This document discusses various staining techniques used to visualize microorganisms under a microscope. It begins by explaining why staining is necessary since microorganisms cannot be seen with the naked eye. It then covers different types of staining including simple staining, Gram staining, acid-fast staining, negative staining, and specialized staining techniques for flagella, capsules, and spores. Each staining method is described in 1-2 sentences and includes the basic procedure and results.

1. Title Layout
Subtitle

2. Staining
 Micro organism cannot beseenbynakedeyes,that why we must magnifythe
sizeof micro-organism.
 Forthat reason we needa magnifier which ismicroscopewhich enlargesthe
organismsalmost 1000more than its actualsize.
 Thesizeof bacteria ismeasuresby micronmeter(µm)
 Thesizeof most bacteria ragesfrom 1to 3µm.
 Thesmallestbacteria isMycoplasma0.2µm, where asthe largestisBorrelia
10µm

3. • Most of the micro-organism are enableto seenevenby microscope,we
need to stain theorganism
• According to pHall stainsare acidic,alkali or neutral.
• Generallyacidic materials are stainswith alkali or in contrast alkali materials
are stainswith acidic.
• In general we classify the staining methodsasfollow.
1. Simplestaining
2. Differentialstaining ( Gramstaining)
3. Acidfast staining
4. Negativestaining
5. Flagellastaining
6. Capsulestaining
7. Sporestaining

4. 1.Simple staining
In simple staining we useasingledye(methylene blue, carbol
fuchsin, Safranin).
Here just observe the shapeof organismor bacteria either its cocci,
rods orspirochetes
• Procedure;
1. Makeathin smearon aglassslide of agivensample
2. Air dry it and cover with Methylene blue.
3. Washwith tap water and againair dryit
4. Observeunder the microscope ,
5. if the given sampleisbacteria then we will seeBacilli or Coccishapesof
bacteria

5. Safranin stainedbacilli
Methylene Blue stained
cocci

6. 2.Differential staining(Gram Staining)
 Gramstain isthe most important staining procedure which differentiate
all the bacteria in two main categories either gramnegativeor gram
positive bacteria.
 Thismethod wasdiscoveredby HansChristianGramin Germanyin 1884
 In this staining method we usemore then one type ofdyewhich include
 Crystalviolet
 Safranin
 Themechanismof grampositive & gramnegative bacteria isbasedon
bacterialcellwall which gramnegative bacteria haveathin peptidoglycan
coveredby anouterlipid-containingmembrane, whereas gram-positive
bacteria haveathickpeptidoglycanand noouter membrane.

10. • Gramstaining Procedure
1. Makeasmearof bacterial specimenon aglassslide.
2. Air dry it ancoverwith crystal violet for 2-3 minutes.
3. Washwith tap water and cover withgram’siodine for 2 minutes.
4. Washwith tap water and cover with gram’salcohol (95%)for 30seconds
to1minute.
5. Washwith tap water and cover with Safraninfor2 minutes.
6. Washwith tap water and observed under the microscope which will
results either purple or pink, red coloredbacteria.
7. ThePurplecolored bacteria isnamed grampositivebacteria & the Pinkor
redcolored bacteria named gramnegative bacteria.

13. Acidfaststaining (Ziehl–Neelsenstain)
 Ziehl-Neelsen stain was discovered by the bacteriologist Franz Ziehl
(1859–1926)andthe pathologist FriedrichNeelsen(1854 – 1898).
 It is a special bacteriological stain used to identify acid fast
organisms.
 The mycobacterium tuberculosis is deferent from other bacteria in the
composition of cell wall which in addition to peptidoglycan, the outer
membrane or envelope of the acid-fast cell wall of contains large amounts of
glycolipids,especially mycolicacidsthat in the genusMycobacterium, makeup
approximately 60%of the acid-fast cellwall.

14. Structure of Mycobacteriumtuberculosis

15. • Procedure
1. Makeasmearof sputum on glassslide andfixe with ethanol &air
dry it
2. Coverslide with Carbolfuchsinandheated for 5minutes
3. Washwith tap water & cover withacidalcoholfor 1-2 minutes
4. Washwith tap water & coverthe slide with Methylene bluefor 2
minutes
5. Washthe slide and observed the redrodsunder microscope which
will be mycobacterium tuberculosis

17. TBslideafterAFB stain

18. Negativestaining (IndiaInk)
Negativestainingisarecognizedmethod, often usedin diagnosticmicroscopy, for contrasting a
thin specimenwith anoptically opaquefluid.
In this technique, the backgroundisstained,leavingthe actualspecimen untouched, andthus
visible
• Procedure
1. Makeasmearfrom agivensample& mix with Negrosinor Congreddye
2. Air dry itandobservedunder Bright field microscope
• Result
Thebackground will takethe colorbut the bacteriawill not , which the bacteria will appear
bright
Note:Mostly usedfor Spirillaor for electron microscopy

20. Flagellastain
• Theflagellastainallows observation of bacterial flagellaunder
the light microscope.
• Bacterial flagellaare normally too thin to beseen
• Therefore the flagellated bacteria are stained with specialdyeis
commercially available.

21. Capsulestaining
• Acapsuleisagelatinous outer layer secreted by bacterial cell and
that surrounds and adheresto the cellwall
• Themain purpose of capsulestain isto distinguish capsularmaterial
from the bacterialcell.
• Most capsulesare composedof polysaccharides, but someare composed
of polypeptides.
• Thecapsulestain employsanacidic stain and abasicstain to detect
capsule production

22. • Procedure;
1. Placeasmall drop of anegativestain(India Ink, CongoRed,
Nigrosin, or Eosin)on the slide.
2. Add aloopful of bacterial culture to slide, smearingit in the dye.
3. Usethe other slideto dragthe ink-cell mixture into athin film
along the first slide and let stand for 5-7minutes.
4. Allow to air dry (do not heat fix).
5. Flood the smear with crystalviolet stain(this will stain the
cells but not the capsules)for about 1minutes.
6. Coverthe slide with copper sulfate(20%).
7. Drain the crystal violet by tilting the slideat a45 degreeangleand
let stain run off until itair dries .
8. Examinethe smear microscopically (100X)for the presenceof
encapsulatedcells asindicated by clear zonessurrounding the
cells.

25. Sporestaining
 Theendosporestainisadifferential stainusedto visualizebacterial
endospores.
 Endosporesare formed by afew generaof bacteria, suchasBacillus
 Byforming spores,bacteria cansurvive in hostileconditions.
• Sporesare resistant to heat, dryness,chemicals,andradiation

26. • Procedure;
1. Preparesmearsof organisms to be tested for presenceofendospores on aclean
microscope slide and air dry it.
2. Heat fix the smear.
3. Placeasmall piece of blotting paper (absorbent paper) over the smear and place the
slide (smear side up) on awire gauzeon aring stand.
4. Heat the slide gently till it starts to evaporate (either by putting the slide on a
staining rack that hasbeen placed over aboiling water bath or via bunsenburner).
5. Removethe heat and reheat the slide asneeded to keepthe slidesteamingfor about 3-
5 minutes.Asthe paper begins to dry add adrop or two of malachitegreento keep it
moist, but don’t add somuch at one time that the temperatureis appreciably
reduced.
6. Remove the blotting paper and allow the slide to cool to room temperature for 2
minutes.
7. Washthe slide with tap water (to wash the malachite green from both sidesof the
microscope slide).
8. Stain the smear with safraninfor 2minutes.
9. Washboth side of the slide to remove the secondarystain and blot the slide/ air dry.

28. The vegetative forms arepink color
The spores forms aregreenish bluecolor