Chapter 10 Microorganisms Flashcards
1
Q
Bacteria
A
Single celled organisms widely distributed in nature
Each cell is a complete organism
Peptidoglycan cell walls (mucopolysaccharide)
2
Q
Cocci
A
Spherical or ovoid in shape
diplococci: pairs
staphylococci: grape-like clusters MRSA (methicillin resistant staphylococcus aureus)
Streptococci: chains (streptococcus pneumoniae)
3
Q
Bacilli
A
Rod shaped bacteria
Clostridia tetani, Clostridia botulinum, and Bacillus anthracis)
4
Q
Spirochetes
A
Spiral or corkscrew shaped bacteria Treponema pallidum (syphilis), Borrellia burgdorferi (Lyme disease)
5
Q
Mycobacteria
A
Do not form cell walls
rod shaped, sometimes exhibit fungus-like growth
large amounts of lipid in the cell wall
acid fast (because they resist acid decolorization)
Mycobacterium tuberculosis, and leprae
6
Q
Fungi
A
Unicellular or multi-cellular primitive “plants” that have a distinct membrane bound nucleus containing genetic material
Filamentous or molds (Aspergillus fumigatus)
Yeasts (Cryptococcus neoformans)
Yeast-like (Candida albicans)
7
Q
Viruses
A
Made of DNA/RNA and a protein coat that infect living cells and hijack cellular machinery to replicate
8
Q
Yeasts
A
single round or oval cells that reproduce by budding
Cryptococcus neoformans
9
Q
Protozoans
A
Single-celled microorganisms that are functionally complex structures
Classified by type of locomotion
Amebae via pseudopodia (Entamoeba histolytica)
Others via cilia or flagella (Giardia lamblia, Toxoplasma gondii)
10
Q
Kinyoun AFB Purpose
A
Detect acid-fast mycobacteria
11
Q
Kinyoun AFB Principle
A
Mycobacteria are not easily Gram stained because their cells walls are not permeable with basic fuchsin due to high lipid content
Alcoholic acid solutions are used to give a more consistent stain decolorization
Staining is enhanced by both phenol and alcohol in the carbofuchsin solution
12
Q
Kinyoun AFB Preferred Fixative
A
10% NBF, NOT Carnoy which will overdifferentiate slides due to alcohol acetic acid, and chloroform
Control: uterus
13
Q
Kinyoun AFB Basic Procedure
A
- Deparaffinize, hydrate. Remove mercury precipitate with Iodine and hypo solutions if necessary (if mercury fixative used)
- Stain in Kinyoun carbol-fuchsin for 1 hour (stain can be re-used)
- Wash in running tap water
- Differentiate in 1% acid alcohol until tissue is pale pink
- Wash in running tap water
- Counterstain in working methylene blue solution until sky blue (DON’T overstain)
- Rinse in tap water
- Dehydrate, clear, coverslip
14
Q
Kinyoun AFB Results
A
Acid-fast bacteria: bright red
Background: light blue
15
Q
Kinyoun AFB Technical Notes
A
Don’t use tap water which can contain acid-fast organisms
Do no overcounterstain or it will mask microorganisms
Must wash acid out of tissue before counterstaining or it won’t work
16
Q
Ziehl Neelson AFB Purpose
A
Detection of acid-fast bacteria
17
Q
Ziehl Neelson AFB Principle
A
Mycobacteria are not easily Gram stained because their cells walls are not permeable with basic fuchsin due to high lipid content
Alcoholic acid solutions are used to give a more consistent stain decolorization
Staining is enhanced by both phenol and alcohol in the carbofuchsin solution
18
Q
Ziehl Neelson AFB Preferred Fixative
A
Any but Carnoy (because of alcohol and acetic acid content that causes overdifferentiation)
Control: tissue containing acid-fast organisms
19
Q
Ziehl Neelson AFB Basic Procedure
A
- Deparaffinize, hydrate.
- Stain with freshly filtered carbol-fuchsin
- Wash in running tap water
- Decolorize in 1% acid alcohol until tissue is pale pink
- Wash in running tap water
- Counterstain in working methylene blue solution until sky blue (DON’T overstain)
- Rinse in tap water
- Dehydrate, clear, coverslip
20
Q
Ziehl Neelson AFB Results
A
Acid-fast bacteria: bright red
Background: light blue
21
Q
Ziehl Neelson AFB Technical Notes
A
Don’t use tap water which can contain acid-fast organisms
Do no overcounterstain or it will mask microorganisms
Must wash acid out of tissue before counterstaining or it won’t work
22
Q
Fite Purpose
A
Detection of Mycobacterium leprae
23
Q
Fite Principle
A
Lipoid capsule of the organism takes up carbol-fuchsin and resists decolorization with dilute mineral acid
24
Q
Fite Preferred Fixative
A
10% NBF, NO CARNOY
Control: tissue containing leprosy organisms
25
Fite Basic Procedure
1. Deparaffinize with Xylene-peanut oil mixture
2. Drain sections, wipe off excess oil, and blot to opacity. Residual oil helps prevent shrinkage and injury to the sections
3. Stain in Ziehl Neelsen carbol-fuchsin
4. Wash in water
5. Differentiate in 1% acid alcohol
6. Wash in water
7. Counterstain with methylene blue
8. Rinse in water
9. Blot sections and let air dry completely
10. Mount with synthetic resin such as Permount (Do not use alcohol and xylene!)
26
Fite Results
M. leprae and other acid-fast bacteria: bright red
| Background: light blue
27
Fite Technical Notes
To demonstrate Nocardia species: stain in carbol-fuchsin for 10 minutes (time is critical), Decolorize in sulfuric acid to remove background color (also uses a weaker acid alcohol for differentiation)
Alcohol must be avoided because leprae and Nocardia are weakly acid-fast and not alcohol-fast
Acid-fastness of leprosy is enhanced when the waxy capsule is protected by a mixture of peanut oil and xylene and also by avoiding dehydrating solutions (alcohol)
28
Auramine-Rhodamine Purpose
Detection of Mycobacterium tuberculosis and other acid-fast organisms
29
Auramine-Rhodamine Principle
Mycolic acid in cell walls has an affinity for the fluorochromes auramine and rhodamine. These dyes bind to mycobacteria and fluoresce. Both are basic dyes and fluoresce at short wavelengths. Stain better together than individually
30
Auramine-Rhodamine Preferred Fixative
10% NBF
| Control: tissue containing acid-fast bacteria
31
Auramine-Rhodamine Basic Procedure
1. Deparaffinize, hydrate
2. place slides in stain solution and microwave
3. Rinse in distilled water
4. Differentiate in acid alcohol
5. Rinse in distilled water
6. Stain in eriochrome Black T (quench?)
7. rinse in water
8. Stain slides on end and let air dry
9. Dip in xylene and coverslip
10. Examine with fluorescent microscope
32
Auramine-Rhodamine Results
Acid-fast organisms: reddish-yellow
| Background: black
33
Auramine-Rhodamine Technical Notes
Extremely sensitive method that is highly specific for mycobacteria, but there is an increased chance of false positives
Slides can be re-stained with carbol-fuchsin for confirmation if the results are questionable, but carbol fuchsin stains can't be followed by auramine-rhodamine
Au-Rho is more likely to stain dead and dying organisms than carbol fuchsin
34
Brown-Hopps Gram Stain Purpose
Demonstrate gram + and gram - bacteria in tissue
35
Brown-Hopps Gram Stain Principle
1. Crystal violet stain followed by iodine mordant to form lake (both + and - organisms are stained) + peptidoglycan walls are thicker than -
2. The large crystal violet-iodine complex only washes out of gram - because the thinner wall is easily disrupted by alcohol or acetone. If a gram + cell wall is disrupted it will stain gram -
3. After decolorization counterstain is applied to gram - organisms
36
Brown-Hopps Gram Stain Preferred Fixative
10% NBF
| Control: sections with both gram + and - bacteria
37
Brown-Hopps Gram Stain Basic Procedure
1. Deparaffinize, hydrate
2. Stain with crystal violet
3. Rinse in distilled water
4. Stain slides with Gram Iodine
5. Rinse slides in distilled water to remove excess iodine
6. blot one side at a time with slightly damp filter paper and decolorize in acetone
7. Rinse slides in water
8. Stain with working basic fuchsin
9. Rinse in distilled water
10. Differentiate with Gallego solution (formalin+glacial acetic acid)
11. Rinse in distilled water and blot sections, but do not blot to dryness
12. Quickly dip slides in acetone
13. Quickly dip slides in periodic-acid acetone
14. quickly dip slides in acetone
15. Pass slides through acetone-xylene mix 1:2 then clear with xylene
16. Coverslip
38
Brown-Hopps Gram Stain Results
Gram positive bacteria: blue
Gram negative bacteria: red
Background tissue: yellow
Nuclei: light red
39
Brown-Hopps Gram Stain Technical Notes
Modification is preferred method for gram - and rikettsia, while original Brown and Brenn is preferred for gram +
Decolorization works better if picric-acid acetone is near anhydrous, but don't drop below 10% water because of explosiveness
Do not let sections dry or insoluble residue will form
Gram + will not stain correctly if patient is on antibiotics
40
McDonald's Gram Stain Purpose
Demonstrate gram + and gram - bacteria in tissue
41
McDonald's Gram Stain Principle
1. Gentian violet stain followed by iodine mordant to form lake (both + and - organisms are stained) + peptidoglycan walls are thicker than -
2. The large gentian violet-iodine complex only washes out of gram - because the thinner wall is easily disrupted by alcohol or acetone. If a gram + cell wall is disrupted it will stain gram -
3. After decolorization with Gram's decolorizer counterstain (Tartrazine) is applied to gram - organisms
42
McDonald's Gram Stain Preferred Fixative
10% NBF
| Control: sections with both gram + and - bacteria
43
McDonald's Gram Stain Basic Procedure
1. Deparaffinize, hydrate
2. Rinse in distilled water
3. Flood slides with Gentian Violet (stain)
4. Rinse with tap water
5. Flood with universal iodine
6. Rinse in running tap water
7. Decolorize in Gram's Decolorizer
8. Quickly rinse in running tap water
9. Counterstain in Tartrazine
10. Dehydrate, clear, coverslip
44
McDonald's Gram Stain Results
Gram +: blue
Gram -: Red
Other tissue: Yellow
Nuclei: Red
45
McDonald's Gram Stain Technical Notes
Do not let sections dry or insoluble residue will form
Gram + will not stain correctly if patient is on antibiotics
46
Diff-Quik Giemsa Purpose
Identify Heliobacter pylori (gram - in the stomach)
47
Diff-Quik Giemsa Principle
Romanowsky stain combines basic dye methylene blue with acid dye eosin to give a wide color range wen staining tissue and blood smears
48
Diff-Quik Giemsa Preferred Fixative
10% NBF
| Control: Sections containing H. pylori
49
Diff-Quik Giemsa Basic Procedure
1. Deparaffinize, hydrate
2. Dip in Diff-Quik solution 1
3. Dip in Diff-Quik solution 2
4. Rinse in distilled water
5. Differentiate in changes of acetic water
6. Rinse in distilled water. Check microscopically. H. pylori and nuclei should be dark blue, cytoplasm should be pink. Repeat staining if needed
7. Dehydrate, clear, coverslip
50
Diff-Quik Giemsa Results
H. pylori: dark blue
Other bacteria: blue
Nuclei: dark blue
Cytoplasm: pink
51
Diff-Quik Giemsa Technical Notes
Solution 1 is buffered Eosin Y for pink cytoplasm
Solution 2 is cationic dye mixture of azure A and methylene blue which stains nuclei and bacteria blue
H. pylori are associated with gastritis and peptic ulcer disease, also a risk factor for gastric carcinoma and lymphoma
52
Alcian-yellow Toluidine-blue Purpose
Detection of H. pylori
53
Alcian-yellow Toluidine-blue Principle
Alcian yellow acts similarly to alcian blue, staining mucin yellow Toluidine blue is a basic ye that stains the H. pylori organisms and nuclei blue
54
Alcian-yellow Toluidine-blue Preferred Fixative
10% NBF
| Control: Sections containing H. pylori
55
Alcian-yellow Toluidine-blue Basic Procedure
1. Deparaffinize, hydrate
2. Oxidize sections in 1% periodic acid
3. Wash ell with water
4. Place solutions in sodium metabisulfite
5. Wash in water
6. Stain with alcian yellow
7. Wash with water
8. Stain with Toludine blue
9. Wash with water
10. Blot sections dry
11. Dehydrate, clear, coverslip
56
Alcian-yellow Toluidine-blue Results
H. pylori: blue
Mucin: yellow
Background: pale blue
57
Hotchkiss McManus PAS Purpose
To demonstrate fungi
58
Hotchkiss McManus PAS Principle
Polysaccharides present in the fungal cells are oxidized y the periodic acid to aldehydes which are then demonstrated when they react with Schiff reagent to yield rose colored fungi
59
Hotchkiss McManus PAS Preferred Fixative
10% NBF, Bouin, or Zenker
| Control: tissue containing fungi
60
Hotchkiss McManus PAS Basic Procedure
1. Deparaffinize, hydrate
2. Place sections in % periodic acid solution
3. Wash in distilled water
4. Place in Schiff reagent
5. Rinse in sulfurous acid rinse
6. Wash in running water to develop full color
7. Counterstain in fast green
8. Rinse
9. Dehydrate, clear, coverslip
61
Hotchkiss McManus PAS Results
Fungi: rose
Background: green
62
Hotchkiss McManus PAS Technical Notes
Green counterstain provides better contrast than hematoxylin without masking organisms
Diastase digestions are useful on samples that contain glycogen, esp liver
Fresh periodic acid is necessary to obtain good stain results
63
Chromic Acid Schiff Purpose
Identification of fungi
64
Chromic Acid Schiff Principle
Chromic acid oxidizes carbohydrates present in fungal cell walls to aldehydes.
Chromic acid is a strong oxidizer and will eliminate reactive aldehydes except in structures with the greatest concentration of carbohydrates such as: mucin, glycogen, and fungal cell walls.
This stain has less non-specific background compared to PAS
65
Chromic Acid Schiff Preferred Fixative
10% NBF
66
Chromic Acid Schiff Basic Procedure
1. Deparaffinize, hydrate, wash thoroughly
2. oxidize in 5% chromic acid
3. Wash in deionized water
4. Stain in Schiff reagent
5. Sulfurous acid rinse to remove leucofuchsin
6. Wash in running tap water to develop full color
7. Counterstain in Harris Hematoxylin or fast green
8. Wash well, if using hematoxylin blue briefly in ammonia water and rewash
9. Dehydrate, clear, coverslip
67
Chromic Acid Schiff Results
Fungi: deep rose to purple
Nuclei (if hematoxylin is used): blue
Background (if fast green is used): green
68
Chromic Acid Schiff Technical Notes
Make sure Schiff reagent is room temperature before starting
Carefully control time of oxidation step. Too short results in background stain, too long and fungal staining will be poor
Rinse thoroughly after re-hydrating in alcohol to make sure chromic acid hasn't darkened due to reduction with alcohol from the re-hydration step
Sulfurous acid rinse removes unbound leucofuchsin after Schiff reaction
Chromic acid is highly toxic and carcinogenic
69
Gridley for Fungus Purpose
Demonstrate fungi
70
Gridley for Fungus Principle
Chromic acid oxidized glycol groups to aldehydes which then react with Schiff reagent. Reaction is less intense due to strength or chromic acid but background is also reduced.
Aldehyde fuchsin reinforces depth of staining by interacting with the Schiff reagent
71
Gridley for Fungus Preferred Fixative
10% NBF
72
Gridley for Fungus Basic Procedure
1. Deparaffinize, hydrate
2. Oxidize in chromic acid
3. wash in running water
4. Stain in Schiff reagent
5. Wash in running water
6. Rinse in 70% alcohol
7. Stain in aldehyde fuchsin
8. Rinse off excess stain with 95% alcohol
9. Rinse in distilled water
10. Counterstain in metanil yellow
11. Rinse in distilled water
12. Dehydrate, clear, coverslip
73
Gridley for Fungus Results
Mycelia: deep purple
Conidia: deep rose to purple
Background: yellow
Elastic fibers and mucin: deep purple
74
Gridley for Fungus Technical Notes
Old, nonviable fungi and not well stained with this technique or GMS
Pararosaniline, not basic fuchsin, should be used to prepare the aldehyde fuchsin
Sulfurous acid rinse may be used after the Schiff reagent to remove leucofuchsin
75
Grocott Methenamine Silver Purpose
Demonstrate fungi
76
Grocott Methenamine Silver Principle
Chromic acid oxidizes polysaccharides in fungal cell walls to aldehydes. It is a strong oxidizer that breaks down aldehydes so only substances with a high aldehyde concentration will be demonstrated, such as glycogen, mucin, and fungal cell walls, while basement membranes and collagen will not be shown.
Silver nitrate is deposited and reduced to visible metallic silver by methenamine silver, then toned with Gold Chloride
Sodium Thiosulfate removes unreduced silver
77
Grocott Methenamine Silver Preferred Fixative
10% NBF
| QC: section containing fungi
78
Grocott Methenamine Silver Basic Procedure
1. Deparaffinize, hydrate
2. oxidize in chromic acid 1 hour at room temp
3. Wash in running tap water
4. Rinse in 1% sodium bisulfite to remove any residual chromic acid
5. Wash in tap water
6. Wash in distilled water
7. Place slides in preheated methenamine silver solution until sections turn yellowish brown. Fungi should be dark brown at this stage
8. Rinse in distilled water
9. Tone in Gold Chloride
10. Rinse in distilled water
11. Place in sodium thiosulfate to remove any unreduced silver
12.
13.
14.
79
Grocott Methenamine Silver Results
Fungi: cell walls should be crisp black with visible internal structures
Mucin: taupe to dark grey
Background: green
80
Grocott Methenamine Silver Technical Notes
.
81
Warthin Starry Purpose
Demonstrate spirochetes
82
Warthin Starry Principle
Argyrophil method of silver staining, uses hydroquinone as a reducer/developer to reduce silver to its visible metallic form
83
Warthin Starry Preferred Fixative
10% NBF
| Control: must contain spirochetes
84
Warthin Starry Basic Procedure
1. Heat silver nitrate, gelatin, and hydroquinone in separate tubes (developer components)
2. Place in a graduated cylinder and chemically clean coplin jar in oven for at least an hour
3. Deparaffinize, hydrate to acidulated water (slightly acidic)
4. Place in silver nitrate impregnating solution, Do not preheat
5. Prepare the developer
6. Put slides in developer, check frequently
7. Wash slides in distilled water
8. Dehydrate, clear coverslip
85
Warthin Starry Results
Spirochetes: black
Other bacteria: black
Background: pl yellow to light brown
86
Warthin Starry Technical Notes
Overstained sections can be decolorized with iodine and sodium thiosulfate and then restained
All bacteria are non-selectively demonstrated by silver impregnation. This method is especially good for small, weakly gram negative bacteria, and small numbers of bacteria compared to gram staining
Reducing substances, such a formalin pigment, aill also give a positive reaction. This can be removed by treating with alcoholic picric acid or alkaline alcohol
87
Steiner and Steiner Purpose
Demonstrate Spirochetes, H pylori, or Legionella
88
Steiner and Steiner Principle
Spirochetes, H pylori, and Legionella are argyrophilic and need a reducing agent to absorb silver from a silver solution. Hydroquinone is used as the reducing agent or developer in this protocol
89
Steiner and Steiner Preferred Fixative
10% NBF, avoid mercurial and chromate fixatives which causes non-specific silver deposition
90
Steiner and Steiner Basic Procedure
1. Deparaffinize, hydrate
2. Sensitize sections in uranyl nitrate and then heat to just below the melting point. Transfer to distilled water
3. Rinse in distilled water until the possibility of cross contamination is eliminated
4. Place in silver nitrate and heat
5. Rinse in distilled water
6. Rinse in 95% alcohol
7. Rinse in 100% alcohol
8. Place slides in gum mastic
9. Air dry
10. Rinse in distilled water
11. Place in reducing solution until sections have developed with black spirochetes and a light yellow background
12. Rinse in distilled water to stop reduction
13. Dehydrate, clear, coverslip
91
Steiner and Steiner Results
```
Spirochetes: dark brown to black
H. pylori: dark brown to black
L. pneumophilia: dark brown to black
other nonfillamentous bacteria: dark brown to black
Background: light yellow
```
92
Steiner and Steiner Technical Notes
Removal of calcium and formalin pigment is essential when only a few microorganisms are present
Some xylene substitutes will cause rapid fading of silver stained microorganisms
93
Dieterle Purpose
Demonstrate spirochetes or causative organisms of legionellosis (legionaries disease)
94
Dieterle Principle
Spirochetes are argyrophilic and can be demonstrated with Silver staining and a developer/reducer (hydroquinone)
95
Dieterle Preferred Fixative
10% NBF
| Control: tissue with spirochetes
96
Dieterle Basic Procedure
1. Preheat alcoholic uranyl nitrate (sensitizer) and the silver nitrate solution
2. Deparaffinize, hydrate
3. Place in preheated alcoholic uranyl nitrate in an oven
4. Dip in distilled water
5. Dip in 95% alcohol
6. Place in 10% alcoholic gum mastic
7. Dip in 95% alcohol
8. place in distilled water, ten allow slides to drain until almost dry. Can be left overnight if needed
9. Place in preheated silver nitrate in an oven in the dark
10. Dip in distilled water
11. Place in developer (hydroquinone) and dip until sections are tan to gold
12. Dip in distilled water
13. Place in formic acid
14. Dip in distilled water
15. Dip in 95% alcohol
16. Dip in acetone
17. Clear in xylene, and coverslip
97
Dieterle Results
Spirochetes, bacteria: brown to black
| Background: pale yellow or tan
98
What makes acid fast organisms unique?
they contain a large amount of lipid in their cell walls which resists Gram staining. They are called acid-fast because they resist decolorization by dilute mineral acids
99
What are all the shapes of bacteria?
coccus (round), bacillus (rods), spirochetes (corkscrew)
100
what is mycosis?
A disease produced by fungi
101
what is budding?
Method of division for yeasts:
A protuberance is formed on the outer surface of the parent cell and the nucleus of the parent cell divides. The nucleus migrates to the bud, cell wall material is laid down between the parent cell and the bud, then the bud breaks away from the parent cell.
102
what are pseudoyphae?
Filamentous structures of Yeast-like fungi that reproduce by budding, but the buds do not detach from the parent cell and instead elongate into the filamentous pseudohyphae
Ex: Candida albicans
103
What are dimorphic fungi?
Have different morphology dependent on temperature
at 37C they have a yeast-like morphology, but when grown on artificial media at 25C they have a filamentous morphology
Ex: Blastomyces dermatitidis, Coccidoides immitis, and Histoplasma capsulatum
