Lecture 3B - Microscopy and Staining Flashcards
1
Q
Preparation of Specimens for light microscopy
A
- Smear
- Fixing
- Staining
1
Q
- spread a thin film of material containing microorganisms over the slide surface
- allow to air dry
A
smear
2
Q
Smear: spread a __ __ of material containing microorganisms over the slide surface. Allow to __ __
A
- thin film
- air dry
3
Q
- process that kills microorganisms and attaches them to a microscope slide
- preserves and minimizes distortion of cells
A
fixing
4
Q
Fixing: process that __ microorganisms and __ them to a microscope slide. Fixing __ and __ distortion of cells
A
- kills
- attaches
- preserves
- minimizes
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
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
7
Q
coloring microorganisms with a dye that emphasizes certain structures
A
staining
8
Q
Before staining a sample, it must be __
A
fixed
9
Q
Stains are __ composed of __ __ and __ __
A
- salts
- positive ion (cation)
- negative ion (anion)
10
Q
the colored ion is called the __
A
chromophore
11
Q
Two types of dyes
A
- basic dyes
- acidic dyes
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
13
Q
examples of basic dyes
A
- crystal violet
- methylene blue
- safranin (red)
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
15
Q
stain the background
A
negative staining
16
Q
acidic dyes are used to observe the cell __, __ and __
A
- shape
- size
- capsules
17
Q
Example of acidic dyes
A
- eosin
- nigrosin
- India ink
18
Q
eosin
A
bright pink
19
Q
nigrosin
A
blue-black
20
Q
India ink
A
black
21
Q
Types of staining methods
A
- simple stains
- differential stains
- special stains
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
23
Q
purpose of simple stains
A
stain microorganisms to view cell shape and basic structures
24
compound used to hold down molecules of a stain onto a microorganism
mordant
25
simple stain procedure
1. stain is applied for certain time and washed off
2. dried and examined
26
- react differently to different types of bacteria
- can be sued to distinguish among different groups of bacteria
differential stains
27
Two important differential stains used
1. Gram stain
2. Acid-fast stain
28
When and who developed gram stain
1884 by Hans Gram
29
- most useful staining procedure in medical microbiology
- distinguishes bacteria of two large and medically important groups
gram stain
30
Two groups in gram stain
1. gram-positive bacteria
2. gram-negative bacteria
31
Four (4) steps of gram stain
1. primary stain
2. mordant
3. decolorizing
4. counterstain
32
(gram-stain)
- cover a heat fixed smear with a basic dye
- all cells are stained
primary stain
33
dye commonly used in primary stain
crystal violet
34
after smear is rinsed with water, an __ _ solution is applied
iodine mordant
35
(gram-stain) primary stain duration
1 min.
36
(gram-stain) mordant duration
1 min.
37
slide is washed with alcohol, which remove stain from gram-negative but not gram-positive
decolorizing
38
(gram-stain) decolorizing duration
rock back and forth, 12-15 sec.
39
in decolorizing,
- gram-negative cells will be __
- gram-positive cells will remain __
- decolorized
- purple
40
(gram-stain) alcohol is rinsed off, safranin applied which will stain cells that are decolorized
counterstain
41
(gram-stain) what dye is used in counterstaining
safranin
42
(gram-stain) counterstain duration
45 sec. (rinse & blot dry)
43
in counterstain,
- gram-negative cells are stained __
- gram-positive cells remain __
- pink
- purple
44
in gram stain, __ culture cells, at least less than __ __ old are used
- young
- 18-24 hours
45
- gram positive have very __ peptidoglycan cell walls
- gram negative have very __ cell walls
- thick (positive)
- thin (negative)
46
CV-I
crystal violet-iodine complex
47
why is the CV-I not easily removed from the gram-positive cells
due to thick cell wall
48
gram-positive cells with their very thick peptidoglycan cell walls are susceptible to __ and __
- penicillins
- cephalosporins
49
gram-negative cells with their thin cell walls and __ layer are __ to these antibiotics
- lipopolysaccharide
- resistant
50
stain irregularly, and there's a mix of pink- and purple-colored bacteria
gram-variable bacteria
51
acid-fast stain or __ stain
Ziehl-Neelsen stain
52
acid-fast stain is a modification method developed in __ by __
1882 by Paul Ehrlich
53
Paul Ehrlich used acid-fast stain to detect tuberculosis and leprosy causing organisms of the genus __ and pathogens of the genus __
- Mycobacterium
- Nocardia
54
Mycobacterium and Nocardia are bacteria that have __ cell walls, which makes them difficult to stain
waxy
55
Three (3) steps of acid-fast stain
1. primary stain
2. decolorizing
3. counterstain
56
(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
primary stain
57
(acid-fast stain) Primary stain: cover a heat fixed smear with __
carbolfuchsin (red basic dye)
58
(acid-fast stain) Carbolfuchsin steaming duration
5 min.
59
(acid-fast stain) slide is washed with acid-alcohol
decolorizing
60
In decolorizing
- non acid-fast cells will be __
- acid-fast cells will remain __
- decolorized (non acid-fast)
- red (acid-fast)
61
(acid-fast stain) decolorizing duration
1-2 min.
62
(acid-fast stain)
- acid-alcohol is rinsed off
- methylene blue is applied
counterstain
63
in counterstaining,
- non acid-fast cells are stained __
- acid-fast cells remain __
- blue
- red
64
(acid-fast stain) counterstain duration
30 sec - 1 min
65
used to color and isolate specific parts of microorganisms
special stains
66
special stains are used to color and isolate specific parts of microorganisms such as: __ __ __
1. endospore
2. capsules
3. flagella
67
endospores are extremely __, __ structures that are formed by some gram-positive bacteria
- resistant
- dormant
68
endospores are formed by gram-positive bacteria to protect them from __ __ __
harsh environment conditions
69
differential stain used to visualize bacterial endospores
endospore stain
70
most commonly used method in endospore staining
Schaeffer-Fulton Endospore Stain
71
Three (3) steps of Schaeffer-Fulton endospore stain
1. primary stain
2. wash
3. counterstain
72
(Schaeffer-Fulton Endospore Stain)
malachite green is applied to heat fixed smear and steamed for 5-7 minutes
primary stain
73
(Schaeffer-Fulton Endospore Stain)
what is applied in primary stain
malachite green
74
(Schaeffer-Fulton Endospore Stain)
primary stain duration
5-7 min.
75
(Schaeffer-Fulton Endospore Stain)
rinse with water for 30 sec.
wash
76
(Schaeffer-Fulton Endospore Stain)
wash duration
30 sec
77
(Schaeffer-Fulton Endospore Stain)
what is used to wash primary stain
water
78
(Schaeffer-Fulton Endospore Stain)
safranin will stain rest of cell
counterstain
79
(Schaeffer-Fulton Endospore Stain)
counterstain duration
30 sec.
80
(Schaeffer-Fulton Endospore Stain)
appearance of cell with endospore
pink cell with green endospore
81
gelatinous covers, which are important virulence (disease) factors
capsule
82
why are capsules difficult to stain
- repel most stains
- water soluble
- easily disrupted
83
(capsule stain)
used to obtain background
negative stain
84
(capsule stain)
cell is stained with __ __
basic dye
85
(capsule stain)
capsule appearance
- light halo around stained cell
- dark background
86
- organic polymers of microbial origin involved in bacterial cells' interactions with their environment
- comprised of polysaccharides, proteins, extracellular DNA (eDNA), and lipids
Extracellular polymeric substances (EPS)
87
used for locomotion that are too thin to be seen easily with light microscope
flagella
88
(flagella stain)
usually involves using __ and coating the flagellar structure with __ or __
- mordant
- dye or metal (e.g. silver)
89
(flagella stain)
the __ and __ of flagella can be used as diagnostic aids
- number
- arrangement
