Microscopy and Staining Flashcards
1
Q
Define microscopy
A
The use of light / electrons to magnify objects
2
Q
Visible light is part of the ______
A
Electromagnetic radiation spectrum
3
Q
With regard to light, what is the relationship between wavelength and energy?
A
Shorter wavelength = higher energy
4
Q
Define magnification
A
Apparent increase in size of an object
5
Q
How does magnification occur?
A
A beam of radiation refracts as it passes through a lens
6
Q
Curved glass lenses refract ______
A
Light
7
Q
Magnetic lenses refract ______
A
Electron beams
8
Q
Define resolution
A
The ability to distinguish between objects that are close together
9
Q
Define contrast
A
Differences in intensity between two objects / backgrounds
10
Q
What is the most common type of microscope?
A
Bright field microscope
11
Q
What makes a bright-field microscope a ‘bright-field microscope’?
A
The background (field) is illuminated
12
Q
Name the 2 types of bright-field microscopes
A
- Simple microscope
- Compound microscope
13
Q
What is a ‘simple’ microscope?
A
Contains a single magnifying lens
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)
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
16
Q
Which type of objective lens increases magnification and resolution?
A
Oil immersion objective lens
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
18
Q
What is the function of the ocular lens?
A
Magnifies the image created by the objective lens by another 10X
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
20
Q
What is the function of the condenser lens?
A
Directs light emitted from the illuminator upon the specimen
21
Q
What is the function of the iris diaphragm?
A
Controls the amount of light exiting the condenser lens
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
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
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
25
What is the difference between air drying and heat fixation?
- Air drying allows for excess to dry before heating
- Heating allows the smear to adhere to the slide
26
What would happen if a specimen was heated immediately, before air drying?
The smear would overheat / denature
27
When making a heat-fixed smear, what 3 considerations should be kept in mind at all times?
- Adhere cells to the microscope slide
- Avoid shrinkage of cells during staining
- Prepare thin smears
28
Why is it important to adhere cells to the slide?
Ensures that they are not washed off during staining / washing procedures
29
What can result from shrinkage of cells during staining? (2)
- Distortion
- Formation of artifacts
30
What does the thickness of the smear determine? (2)
- Ability to visualize individual cells
- Arrangement of cells
31
Differentiate between the terms morphology and arrangement
- Morphology - appearance of groups of bacteria
- Arrangement - groupings of individual cells
32
What are the 3 most common bacterial morphologies?
- Cocci - spheres
- Bacilli - rods
- Spirilla - spirals
33
Describe diplococci
Daughter cells remain attached after a coccus divides
34
Describe diplobacilli
Daughter cells remain attached after a bacillus divides
35
Describe streptococcus / streptobacillus
Cells continue to divide in the same plane and remain attached to form a chain
36
Tetrads and sarcinae are only seen in ______
Cocci
37
Describe a tetrad
Second division occurs in a plane perpendicular to the first
38
Describe a sarcina
Third plane occurs perpendicular to to the other two
39
Describe the formation resulting from a sarcina
A cube-shaped arrangement of 8 cells
40
Describe staphylococcus
Division planes of a coccus are irregular
41
Describe the formation resulting from staphylococcus
A cluster of cells
42
What is a chromophore?
The colored portion of the dye (at least one of the two ions is colored)
43
Describe the chemical composition of methylene blue regarding chromophores (2)
- Cationic chromophore
- Chloride anion
44
What is the overall net charge of most cells?
Between -1 and 0
45
What is the charge of the chromophore in basic dyes?
Negative
46
Describe the nature by which basic dyes such as methylene blue ‘stain’ microbial cells (2)
- Methylene blue is positively charged
- Ionically bond to negatively charged molecules in cells
47
What are some examples of molecules that methylene blue ionically bonds to? (2)
- DNA
- Proteins
48
What is the definition of simple staining?
The use of a single stain to color bacterial cells
49
Describe the technique of simple staining (2)
- Soak the smear in dye for 30 - 60 seconds
- Rinse the slide with water
50
What is the fundamental difference between a simple stain and a negative stain?
- Simple staining directly stains the bacteria with a positively charged dye
- Negative staining stains the background and leaves the cells colorless
51
What is the charge of the chromophore in acidic dyes?
Negative
52
What is the nature of interaction between acidic dyes and bacterial cells?
Anionic chromophores in acidic dyes are repulsed by the negative charge on the surface of cells
53
Describe the technique of negative staining
Stain cells by attaching to negatively charged molecules within them
54
What are some examples of basic dyes used in negative staining of bacterial cells? (4)
- Crystal violet
- Methylene blue
- Malachite green
- Safranin
55
Name 1 genera associated with acid-fast bacteria
Mycobacterium
56
Name 2 diseases associated with acid-fast bacteria
- Tuberculosis
- Leprosy
57
Describe the differentiation of the acid-fast stain
Differentiation from blue ‘non-acid-fast’ cells
58
What is an example of blue non-acid-fast cells?
Human cells / tissues
59
What unique structure / cellular quality is exploited during the acid-fast staining procedure?
Large amounts of waxy lipid (mycolic acid) in their cell walls - cells do not readily stain
60
What is the primary stain vs. the counterstain in the acid-fast stain?
- Primary stain - red carbolfuchsin
- Counterstain - methylene blue
61
Why is it necessary to use steaming water during the primary stain incubation in the acid-fast stain?
Heat is used to drive the stain through the waxy wall and into the cell - remains trapped
62
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)
- Acid alcohol decolorizer used to rinse the smear
- Ability to resist decolorization from acids
63
What color are acid-fast bacteria vs. non-acid-fast bacteria by the end of the staining procedure?
- Non-acid-fast - no color
- Acid-fast - red
64
Name 2 genera of endospore producers
- Bacillus
- Clostridium
65
What is the difference between a vegetative cell and an endospore?
- Vegetative cell - active form of bacteria (grows and metabolizes)
- Endospore - dormant form of bacteria
66
What is the function of an endospore?
To ensure survival of a bacteria during harsh environmental conditions
67
Describe the conditions under which sporulation is induced
Conditions unfavorable for growth (nutrient depletion)
68
______ are more likely to be enriched with endospores
Young cultures
69
Why are young cultures more likely to be enriched with endospores?
Older gram-positive cells bleach easier than younger cells (stain pink, appearing to be gram-negative cells)
70
Endospores cannot be stained by normal staining procedures because ...
Their walls are impermeable to all chemicals
71
What is the primary stain vs. the counterstain in the endospore stain?
- Primary stain - malachite green
- Counterstain - safranin
72
Why is it necessary to use steaming water during the primary stain incubation in the endospore stain?
Allows for heat to break through the endospore wall
73
What color are endospores vs. vegetative cells by the end of the staining procedure?
- Endospores - green
- Vegetative cells - red
74
What is the fundamental difference between simple stains and differential stains?
- Simple stains use one dye
- Differential stains use more than one dye
75
After performing the gram stain, what color are gram-positive cells vs. gram-negative cells?
- Gram-positive - purple
- Gram-negative - pink
76
What is the differential quality of the gram stain based on?
Chemical and physical properties of the cell walls
77
Of gram-positive cells vs. gram-negative cells, which has a thicker layer of peptidoglycan?
Gram-positive cells
78
What are the 4 reagents (other than water) in the gram staining procedure, and in what order are they used?
- Crystal violet
- Iodine
- Decolorization
- Safranin
79
What is the primary stain vs. the counterstain in the gram staining procedure?
- Primary stain - crystal violet
- Counterstain - safranin
80
What color are gram-positive vs. gram-negative cells after addition of each of the gram-stain reagents?
- Crystal violet - purple
- Iodine - purple
- Decolorization - colorless
- Safranin - gram-negative are pink / gram-positive are purple
81
Name 3 factors that can affect the outcome of a gram-stain
- Age of cell
- Effectiveness of decolorization
- Thickness of the smear
82
What occurs in the case of over decolorization?
Gram-positive cells appear to be gram-negative cells
83
What occurs in the case of under decolorization?
Gram-negative cells appear to be gram-positive cells
84
What occurs if the smear is too thick?
Gram-negative cells can appear to be gram-positive cells
85
1
Line of vision
86
2
Ocular lens
87
3
Path of light
88
4
Prism
89
5
Body
90
6
Objective lens
91
7
Specimen
92
8
Condenser lens
93
9
Illuminator
