each slant
  2. Incubate at 25C
63
Q

UV light falls between:

A

4nm and 400 nm

64
Q

Purpose of UV light effects experiment:

A

To demonstrate bacteriostatic effect

65
Q

3 ways that UV light damages our cells:

A

UVA: tanning beds
UVB: damaging cancer
UVC: germicidal lamps, hoods not seen in nature-> absorbed by O2

66
Q

Procedure for UV light effects experiment:

A
  1. Obtain TSA plate and sterile swab
  2. Swab plate with organism
  3. When it is your exposure time, place plate in biosafety cabinet with a flash card covering half of the plate
  4. Flip over and examine number of colonies exposed to the UV light next lab period
67
Q

Enumeraton:

A

Number of viable and non-viable microbes in a sample

68
Q

Purpose of enumeration of bacteria experiment:

A

Determine the concentration of an organism

69
Q

How is turbidity measured?

A

With a spectrophotometer where % transmittance or absorbance is obtained

70
Q

Why is turbidity considered an indirect method?

A

It uses absorption of light to determine the amount of colloidal material in suspension

71
Q

E. coli charge and shape:

A

Gram (-) rod

72
Q

B. cereus: charge and shape:

A

Gram (+) rod

73
Q

S. aureus charge and shape:

A

Gram (+) cocci

74
Q

Primary lethal effect of UV light:

A

At 260nm, DNA absorbs the UV light and bonds are broken at its maximum-> pyrimidine dimers formed

75
Q

Pyrimidine dimers:

A

Covalent bonds that form 2 adjacent thiamine or cytosine molecules of DNA-> changes shape of DNA-> can’t replicate

76
Q

Purpose of effects of temp on growth exp:

A

To see the effects of temp on growth of organisms

77
Q

Procedure for effects of temp on growth experiment:

A

Day 1

  1. Obtain 6 TSA tubes and label different temps
  2. Inoculate each tube with org
  3. Obtain 2 TSA slants and label different temps
  4. Inoculate with 2nd org

Day 2

  1. Measure %transmittance of each temp tube
  2. Determine OD and pigment production for each temp tube
78
Q

Procedure for antiseptics mouth experiment:

A

Day 1

  1. Swab inside of mouth and place swab into TSA tube
  2. Incubate

Day 2

  1. Obtain TSA plate and divide into 3 quadrants
  2. Label each with scope, listerine, and cepacol
  3. Streak plate with mouth swab
  4. Place disks into each quadrant
79
Q

Procedure for antiseptics assigned org experiment:

A
  1. Obtain TSA plate and divide into 3 quadrants
  2. Label each with: 70% isopropanol, bactine, and povidone iodine
  3. Streak plate with assigned org
  4. Place disks into each quadrant
80
Q

T or F

E. coli is apart of Enterobacteriaceoe

A

T

81
Q

T or F

E. faecalis is apart of streptococci as well as an enterococci:

A

T

82
Q

Most effective antiseptic:

Least effective antiseptic:

A

Scope

Cepacol

83
Q

Most susceptible org:

Least susceptible org:

A

Bactine

70% isopropanol

84
Q

Procedure for antibiotic sensitivity experiment: Kirby-Bauer Method

A

Day 1

  1. Obtain a Mueller-Hinton plate (big plate)
  2. Swab plate with org
  3. Place abx disc on plate

Day 2
1. Measure zone of inhibition for each abx disc on plate

85
Q

Indirect methods of enumeration of bacteria experiment:

A

Turbidity

86
Q

Direct methods of enumeration of bacteria experiment:

A
  1. bacterial count
  2. standard plate count
  3. most probable number
87
Q

Why is turbidity not a good test?

A

Contribution of dead and living cells

88
Q

Formula for OD:

A

OD= 2-log(%T)

89
Q

What is the difference between the methods?

A

Turbidity determines if sample has bacteria in it

Direct methods are a bacterial count

90
Q

Purpose of slant tube:

A

More surface area

BIOL 270 (26 decks)

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