Lab test 2 Flashcards
(80 cards)
1
Q
General uses for simple and negative stains
A
- Provides contrast for tiny colorless bacteiral cells
- Used to identify cell morphology (shape) and arrangement
2
Q
What is a negative stain great for
A
Cell morphology
3
Q
What is stained in negative staining
A
The background (not the cells themselves)
4
Q
Benefit of negative staining
A
No heat fixing, cell is not distorted in any way
5
Q
What is the stain for negative staining
A
Nigrosin, india ink
6
Q
Negative stain steps
A
- Place drop of nigrosin near edge of slide
- Aseptically transfer loopful of culture into dye
- Using another slide, spread out mixture into thin film
- Allow time to airdry completely
7
Q
Purpose of simple stain
A
Idnetify cell shape arrangement and size
Provides needed contrast
8
Q
How many stains in simple stain?
A
One
9
Q
Heat fixing in simple stain?
A
Yes
10
Q
Is the stain basic or acidic
A
Basic
11
Q
Charge of acids and bases
A
Acid –> -
Basic –> +
12
Q
Charge of cell
A
Negative
13
Q
Why are cells stained with positive dye
A
It is attracted to the negative cell
14
Q
Smears
A
Loopful of culture is air dried and then fixed by heat
15
Q
How to make a smear using an culture from solid media
A
Add a drop of water to the slide first
16
Q
Simple stain procedure
A
- Saturate the stain with basic dye for 1 minute
- Rinse the slide gently with water
- Blot dry with bibulous paper
- Observe slide under the microscope
17
Q
Stains that can be used in simple stain
A
Crystal violet, safranin, or methyline blue
18
Q
Smear too thick
A
Difficult to see individual cells
19
Q
Smear too thin
A
May not find any organisms
20
Q
Too much stirring of smear
A
Disrupt cell arrangement
21
Q
Importance of heat fixing
A
- Kills organism
- Adheres organism to the slide
- Increases stain penetration
22
Q
Too much heat fixing
A
Distorts cell shape
23
Q
Who developed gram stain
A
Hans Christian Gram
24
Q
Year gram stain was created
A
1884
25
Significance of gram stain
Fast and inexpensive way to distinguish between bacteria based on their cell wall structure
26
More virulent
Gram negative
27
Gram positive cell wall
Peptidoglycan, protein, phospholipid
28
Gram negative cell wall
Lipoprotein, peptidoglycan, phospholipid `
29
Differential stain
Allows observer to distinguish between two large groups of bacteria based on staining differences
30
Gram stain steps
1. CV
2. Iodine
3. Alcohol wash
4. Safranin
31
Gram negative color
Pink
32
Gram positive color
Purple
33
Limitations of gram stain
1. Staining and decolorizing are time sensitive
2. Must be used on cells less than 48 hours old
3. Does not necessarily indicate phylogenic relationships
34
Stage
Fixed platform with an opening in the center that allows light from an illuminating source below to the lens system above the stage
35
Mechanical stage
Can be moved horizontally or vertically
36
Abbé condenser
Found directly under the stage. Made of two lenses thatt collect and concentrate light as it passes from the light source to the lens system
37
Iris diaphragm
Shutter controlled by a lever that is used to regulate the amount of light controlled by the lens system
38
Body tube
Houses the lens system
39
Resolving power
How far apart two adjacent objects must be before a given lens shows them as discrete entities
40
Resolving power formula
wavelength of light/2x numerical apeture
41
Refractive index
Bending power of light through the air from the glass slide to the objective lens
42
Condensor knob
Adjusts height of the condensor
43
Why not use a lot of light
It may obscure the specimen because there isnt a lot of contrast
44
Magnification of lens increases, distance between objective lens and slide ____, numerical apeture _____
Decrease
| Increase
45
Working distance
Distance between objective lens and slide
46
How to get oil off lenses
Lens paper
47
True or false. Lower the body tube while looking through the ocular lens
False
48
Parfocal
When one lens is in focus, other lenses will have the same focal length and can be rotated into position without further major adjustment
49
How to focus with oil immersion
Fine adjustment only
50
How to remove oil from the stage
Xylol
51
What does a good smear look like
Thin whiteish layer on slide when dry
52
Heat fix for smear?
Yes
53
Why are thick or dense smears less likely to produce a good smear evalutation
Light cannot easily pass through dense smears
54
Why should you be careful not to overheat the smear during the heat fixing process
It can distort cell shape
55
Cocci
Sphere
56
Bacilli
Rod
57
Why does the negative stain not penetrate cells
It is acidic
58
Why cant methylene blue be used for negative staining?
It is basic and will penetrate the cell
59
How many chemical reagants are needed in differential staining
At least 4
60
First reagant
Primary stain
61
Primary stain function
Impart color on all the cells
62
Secondary stain function
Mordant. Intensifies the color of the primary stain
63
Third reagant
Decolorizing agent that may or may not remove the primary stain from some cells
64
What determines if the counter stain is absorbed
If the cell is decolorized, it will absorb. If not, it will not
65
Which cells have a thicker peptidoglycan layer
Gram positive
66
What forms when iodine binds to crystal violet
CV-I complex, appears purple black
67
What does the alcohol do to gram negative cells
Increases the porosity of the cell wall by dissolving lipids. CV-I complex is more easily removed
68
What does the alcohol do the the gram positive cells
Pores become smaller. Retains CV-I
69
Over decolorization
Stain will also be removed from positive cells and they will appear gram negative
70
Under decolorization
Stain will not fully be removed and gram positive cells can appear gram negative
71
Why must young cultures be used for gram stain
Old cultures lose their ability to retain the primary stain and will appear gram variable
72
Why shouldnt the body tube be lowered when looking through the ocular lens
Eliminate the change of the slide hitting the lens
73
Why adjust the iris diaphragm
Regulate the amount of light
74
Why adjust Coarse adjustment knob
Focusing/moving stage
75
Why adjust Fine adjustment knob
Sharpness of focus
76
Why adjust condensor
Focuses light onto specimen
77
Why adjust mechanical stage control
Locate the specimen
78
Inability to bring into sharp focus
Make object sharp under 40x first. Too little or too much oil can cause this
79
Insufficient light while viewing specimen
Adjust diaphragm
80
Artifacts in microscopic field
Clean slide with lens paper
