T E R I A L S T A I N I N G 1 What is a Stain A stain is a substance that adheres to a cell giving the cell color The presence of color gives the cells significant contrast so are much more visible ID: 410563
Download Presentation The PPT/PDF document "B A C" is the property of its rightful owner. Permission is granted to download and print the materials on this web site for personal, non-commercial use only, and to display it on your personal computer provided you do not modify the materials and that you retain all copyright notices contained in the materials. By downloading content from our website, you accept the terms of this agreement.
Slide1
BACTERIAL STAINING
1Slide2
What is a StainA stain is a substance that adheres to a cell, giving the cell color.The presence of color gives the cells significant contrast so are much more visible. Different stains have different affinities for different organisms, or different parts of organismsThey are used to differentiate different types of organisms or to view specific parts of organisms 2Slide3
Why we should be Stain BacteriaBacteria have nearly the same refractive index as water, therefore, when they are observed under a microscope by wet mount they are opaque or nearly invisible to the naked eye.Different types of staining methods are used to make the cells and their internal structures more visible under the light microscope.3Slide4
stains composes fromStains are a mixture of chromogen and auxochrome Chromogen = benzene derivative + chromophore (coloring agent)Auxochrome: give +ve or –ve charge to the chromogenThe ionized stain is capable of binding to cell structures with opposite charges.
4Slide5
Basic stains are cationic; when ionized, the chromogene exhibits a positive charge. Basic stains bind to negatively charged cell structures like nucleic acids. Methylene blue, crystal violet and carbolfuchsin are common basic stains.Acidic stains are anionic; when ionized, the chromogen exhibits a negative charge.
Acidic stains bind to positively charged cell structures like proteins
. Picric acid, eosin and
nigrosin
are common
acidic
stains.
Bacteria are slightly negatively charged at pH 7.0
Basic dye stains
bacteria
Acidic dye stains
background
5Slide6
There are three type of staining in Microbiological lab.1. Simple stain.2. Differential Stain: (Gram stain , Acid fast Stain)3. Special stain: (Capsular stain, Endospore stain, Flagellar stain). 6Slide7
Simple stain 7Slide8
Simple stainUsed to show the general structures (shape and arrangement) of some bacteria.Aqueous or alcohol solution of single (usually basic) stain is used in this procedure 8Slide9
procedure1. Obtain broth cultures of the bacteria.Using an inoculating loop, remove a loopful of suspension from one of the tubes. Remember to use sterile technique. 3. Smear the bacteria across the center of the slide with the loop. If the bacterial suspension is very thick, add a drop of water and mix the bacteria and the water on the slide.
9Slide10
4. Allow the smear to completely air dry.Air dry first to prevent lysis (boiling)5. Heat-fix the smear by quickly passing the slide through a Bunsen burner flame three times. This causes partial melting of the cell walls and membranes of the bacteria, and makes them stick to the slide. Do not overheat the slide as this will destroy the bacteria. Heat FixingKill.Stops autolysis.Adherence to slide.Increase dye taking
10Slide11
116. Cover the smear with a few drops of one of the stains. Allow the stain to remain for the following periods of time: Carbolfuchsin- 15-30 seconds. Methylene blue- 1-2 minutes.
Nigrosin
- 20-60 seconds.
. The stain can be poured drop by drop on the slide
7. Gently rinse the slide by holding its surface parallel to a gently flowing stream of water.
8. Gently blot the excess water from the slide with bibulous paper. Do not wipe the slide. Allow the slide to air dry.
Observe the slide under the microscope with air and oil lenses
. Slide12
Simple Stains 12Slide13
Bacterial shape arrangementClusters (group).Chains.Pairs (diploids). No special arrangement. 13
Cocci
BacilliSlide14
Differential stain 14Slide15
Gram stainDifferential stain (Hans Christian Gram, a Danish doctor ). He developed a new method to stain bacteria so they can be visible in specimen samples.Differentiate bacteria into two large groups (the Gram Positive and the Gram negative)Gram status is important in medicine; the presence or absence of a cell wall will change the bacterium's susceptibility to some antibioticsAlmost all bacteria are described by their Gram stain characteristics.
Based on differences of Cell wall structuresSlide16
Reagents for Gram StainCrystal Violet (purple).Primary stain; positive stainStains cell wall purpleIodineMordantCombines with primary stain to form an insoluble complex that gets trapped in
bacterial cell wall
Ethanol
Decolorizer
CV-I complex washed out of Gram negative organisms because it cannot be trapped
by
lippopolysaccharide
layer
; flows right through outer membrane
Safranin
(pink)
Counterstain
Simple positive stain that provides contrasting dye for decolorized cells (Gram negative)
Stains all cells, but only the negative ones actually appear pink.Slide17
17Slide18
Slide19
Gram Stain colors 19Color of
Gram + cells
Color of
Gram – cells
Primary stain:
Crystal violet
Purple
Purple
Mordant:
Iodine
Purple
Purple
Decolorizing agent:
Alcohol-acetone
Purple
Colorless
Counterstain:
Safranin
Purple
RedSlide20
Gram Staining Procedure 20
(after air drying and heat fixation)Slide21
Gm-ve bacilli 21Gm+ve
cocciSlide22
Errors during stainingNever ever used old culture. Never ever used sample for patient take antibiotic.Time of Decolorizer:Over: G + see as G -.Low: G- see as G +.Time of fixation:Over: G + see as G -.Low: no sample on slide.