Staining procedure Flashcards
CLASSES OF IONIZABLE DYES:
Basic Dye
- commonly used
- CATIONIC : positively charged groups (pentavalent nitrogen) bind to nucleic acid and proteins *remember: bacteria’s cell wall is negatively charged (LPS and teichoic acid)
- Methylene blue, Basic fuchsin, Crystal violet, Safranin and Malachite Green
CLASSES OF IONIZABLE DYES:
Acid Dye
- ANIONIC : negatively charged groups (carboxyls and phenolic)
- Eosin, Rose bengal, Acid fuchsin
- single stain is used
- directed towards colouring the forms and shapes present
- Malachite green, Methylene blue, Crystal violet, Carbolfuchsin, Safranin
STAINING TECHNIQUE:
SImple staining
- divides bacteria into separate groups
- GS, AFBS
- 10^5 organisms/mL is necessary to observe one organism/ OIO
STAINING TECHNIQUE:
Differential Staining
- Directed specifically at an organism identification
- Specific ID of selected pathogens (Chlamydia trachomatis, Bordetella pertussis, Legionella pneumophilia, HSV, varizella-zoster virus, CMV, adenovirus and respiratory virus)
STAINING TECHNIQUE:
Diagnostic Antibody or DNA probe-mediated staining
Chlamydia, Rickettsia, Bordetella, Legionella
Intracellular
- Demonstrate the presence of diffuse capsule
- cell wall repels the acidic stain-kasi nga negatively charged siya di ba
- Excellent: GAS VACOULES and VIRAL morphology
- India ink/ Nigrosin dye (acidic stain)
STAINING TECHNIQUE:
Negative staining
Responsible for retaining crystal violet-iodine complex
TEICHOIC ACID
Why do G+ become G-?
1 Removal of MgRNA
2 Aging, dying and autolyzing cells (G variable)
3 Using acidic iodine : G+ will lose affinity with an acidic iodine
4 Technical error
Exceptions in GS
-organisms that exist almost exclusively within host cell (chlamydia)
-lack cell wall (mycoplasma and ureaplasma : sterols yung moron siya)
-have insufficient dimensions to be resolved by light microscopy (Spirochetes)
“CHLAMYDIA, MYCOPLASMA, UREAPLASMA, SPIROCHETES”
primary stain; binds to mycelia acid
STAINING TECHNIQUE:
Acid Fast Stain
HOT METHOD AFB
ACID FAST STAINING METHOD:
Ziehl-Neelsen (HOT)
COLD METHOD AFB
ACID FAST STAINING METHOD:
Kinyoun’s
AFS differentiates M. smegmatis from M. tuberculosis
ACID FAST STAINING METHOD:
Pappenheim’s method
AFS differentiates M. leopard from M. tuberculosis
ACID FAST STAINING METHOD:
Baumgarten’s Method
ACID FAST STAIN +
Bacilli: Mycobacteria
Other: Nocardia, Rhodococcus, Legionella, Cryptosporidia, Isospora
Ways to facilitate AFS Rxns
1 Steaming: temporarily remove mycelia acid
2 Increase [dye and phenol]
-ZN: 3g Carbolfuchsin + 5% phenol
-K: 4g Carbolfuchsin + 9% phenol
3 Prolonging contact with primary stain
4 Addition of wetting agent : TERGITOL
ACID FAST : cells are resistant to decolorisation due to…
Mycolic Acid
ACID FASTNESS IS AFFECTED BY…
colonial age, medium for growth, ultraviolet light
Stain for CELL WALL
SPECIAL STAINS:
Dyar stain
Stain for CAPSULE
SPECIAL STAINS:
Hiss, Gin, Anthony and Welch
Stain for METACHROMATIC GRANULES
SPECIAL STAINS:
Lamb, Neisser, Albert, Ljubinsky
Stain for ENDOSPORE
SPECIAL STAINS:
Dorner, Wirtz and Conklin, Schaeffer-Fulton
Stain for FLAGELLA
SPECIAL STAINS:
Gray, Leifson, Fisher and Conn
Stain for YEAST CELLS (capsule)
SPECIAL STAINS:
India Ink/Borris method and Nigrossin
Stain for DNA
SPECIAL STAINS:
Feulgen Stain
Stain for Spirochetes
SPECIAL STAINS:
Fontana Tribondeau and Levadite Silver Impregnation
