STAINING TECHNIQUES Flashcards
-> organic or aqueous preparations of dyes that impart a variety of color to tissue or microorganisms
• Differentiate or identify structures and characteristics of bacteria
Dyes or stains
- ion that imparts the color
Chromophore
The chromophore is a positive ion -›____
• methylene blue, crystal violet, carbol fuchsin, malachite green, safranin
BASIC STAIN
- The chromophore is a negative ion -› _______
eosin, nigrosin, picric acid
ACIDIC STAIN
• -> ion not giving off the color
Counterion
• directly stains bacteria and bacterial structure
bacteria absorbs the stain
DIRECT STAINING
• No special structures are seen
Stain evenly dyes the bacteria
The stain is flooded across the smear
Simple Staining
• Only one type of dye is used
• No special structure of a cell is distinguished
• eg. methylene blue
Simple Staining
• Can visualize bacterial structures
• Can isolate specific parts of bacterial structure
Selective/Special Staining
-> Not easily colorized, not easily decolorized
-> Impenetrable
Endospore Stain or Schaeffer-Fulton stain
Differential Staining
• Two Types:
Gram Stain
Acid Fast Stain
-> The first stain used in the procedure
-> A stain that will impart the color
Initial/Primary Stain
•
-> allow stains to stick to the bacteria
- for a bridge between cell and stain
Mordant
-› no chemical eg. heat or cold
-› ferrous sulfate, iodine, tannic acid
Physical Mordant
Chemical Mordant
-> Removes color
-> Bacteria in question retain the color
-> Bacteria without the specific structure or composition in them will not retain the color
Decolorizer
- Bacteria that were decolorized will absorb the color
Counterstain/Secondary Stain
• Negative/Relief Staining
• The background is stained instead for contrast
- Demonstrate the HALO EFFECT
• DRY INDIA INK/NIGROSINE STAIN
INDIRECT STAINING
TYPES OF IONIZABLE DYES USED IN STAINING BACTERIA
Basic dyes
Acidic dyes
▪ Commonly used in bacteriology
▪ These are cationic dyes with positively charged groups that adhere to negatively charged molecules.
BASIC DYES
positive
Basic dyes
Includes:
METHYLENE BLUE
MALACHITE GREEN
BASIC FUCHSIN
CRYSTAL VIOLET
SAFRANIN
▪ These are anionic dyes with negatively charged group that bind to positively charged cell structures
ACIDIC DYES
ACIDIC DYES
EOSIN
ACID FUCHSIN
ROSE BENGAL
▪ Example: METHYLENE BLUE, IODINE
SIMPLE STAINING
▪ Directed towards the forms and shapes of the cells
SIMPLE STAINING
▪ Basic dyes have greater affinity for nuclei; Acid dyes have greater affinity for cytoplasm
SIMPLE STAINING
▪ Application of single staining solution
SIMPLE STAINING
▪ Example: GRAM STAINING, ACID FAST STAINING
DIFFERENTIAL STAINING
▪ Makes use of primary stain, mordant, decolorizer and counterstain
DIFFERENTIAL STAINING
▪ Application of 2 or more stains
DIFFERENTIAL STAINING
▪ Example: INDIA INK, NIGROSIN STAIN
▪ NEGATIVE STAINING
▪ Results the bacteria appearing as light colored bodies against the dark background
▪ NEGATIVE STAINING
GRAM STAINING
▪ Named after________ (Danish Scientist), worked with______ in City hospital morgue in Berlin.
HANS CHRISTIAN GRAM
CARL FRIEDLANDER
▪ PRINCIPAL STAIN in the laboratory for microscopic examination of bacteria
Gram stain
▪ Divides bacteria into two large groups: GRAM POSITIVE and GRAM NEGATIVE.
Gram stain
Gram staining
PRINCIPLE:
Based on the differences in composition between gram positive cell walls which contain thick_____ with NUMEROUS_______ and gram negative cell walls which consist of______.
peptidoglycan; TEICHOICE ACID CROSS-LINKAGES
THINNER LAYER OF PEPTIDOGLYCAN
If the______ is rinsed too vigorously prior to the application of iodine, it will not be retained and will leave the gram negative bacteria unstained
crystal violet dye
If the decolorization is prolonged, the ———-will be removed and the gram positive bacteria will not be stained.
Gram – positive complex
If the decolorization is insufficient, the organism may_______
falsely appear as gram positive cells.
If the______ is left applied for more than a minute, the ________will be eliminated from the previously stained cells
safranin dye
Gram positive complex
Failure to leave the safranin dye for a sufficient time will result in ______and background materials.
unstained gram negative bacteria
All cocci are gram – positive except for
Neisseria
Moraxella
Veilonella
All bacilli are gram – negative except for
Actinomadura, Arcanobacterium, Bacillus, Clostridium, Corynebacterium, Erysipelothrix, Gordonia, Kuthria, Listeria, Mycobacterium, Nocardia, Rhodococcus, Streptomyces, Tropheryma whipplei, and Tsukamurella.
All Awesome Biologists Collect Cool Exotic Germs Knowing Lab Microbes Need Real Scientific Testing and Training.
Actinomadura
Arcanobacterium
Bacillus
Clostridium
Corynebacterium
Erysipelothrix
Gordonia
Kuthria
Listeria
Mycobacterium
Nocardia
Rhodococcus
Streptomyces
Tropheryma whipplei
Tsukamurella
REASONS WHY GRAM – POSITIVE BACTERIA BECOME GRAM NEGATIVE BACTERIA
- Removal of MgRNA
- Aged, dying, and autolyzing cells
- By using acidic iodine during staining
- Due to technical error or the wrong use of stains
EXCEPTIONS IN GRAM STAINING
- Organisms that exist almost exclusively within host cells (Chlamydia)
- Organisms that lack cells walls (Mycoplasma and Ureaplasma)
- Organisms with insufficient dimension to be resolved by light microscope (Spirochetes)
Acid fast
▪ Heat is applied as a mordant in the ______while tergitol is used in the______
Ziehl Neelsen Method
Kinyoun Method
▪ In this procedure, the cell wall of acid – fast bacteria resists the acid –
alcohol (hydrochloric acid – ethanol mixture) decolorization step.
ACID FAST STAINING
▪ It utilizes carbolfuchsin as the primary stain and methylene blue or malachite green as the secondary stain.
ACID FAST STAINING
▪ It is used to stain bacteria that have high lipid contents in their cell walls.
ACID FAST STAINING
Acid fast
▪ It utilizes______ as the primary stain and _____ or _____ as the secondary stain.
carbolfuchsin
methylene blue or malachite green
Acid fast principle
PRINCIPLE:
• The primary stains binds to mycolic acid in the cell walls of acid fast bacteria, and is retained after decolorizing with acid alcohol.
• Acid Fast Bacilli (AFB) retain the primary stain and deep-pink or red-colored while non – AFB are either blue or green colored.
– Hot staining method
Ziehl Neelsen
– Cold staining method
Kinyoun
– differentiates Mycobacterium smegmatis from Mycobacterium tuberculosis.
Pappenheim Method
– differentiates Mycobacterium leprae from Mycobacterium tuberculosis.
Baumgarted Method
– selective for the cell wall of AFB.
Auramine – rhodamine Method
WAYS TO FACILITATE ACID – FAST STAINING
- The use of heating or steaming process for five to seven minutes to temporarily remove the mycolic acid, while the smear is flooded with stain.
- By increasing the concentration of dye and phenol in the staining reagent.
- A prolonged contact of the specimen with the primary stain.
- The addition of wetting agent like tergitol
