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z - BIO103 - Microbiology > Lecture 3B - Microscopy and Staining > Flashcards

Lecture 3B - Microscopy and Staining Flashcards

1
Q

Preparation of Specimens for light microscopy

A
  1. Smear
  2. Fixing
  3. Staining
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1
Q
  • spread a thin film of material containing microorganisms over the slide surface
  • allow to air dry
A

smear

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2
Q

Smear: spread a __ __ of material containing microorganisms over the slide surface. Allow to __ __

A
  • thin film
  • air dry
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3
Q
  • process that kills microorganisms and attaches them to a microscope slide
  • preserves and minimizes distortion of cells
A

fixing

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4
Q

Fixing: process that __ microorganisms and __ them to a microscope slide. Fixing __ and __ distortion of cells

A
  • kills
  • attaches
  • preserves
  • minimizes
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5
Q

technique to fix a specimen with chemicals to prevent autolysis by the action of enzymes and deformation of morphologies during specimen preparation

A

Chemical fixation

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6
Q
  • bind the specimen to the slide so that it does not wash off during staining
  • Killing the cells also increases their permeability to the dyes used in staining
A

heat fixation

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7
Q

coloring microorganisms with a dye that emphasizes certain structures

A

staining

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8
Q

Before staining a sample, it must be __

A

fixed

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9
Q

Stains are __ composed of __ __ and __ __

A
  • salts
  • positive ion (cation)
  • negative ion (anion)
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10
Q

the colored ion is called the __

A

chromophore

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11
Q

Two types of dyes

A
  1. basic dyes
  2. acidic dyes
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12
Q
  • chromophore is in positive ions
  • most commonly used dyes
  • bacteria are slightly negatively charged at pH 7, therefore they stain
A

basic dyes

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13
Q

examples of basic dyes

A
  1. crystal violet
  2. methylene blue
  3. safranin (red)
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14
Q
  • color is in negative ions
  • stain the background
  • bacteria do not stain
  • used to observe cell shape, size, and capsules
  • minimal distortion because heat fixing is not necessary and dye is not taken up by cells
A

acidic dyes

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15
Q

stain the background

A

negative staining

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16
Q

acidic dyes are used to observe the cell __, __ and __

A
  • shape
  • size
  • capsules
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17
Q

Example of acidic dyes

A
  1. eosin
  2. nigrosin
  3. India ink
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18
Q

eosin

A

bright pink

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19
Q

nigrosin

A

blue-black

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20
Q

India ink

A

black

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21
Q

Types of staining methods

A
  1. simple stains
  2. differential stains
  3. special stains
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22
Q
  • aqueous or alcohol solution of a single basic dye
  • primary purpose is to stain entire microorganisms to view cell shape and basic structures
A

simple stains

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23
Q

purpose of simple stains

A

stain microorganisms to view cell shape and basic structures

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24
Q

compound used to hold down molecules of a stain onto a microorganism

A

mordant

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25
Q

simple stain procedure

A
  1. stain is applied for certain time and washed off
  2. dried and examined
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26
Q
  • react differently to different types of bacteria
  • can be sued to distinguish among different groups of bacteria
A

differential stains

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27
Q

Two important differential stains used

A
  1. Gram stain
  2. Acid-fast stain
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28
Q

When and who developed gram stain

A

1884 by Hans Gram

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29
Q
  • most useful staining procedure in medical microbiology
  • distinguishes bacteria of two large and medically important groups
A

gram stain

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30
Q

Two groups in gram stain

A
  1. gram-positive bacteria
  2. gram-negative bacteria
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31
Q

Four (4) steps of gram stain

A
  1. primary stain
  2. mordant
  3. decolorizing
  4. counterstain
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32
Q

(gram-stain)
- cover a heat fixed smear with a basic dye
- all cells are stained

A

primary stain

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33
Q

dye commonly used in primary stain

A

crystal violet

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34
Q

after smear is rinsed with water, an __ _ solution is applied

A

iodine mordant

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35
Q

(gram-stain) primary stain duration

A

1 min.

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36
Q

(gram-stain) mordant duration

37
Q

slide is washed with alcohol, which remove stain from gram-negative but not gram-positive

A

decolorizing

38
Q

(gram-stain) decolorizing duration

A

rock back and forth, 12-15 sec.

39
Q

in decolorizing,
- gram-negative cells will be __
- gram-positive cells will remain __

A
  • decolorized
  • purple
40
Q

(gram-stain) alcohol is rinsed off, safranin applied which will stain cells that are decolorized

A

counterstain

41
Q

(gram-stain) what dye is used in counterstaining

42
Q

(gram-stain) counterstain duration

A

45 sec. (rinse & blot dry)

43
Q

in counterstain,
- gram-negative cells are stained __
- gram-positive cells remain __

A
  • pink
  • purple
44
Q

in gram stain, __ culture cells, at least less than __ __ old are used

A
  • young
  • 18-24 hours
45
Q
  • gram positive have very __ peptidoglycan cell walls
  • gram negative have very __ cell walls
A
  • thick (positive)
  • thin (negative)
46
Q

CV-I

A

crystal violet-iodine complex

47
Q

why is the CV-I not easily removed from the gram-positive cells

A

due to thick cell wall

48
Q

gram-positive cells with their very thick peptidoglycan cell walls are susceptible to __ and __

A
  • penicillins
  • cephalosporins
49
Q

gram-negative cells with their thin cell walls and __ layer are __ to these antibiotics

A
  • lipopolysaccharide
  • resistant
50
Q

stain irregularly, and there’s a mix of pink- and purple-colored bacteria

A

gram-variable bacteria

51
Q

acid-fast stain or __ stain

A

Ziehl-Neelsen stain

52
Q

acid-fast stain is a modification method developed in __ by __

A

1882 by Paul Ehrlich

53
Q

Paul Ehrlich used acid-fast stain to detect tuberculosis and leprosy causing organisms of the genus __ and pathogens of the genus __

A
  • Mycobacterium
  • Nocardia
54
Q

Mycobacterium and Nocardia are bacteria that have __ cell walls, which makes them difficult to stain

55
Q

Three (3) steps of acid-fast stain

A
  1. primary stain
  2. decolorizing
  3. counterstain
56
Q

(acid-fast stain)
- cover a heat fixed smear with carbolfuchsin, a red basic dye
- gently heat for several minutes to increase penetration and retention of dye
- allow to cool and rinse with water

A

primary stain

57
Q

(acid-fast stain) Primary stain: cover a heat fixed smear with __

A

carbolfuchsin (red basic dye)

58
Q

(acid-fast stain) Carbolfuchsin steaming duration

59
Q

(acid-fast stain) slide is washed with acid-alcohol

A

decolorizing

60
Q

In decolorizing
- non acid-fast cells will be __
- acid-fast cells will remain __

A
  • decolorized (non acid-fast)
  • red (acid-fast)
61
Q

(acid-fast stain) decolorizing duration

62
Q

(acid-fast stain)
- acid-alcohol is rinsed off
- methylene blue is applied

A

counterstain

63
Q

in counterstaining,
- non acid-fast cells are stained __
- acid-fast cells remain __

64
Q

(acid-fast stain) counterstain duration

A

30 sec - 1 min

65
Q

used to color and isolate specific parts of microorganisms

A

special stains

66
Q

special stains are used to color and isolate specific parts of microorganisms such as: __ __ __

A
  1. endospore
  2. capsules
  3. flagella
67
Q

endospores are extremely __, __ structures that are formed by some gram-positive bacteria

A
  • resistant
  • dormant
68
Q

endospores are formed by gram-positive bacteria to protect them from __ __ __

A

harsh environment conditions

69
Q

differential stain used to visualize bacterial endospores

A

endospore stain

70
Q

most commonly used method in endospore staining

A

Schaeffer-Fulton Endospore Stain

71
Q

Three (3) steps of Schaeffer-Fulton endospore stain

A
  1. primary stain
  2. wash
  3. counterstain
72
Q

(Schaeffer-Fulton Endospore Stain)
malachite green is applied to heat fixed smear and steamed for 5-7 minutes

A

primary stain

73
Q

(Schaeffer-Fulton Endospore Stain)
what is applied in primary stain

A

malachite green

74
Q

(Schaeffer-Fulton Endospore Stain)
primary stain duration

75
Q

(Schaeffer-Fulton Endospore Stain)
rinse with water for 30 sec.

76
Q

(Schaeffer-Fulton Endospore Stain)
wash duration

77
Q

(Schaeffer-Fulton Endospore Stain)
what is used to wash primary stain

78
Q

(Schaeffer-Fulton Endospore Stain)
safranin will stain rest of cell

A

counterstain

79
Q

(Schaeffer-Fulton Endospore Stain)
counterstain duration

80
Q

(Schaeffer-Fulton Endospore Stain)
appearance of cell with endospore

A

pink cell with green endospore

81
Q

gelatinous covers, which are important virulence (disease) factors

82
Q

why are capsules difficult to stain

A
  • repel most stains
  • water soluble
  • easily disrupted
83
Q

(capsule stain)
used to obtain background

A

negative stain

84
Q

(capsule stain)
cell is stained with __ __

85
Q

(capsule stain)
capsule appearance

A
  • light halo around stained cell
  • dark background
86
Q
  • organic polymers of microbial origin involved in bacterial cells’ interactions with their environment
  • comprised of polysaccharides, proteins, extracellular DNA (eDNA), and lipids
A

Extracellular polymeric substances (EPS)

87
Q

used for locomotion that are too thin to be seen easily with light microscope

88
Q

(flagella stain)
usually involves using __ and coating the flagellar structure with __ or __

A
  • mordant
  • dye or metal (e.g. silver)
89
Q

(flagella stain)
the __ and __ of flagella can be used as diagnostic aids

A
  • number
  • arrangement

z - BIO103 - Microbiology (18 decks)

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