DIFFERENTIAL STAININGIDENTIFYING STRUCTURAL FEATURES OF BACTERIA

Seema sangwan M.sc. M.D.University , rohtak

CONTENT:IntroductionGram stainAcid fast stainEndospore stainCapsuler stain

Gram stainingThe gram stain developed by Christian Gram in the 1800’s was the first differential staining technique in use and is still an important tool for distinguish between two main type of bacteria Gram-ve Gram+ve.

PROCEDURE:-

  Place slide with heat fixed smear on staining tray.

  Gently flood smear with crystal violet and let stand for 1 minute.

  Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle.

  Gently flood the smear with Gram’s iodine and let stand for 1 minute.

  Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle. The

smear will appear as a purple circle on the slide. 

Decolorize using 95% ethyl alcohol or acetone. Tilt the slide slightly and apply the alcohol drop by drop for 5 to 10 seconds until the alcohol runs almost clear. Be careful not to over-decolorize. 

Immediately rinse with water. 

Gently flood with safranin to counter-stain and let stand for 45 seconds. 

Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle. 

Blot dry the slide with bibulous paper. 

View the smear using a light-microscope under oil-immersion.  

Gram+ve cell will appear purple and Gram-ve cell

appear pink

Acid Fast stainingAcid Fast staining is also known as Ziehl Neelsen stain is used to

identify specialized bacteria that have waxy mycolic acid in there cell wall.

PROCEDURE1 A slide shaped piece of blotting paper is placed over the

bacterial smear. 2 Apply bright pink dye (Ziehl’s carbolfuchsin) on blotting paper

and place over a water bath for 3-5 minutes and rinse the slide.3 Decolorize with acid alcohal4 Lastly purple counter stain(crystal violet) is applied and slide is

rinsed.5 Acid-fast(waxy)bacterial cell are bright pink and non acid fast

bacterial cell are purplish blue.

ENDOSPORE STAININGThe endospore stain is used to identify bacteria

that can produce endospore or not. The endospore stain is used to identify bacteria

that can produce endospore (small,dormant structure alike to bacterial seeds).

Forming endospore is very advantages to the bacteria allow to survive under difficult circumstances, including dessication , starvation high heat , exposure to chemicals and radiation.

This stain is designed to distinguish between vegetative cell of bacteria from spore.

PROCEDURE:1 Place bacterial smear on water bath and

stain with malachite green.2 Heat on water bath for 5 minutes.3 Rinse the slide and a pink counter stain

(SAFRANIN) is applied for one minute.4 After final rinse when viewing slide under oil

immersion with a compound light microscope , vegetative cells will appear pink and endospore are stained a bluish green.

 .

CAPSULAR STAININGBacterial capsular are composed of high

molecular weight polysccharides and are associated with virulent and bioflim formation. Capsule are not stain well with crystal violet, methylene blue or other simple stain. Here two method of capsular staining are present following :-

1 Manavel’s staining 2 Hiss method of capsular staining