Microbial staining– principle – simple staining and differential staining

By

Devic

Introduction

• Microorganisms in general cannot be studied properly because they are transparent and practically colourless.

• To increase contrast and to reveal additional information, they need to be stained.

• Substances commonly used to stain bacteria are known as dyes. Chemically a dye is an organic compound containing a benzene ring plus a chromophore and auxochrome group.

Acidic and basic dyes

• Dyes are acidic, basic or neutral. • The acidic dyes (eg. Picric acid, Acid fuchsin,

Eosin) are anionic and stain the cytoplasmic components of the cells which are more alkaline.

• The basic dyes (eg. methylene blue, crystal violet, safranin) are cationic and combine with those cellular components, which are acidic in nature (nucleic acids).

Simple and differential staining

• There are two kinds of staining procedures:

Simple and differential.• Simple Stain : It employs a single dye.

The cell and its structures will attain the color of the stain.

• Differential Stain : It employs more than one dye. It is used to differentiate organisms or used to distinguish the structures within a cell.

Preparation of smear

• Place 1-2 loopfuls of the cells suspension on the clean slide.

• Spread into a small area to form a thin film.• Allow the smear to dry completely.• Heat fix the smear, by passing the slide over

the flame two or three times.• Heat fixation is done to avoid washing away

of smear during the staining procedure.

Direct staining

• A simple stain that stains the bacterial cell is called as direct staining.

• Many of the bacterial stains are basic chemicals and they react with negatively charged bacterial components to colour the cell.

• Methylene blue, • crystal violet, • basic fuchsin.

Procedure

• A bacterial smear is prepared on a clean slide and heat fixed.

• Drops of the stain is added and kept as such for 2-3 min.

• Then the smear is washed gently in running tap water.

• After blot drying with blotting paper, the cells can be seen under the high power objective of a microscope.

Negative staining / indirect staining

• Here the cells are not stained, but the background is stained.

• An acidic dye like nigrosin or Indian ink is used.

• Acidic stain carries a negative charge and repelled by the bacteria, which also carry a negative charge on their surface.

• Hence they appear transparent upon examination.

Procedure

• Place a drop of nigrosin / Indian ink at one end of a clean glass slide.

• Add a drop of culture to the dye and mix gently with the loop.

• Spread the mixture of the stained inoculums into a thin wide smear by pushing the top slide along the entire surface of the bottom slide.

• Air dry and observe under oil immersion.

Gram staining

• Dr. Hans Christian Gram, a Danish physician in 1884, developed gram Staining.

• It is the most important and widely used differential staining technique.

• Gram staining has its greatest use in characterizing bacteria but it is not applicable for other groups of microorganisms such as protozoa and fungi but yeast consistently stain gram positive.

Gram staining

• The bacterial smear is subjected to four different reagents.

• Crystal violet (primary stain), iodine solution (mordant), alcohol (decolourising agent) and safranin (counter stain).

• The bacteria which retain the primary stain (appear violet) are called gram positive and those which are counter stained by safranin (red) are referred to as gram negative.

Gram staining

• Gram -ve bacterial cell wall is thin, complex, multilayered with relative high lipid content.

• Lipid is readily dissolvable by alcohol, forming large pores in the cell wall resulting in the easy decolorization of crystal violet - iodine (CV-1) complex. Later they take up the counter stain and appear red.

• In contrast, the Gram +ve cell walls are thick with less of lipids, so will not loose CV-1 and cells remain blue.

Procedure

• Make a thin smear of the bacterial suspension.• Stain the smear with ammonium oxalate crystal

violet for a minute. Wash in tap water for 3-4 sec.• Flood the smear with lugol's iodine solution for 30

sec. Wash in tap water for 10 sec. and blot dry.• Decolourize with 95% ethanol for 30 sec.• Counter stain with safranin solution for 10 sec. Wash

gently in tap water.• Blot dry and examine under high power dry (45x)

objective.

Factors that affect Gram Reaction

• Improper heat fixing of the smear. Over heating will rupture the cell wall which will result in the uptake of counter stain by the G +ve cells.

• Cell density of the smear. • Proper decolourization will not taken place

with thick smear.• Always use fresh and young cultures

Acid-fast staining

• Paul Ehrlich developed the acid fast staining in the year 1882, which was later modified.

• Carbolfuchsin is the primary stain/dye and Loeffler's methylene blue is the counter stain.

• The decolourizer is 3% hydrochloric acid in 95% ethanol.

• Bacteria are classified acid-fast if they retain the primary stain after washing with the decolourizer.

Acid-fast staining

• The property of acid fastness appears to be due to the present of high concentration of a lipid called mycolic acid in the cell wall.

• Most genera are not acid-fast, except Mycobacterium and Nocardia.

• Best known acid fast pathogenic bacteria are Mycobacterium tuberculosis and M. leprae.

• This staining procedure is used for the identification of members of Mycobacterium.

Procedure

• Prepare smears, air dry and heat fix.• Flood smears with carbolfuchsin.• Heat the slides by steaming for 3-5 mins.• Add dye periodically to avoid drying of smear• Cool the slides and wash with distilled water.• Decolorize the smear with acid alcohol for

10-30 seconds or until the smear is faint pink in colour.

• Wash the slides in distilled water.

Procedure

• Counter stain the smear with methylene blue for 1-2 min.

• Wash in distilled water.• Blot dry with blotting paper• Examine under oil immersion objective.• Precautions• Do not allow carbolfuchsin stain to evaporate

and dry, add more stain, if necessary• Prevent carbolfuchsin stain from boiling

Structural staining: Endospore staining

• Some bacteria are capable of changing into dormant structures, that are metabolically inactive under unfavourable conditions.

• These structures are called endospores. • An endospore is developed in a

characteristic position within a cell i.e. either central or sub terminal.

• Members of the genera viz., Bacillus and Clostridium form endospore.

Structural staining: Endospore staining

• The spores are differentially stained using special procedures that help dyes to penetrate the spore wall with the stain malachite green.

• Once stained, the endospores do not readily decolorize and appear green within red cells.

Procedure

• Air dry and heat fix the smear of Bacillus.• Flood the smears with malachite green and

steam it over a water bath for 5 minutes.• Add stain from time to time, so that the stain

doesn't dry.• Wash the slides in slowly running tap water.• Counter stain with safranin for 30 seconds.

Wash in distilled water.• Examine under oil immersion objective.

Structural staining: Capsule staining

• Capsules are mucilaginous substance forming a viscous coat around certain bacterial cells.

• It is referred as capsule when round or oval in shape and slime layer when irregularly shaped and loosely bound to the bacterium.

• Although external to the cell, they are synthesized partially in the cytoplasm and is composed of a polysaccharide, a glycoprotein or a polypeptide.

Procedure

• Prepare a thick smear• Apply nigrosin stain• Allow smear to air dry• Treat the smear with acid alcohol for 15 seconds• Wash the smear with distilled water• Flood the smear with crystal violet for 60 seconds• Wash with distilled water• Blot dry with blotting paper• Examine under oil immersion objectives.

Structural staining: Flagella staining

• Many bacteria are motile due to the presence of one or more fine thread like, filamentous appendages called flagella.

• The presence, location and the numbers possessed by the microorganisms are some of the important criteria used in the identification and classification of bacteria.

• To view them with a microscope, they are to be thickened first by the use of mordents and later stained with a dye.

Procedure

• Make bacterial suspension in distilled water.• Incubate the suspension at room

temperature for 10-15 minutes.• Place one loopful of the suspension on one

end of the slide and allow the drop to spread to form a thin film by tilting the slide.

• Allow the film to air dry. Flood the smear with flagella mordant for 10 minutes.

• Wash gently with distilled water.

Procedure

• Flood the slide with carbolfuchsin for 5 min.• Wash the slide gently with distilled water.• Air dry the slide and examine under oil

immersion objective.• Precautions• Do not heat fix the smears• Do not blot dry the smears• Use reduced illumination of the microscope

to seed flagella clearly