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MICRB 107 - Exam 1 > Microscopy and Staining > Flashcards

Microscopy and Staining Flashcards

1
Q

Define microscopy

A

The use of light / electrons to magnify objects

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2
Q

Visible light is part of the ______

A

Electromagnetic radiation spectrum

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3
Q

With regard to light, what is the relationship between wavelength and energy?

A

Shorter wavelength = higher energy

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4
Q

Define magnification

A

Apparent increase in size of an object

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5
Q

How does magnification occur?

A

A beam of radiation refracts as it passes through a lens

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6
Q

Curved glass lenses refract ______

A

Light

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7
Q

Magnetic lenses refract ______

A

Electron beams

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8
Q

Define resolution

A

The ability to distinguish between objects that are close together

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9
Q

Define contrast

A

Differences in intensity between two objects / backgrounds

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10
Q

What is the most common type of microscope?

A

Bright field microscope

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11
Q

What makes a bright-field microscope a ‘bright-field microscope’?

A

The background (field) is illuminated

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12
Q

Name the 2 types of bright-field microscopes

A
  • Simple microscope
  • Compound microscope
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13
Q

What is a ‘simple’ microscope?

A

Contains a single magnifying lens

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14
Q

What is a ‘compound’ microscope – how does it differ from a simple microscope? (2)

A
  • Uses a series of lenses for magnification
  • Light passes through a specimen and into an ‘objective lens’ (immediately above the object)
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15
Q

What are the names and magnifications of the four objective lenses in our compound microscopes? (4)

A
  • Scanning objective lens - 4X
  • Low-power objective lens - 10X
  • High dry objective lens - 40X
  • Oil immersion objective lens - 100X
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16
Q

Which type of objective lens increases magnification and resolution?

A

Oil immersion objective lens

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17
Q

Explain the principle of using immersion oil when appropriate (3)

A
  • Light refracts as it travels from glass into air
  • Some light passing out of a glass slide is bent so much that is bypasses the lens
  • Immersion oil enables the lens to capture the light - increases resolution
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18
Q

What is the function of the ocular lens?

A

Magnifies the image created by the objective lens by another 10X

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19
Q

How does one calculate total magnification for a compound microscope?

A

Multiply the magnification of the objective lens by the magnification of the ocular lens

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20
Q

What is the function of the condenser lens?

A

Directs light emitted from the illuminator upon the specimen

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21
Q

What is the function of the iris diaphragm?

A

Controls the amount of light exiting the condenser lens

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22
Q

What effect does opening the iris diaphragm have upon the field of view? (2)

A
  • Increases the amount of light passing through
  • Specimen becomes more illuminated
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23
Q

What is a heat-fixed smear and how is one made? (2)

A
  • The slide it passed (smear-side up) through the flame of a Bunsen burner 2-3 times
  • Ensures that cells are not washed off
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24
Q

When making a heat-fixed smear, what considerations should be made depending upon whether the culture comes from broth vs. solid media? (2)

A
  • If the organisms are growing in a liquid, a small drop is spread across the surface of the slide
  • If the organisms are growing on a solid surface (such as an agar plate), they are mixed into a small drop of water on the slide
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25
Q

What is the difference between air drying and heat fixation?

A
  • Air drying allows for excess to dry before heating
  • Heating allows the smear to adhere to the slide
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26
Q

What would happen if a specimen was heated immediately, before air drying?

A

The smear would overheat / denature

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27
Q

When making a heat-fixed smear, what 3 considerations should be kept in mind at all times?

A
  • Adhere cells to the microscope slide
  • Avoid shrinkage of cells during staining
  • Prepare thin smears
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28
Q

Why is it important to adhere cells to the slide?

A

Ensures that they are not washed off during staining / washing procedures

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29
Q

What can result from shrinkage of cells during staining? (2)

A
  • Distortion
  • Formation of artifacts
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30
Q

What does the thickness of the smear determine? (2)

A
  • Ability to visualize individual cells
  • Arrangement of cells
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31
Q

Differentiate between the terms morphology and arrangement

A
  • Morphology - appearance of groups of bacteria
  • Arrangement - groupings of individual cells
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32
Q

What are the 3 most common bacterial morphologies?

A
  • Cocci - spheres
  • Bacilli - rods
  • Spirilla - spirals
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33
Q

Describe diplococci

A

Daughter cells remain attached after a coccus divides

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34
Q

Describe diplobacilli

A

Daughter cells remain attached after a bacillus divides

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35
Q

Describe streptococcus / streptobacillus

A

Cells continue to divide in the same plane and remain attached to form a chain

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36
Q

Tetrads and sarcinae are only seen in ______

A

Cocci

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37
Q

Describe a tetrad

A

Second division occurs in a plane perpendicular to the first

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38
Q

Describe a sarcina

A

Third plane occurs perpendicular to to the other two

39
Q

Describe the formation resulting from a sarcina

A

A cube-shaped arrangement of 8 cells

40
Q

Describe staphylococcus

A

Division planes of a coccus are irregular

41
Q

Describe the formation resulting from staphylococcus

A

A cluster of cells

42
Q

What is a chromophore?

A

The colored portion of the dye (at least one of the two ions is colored)

43
Q

Describe the chemical composition of methylene blue regarding chromophores (2)

A
  • Cationic chromophore
  • Chloride anion
44
Q

What is the overall net charge of most cells?

A

Between -1 and 0

45
Q

What is the charge of the chromophore in basic dyes?

46
Q

Describe the nature by which basic dyes such as methylene blue ‘stain’ microbial cells (2)

A
  • Methylene blue is positively charged
  • Ionically bond to negatively charged molecules in cells
47
Q

What are some examples of molecules that methylene blue ionically bonds to? (2)

A
  • DNA
  • Proteins
48
Q

What is the definition of simple staining?

A

The use of a single stain to color bacterial cells

49
Q

Describe the technique of simple staining (2)

A
  • Soak the smear in dye for 30 - 60 seconds
  • Rinse the slide with water
50
Q

What is the fundamental difference between a simple stain and a negative stain?

A
  • Simple staining directly stains the bacteria with a positively charged dye
  • Negative staining stains the background and leaves the cells colorless
51
Q

What is the charge of the chromophore in acidic dyes?

52
Q

What is the nature of interaction between acidic dyes and bacterial cells?

A

Anionic chromophores in acidic dyes are repulsed by the negative charge on the surface of cells

53
Q

Describe the technique of negative staining

A

Stain cells by attaching to negatively charged molecules within them

54
Q

What are some examples of basic dyes used in negative staining of bacterial cells? (4)

A
  • Crystal violet
  • Methylene blue
  • Malachite green
  • Safranin
55
Q

Name 1 genera associated with acid-fast bacteria

A

Mycobacterium

56
Q

Name 2 diseases associated with acid-fast bacteria

A
  • Tuberculosis
  • Leprosy
57
Q

Describe the differentiation of the acid-fast stain

A

Differentiation from blue ‘non-acid-fast’ cells

58
Q

What is an example of blue non-acid-fast cells?

A

Human cells / tissues

59
Q

What unique structure / cellular quality is exploited during the acid-fast staining procedure?

A

Large amounts of waxy lipid (mycolic acid) in their cell walls - cells do not readily stain

60
Q

What is the primary stain vs. the counterstain in the acid-fast stain?

A
  • Primary stain - red carbolfuchsin
  • Counterstain - methylene blue
61
Q

Why is it necessary to use steaming water during the primary stain incubation in the acid-fast stain?

A

Heat is used to drive the stain through the waxy wall and into the cell - remains trapped

62
Q

What is the nature of the decolorizer used in the acid-fast staining procedure, and how is it different from the decolorizer used in the gram stain? (2)

A
  • Acid alcohol decolorizer used to rinse the smear
  • Ability to resist decolorization from acids
63
Q

What color are acid-fast bacteria vs. non-acid-fast bacteria by the end of the staining procedure?

A
  • Non-acid-fast - no color
  • Acid-fast - red
64
Q

Name 2 genera of endospore producers

A
  • Bacillus
  • Clostridium
65
Q

What is the difference between a vegetative cell and an endospore?

A
  • Vegetative cell - active form of bacteria (grows and metabolizes)
  • Endospore - dormant form of bacteria
66
Q

What is the function of an endospore?

A

To ensure survival of a bacteria during harsh environmental conditions

67
Q

Describe the conditions under which sporulation is induced

A

Conditions unfavorable for growth (nutrient depletion)

68
Q

______ are more likely to be enriched with endospores

A

Young cultures

69
Q

Why are young cultures more likely to be enriched with endospores?

A

Older gram-positive cells bleach easier than younger cells (stain pink, appearing to be gram-negative cells)

70
Q

Endospores cannot be stained by normal staining procedures because …

A

Their walls are impermeable to all chemicals

71
Q

What is the primary stain vs. the counterstain in the endospore stain?

A
  • Primary stain - malachite green
  • Counterstain - safranin
72
Q

Why is it necessary to use steaming water during the primary stain incubation in the endospore stain?

A

Allows for heat to break through the endospore wall

73
Q

What color are endospores vs. vegetative cells by the end of the staining procedure?

A
  • Endospores - green
  • Vegetative cells - red
74
Q

What is the fundamental difference between simple stains and differential stains?

A
  • Simple stains use one dye
  • Differential stains use more than one dye
75
Q

After performing the gram stain, what color are gram-positive cells vs. gram-negative cells?

A
  • Gram-positive - purple
  • Gram-negative - pink
76
Q

What is the differential quality of the gram stain based on?

A

Chemical and physical properties of the cell walls

77
Q

Of gram-positive cells vs. gram-negative cells, which has a thicker layer of peptidoglycan?

A

Gram-positive cells

78
Q

What are the 4 reagents (other than water) in the gram staining procedure, and in what order are they used?

A
  • Crystal violet
  • Iodine
  • Decolorization
  • Safranin
79
Q

What is the primary stain vs. the counterstain in the gram staining procedure?

A
  • Primary stain - crystal violet
  • Counterstain - safranin
80
Q

What color are gram-positive vs. gram-negative cells after addition of each of the gram-stain reagents?

A
  • Crystal violet - purple
  • Iodine - purple
  • Decolorization - colorless
  • Safranin - gram-negative are pink / gram-positive are purple
81
Q

Name 3 factors that can affect the outcome of a gram-stain

A
  • Age of cell
  • Effectiveness of decolorization
  • Thickness of the smear
82
Q

What occurs in the case of over decolorization?

A

Gram-positive cells appear to be gram-negative cells

83
Q

What occurs in the case of under decolorization?

A

Gram-negative cells appear to be gram-positive cells

84
Q

What occurs if the smear is too thick?

A

Gram-negative cells can appear to be gram-positive cells

85
Q

1

A

Line of vision

86
Q

2

A

Ocular lens

87
Q

3

A

Path of light

88
Q

4

89
Q

5

90
Q

6

A

Objective lens

91
Q

7

92
Q

8

A

Condenser lens

93
Q

9

A

Illuminator

MICRB 107 - Exam 1 (3 decks)

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