Staining Flashcards
TYPES OF DYES
1. cationic with positive charged
- adheres to negative charged structures like ___ & ____
2. ionic with negative charged
- adheres to positive charged structures like ____, ____, ____
- Basic
- nucleic acids, proteins - Acidic
- mitochondria, collagen , secondary granules
NOTE: not performed on _____, ____, or _____ specimens as there are lot of normal flora on these sites
- to determine biologic activity of
microorganisms, including motility or reactions to certain chemicals or serologic reactivity in specific antisera - less distortion from the weight
of coverslip & deeper field focus into the drop can be achieved
throat, nasopharyngeal, or stool specimens
- Saline Wet Mount
- Hanging Drop Procedure
STAINING TECHNIQUES
Simple/ Direct Staining
1. example stains (3)
2. Inoculum size
Differential (Regressive)
1. example staining (2)
2. this will produce insoluble stain precipitation
3. most important step
Negative
1. demonstrates presence of ______
2. example stains (2)
3. appears ____ against dark bg as cell surface repels ____ stain
Simple/ Direct Staining
1. methylene blue, safranin, crystal violet
2. 10^5 CFU/mL
Differential (Regressive)
1. Gram & AFB Staining
2. mordant
3. decolorizing agent
Negative
1. diffuse capsules
2. India ink/ Nigrosine Dye
3. light-colored bodies; acidic stain precipitation
Principle of Gram Staining
- bacteria w/ thick peptidoglycan layer
- w/ ____ & ____
- _____ cross-links prevent decolorization in gram stain
- GRAM _______ - bacteria w/ thin peptidoglycan layer
- w/ ____,_____, & ______
- _____ cause increased permeability of lipid-rich cell wall and primary stain (crystal violet) washes out
- GRAM ______
- bacteria w/ thick peptidoglycan layer
- teichoic acid & lipoteichoic acid
- teichoic acid
- POSITIVE - bacteria w/ thin peptidoglycan layer
- proteins, phospholipids & lipopolysaccharides
- decolorizer
- NEGATIVE
Exceptions in Gram Staining
- exist almost exclusively within host cells
- organisms that lack cell
- high concentration of mycolic acid
- insufficient dimensions to be resolved by light
- chlamydia
- mycoplasma & ureaplasma
- mycobacterium
- spirochetes
General Rule in Gram Staining
1. all cocci are gram positive (purple) except: (3)
2. all bacilli are gram negative (pink) except: (8)
3. all spiral organisms are reported as
4. yeast are
5. mycoplasma/ureaplasma are
6. mycobacterium are
- NVB: Neisseria, Veilonella, Branhamella
- MEBR C2L2 = Mycobacterium, Erysipelothrix, Bacillus, Rothia, Clostridium, Corynebacterium, Listeria
- gram negative
- gram positive
- gram negative
- gram positive
Errors in Gram Staining
Gram positive -> Gram negative
1. ___ decolorization
2. use of ____ as mordant
3. these attacks the cell wall
4. omit ____
Gram negative -> Gram positive
1. ___ decolorization
2. ___ smear
Gram positive -> Gram negative
1. over decolorizing
2. acidic iodine
3. antibiotics
4. iodine
Gram negative -> Gram positive
1. under decolorizing
2. thick smear
Modification of Gram Staining
primary
1. Metachromatic granules
2. Hucker’s Modification
3. Burke’s Modification
decolorizer
1. Metachromatic granules
2. Hucker’s Modification
3. Burke’s Modification
primary
1. methyl violet- sodium bicarbonate
2. Crystal violet + ammonium oxalate
3. crystal violet
decolorizer
1. acetone
2. n/a
3. sodium bicarbonate + ether acetone
ACID-FAST
- separates species of _____ from other bacteria
- hydrophobic which renders the resistance of acid fast to decolorization
- acid fast organisms: Mycobacteria, ______ (partially), ____, ____, ______
- reagents used:
- primary stain
- mordant: _____ (ziehl); ____ (kinyoun)
- decolorizer
- counterstain
- Mycobacterium
- mycolic acid/ hydroxymethoxyl acid
- Mycobacteria, Nocardia (partially acid fast), Cryptosproridium, Isospora, Cyclospora
- reagents used:
- carbon fuchsin
- heat (ziehl-neelsen) or detergent (kinyoun’s method)
- acid alcohol
- methylene blue or malachite green
Ziehl-Neelsen Method (Hot)
1. carbol fuchsin conc
- time
2. mordant
3. decolorizer conc
4. counterstain time
Kinyoun’s Method (Cold)
1. carbol fuchsin conc
- time
2. mordant
3. decolorizer conc
4. counterstain time
Modified Kinyoun’s Method
1. carbol fuchsin conc
2. decolorizer conc
Ziehl-Neelsen Method (Hot)
1. 3% phenol
- 4-5mins
2. heat
3. 3% hydrochloric acid + 5% ethanol
4. 1min
Kinyoun’s Method (Cold)
1. 9% phenol
- 5mins
2. tergitol
3. 3% sulfuric acid + 95% ethanol
4. 1-3mins
Modified Kinyoun’s Method
1. 9% phenol
2. 1% sulfuric acid + 70% ethanol
acid bacilli color =
non AFB color
deep pink/ red
blue/ green
SPECIAL STAINS
1. Capsule
2. Cell wall
3. DNA
4. Endospore
5. Flagella
6. Metachromatic granules
- Welch stain
- Dyar stain
- Feulgen
- Schaeffer-Fulton
- Leifson stain
6.Albert stain, Burker’s Modification
