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INTRODUCTION

Bacteria cannot be seen under the light microscope without staining due to the lack of contrast, until special methods of illumination are used.


IMPORTANCE OF STAINING

1. The microscopic examination of stained smear enables the morphology, relative sizing &

arrangement of microorganism to be seen clearly. It also assists in the detection of cells, esp. pus cells.

2. Bacteria can also be differentiated by their staining reactions, e.g. gram positive from gram negative organisms.

DEFINITIONS

Supravital Stain: Stains which are toxic and kill the cell on staining.


Differential Stains: They impart different colours to different bacteria or their structures.

Example: Gram stain and ZN stain.

Use: Gram stain differentiates bacteria into gram positive and gram negative. Acid fast stain (ZN stain) differentiates bacteria into acid fast and non acid fast.

Special Stains

Definition: They are devised for some special purpose, to identify some particular bacteria or its component.

Example: Albert’s stain, Neisser’s stain and Ponder’s stain for Corynebacterium diphtheriae.

Use: They demonstrate metachromatic granules of corynebacterium diphtheriae.

LABELLING OF SLIDES

Every slide should be labelled clearly with number or letters by diamond or grease pencil. Stick—on paper labels should not be used as they are liable to be soiled or washed off in staining procedures.

PREPARATION OF SMEAR

A bacterial smear is a dried preparation of bacterial cells on a glass slide. In a bacterial smear that has been properly processed, (1) the bacteria are evenly spread out on the slide in such a concentration that they are adequately separated from one another, (2) the bacteria

are not washed off the slide during staining, and (3) bacterial form is not distorted.

Smears should be spread evenly covering an area of about 15-20mm diameter on a slide.

1. From purulent specimen (pus, turbid urine etc.).

Take a loopful of specimen with the sterile inoculating wire and spread thinly on the slide.

2. From non purulent specimen.

Centrifuge the specimen & make a smear from a drop of the well mixed deposit.

3. From Sputum.

Use a piece of clean stick to transfer and spread purulent & caseous material on a slide. Soak the stick in phenol before discarding it.

4. From swab.

Roll the swab on the slide.

5. From culture plate.

Put a drop of saline on a clean slide. With the help of sterile inoculating wire, a individual colony is picked up & mixed with saline drop to prepare a thin emulsion.

In making a smear from culture, bacteria from either a broth culture or an agar slant or plate may be used. If a slant or plate is used, a small amount of bacterial growth is transferred to a drop of water on a glass slide and mixed. The mixture is then spread out evenly over a large area on the slide.

FIXATION OF SMEAR

PURPOSE

The purpose of fixation is to preserve microorganisms, and to prevent smears being washed from slide during staining. Smears are fixed by heat or alcohol.

NOTE: Microorganisms are not always killed by heat fixation. So please handle them carefully, considering them to be infective. Discard the slides after use in 5% phenol.


HEAT FIXATION

1. Allow the smear to air-dry completely.

2. Rapidly pass the slide; smear uppermost, three times through the blue flame of the spirit lamp or pilot Flame of Bunsen burner.

3. Allow the smear to cool before staining.

Disadvantage

1. Integrity and morphology of the organism may be disturbed & staining reactions altered especially where excessive heat is used.

2. It also damages leucocytes. So it is unsuitable for fixing smear which contains intracellular organism such as N. gonorrhoea & N. meningitides.

ALCOHOL FIXATION

1. Allow the smear to air dry completely

2. Add one or two drops of absolute methanol or ethanol

3. Leave the alcohol on smear for a minimum 2min. or until the alcohol dries on the smear.

Advantages

1. Less damaging to micro organism than heat.

2. Pus cells are well preserved. So alcohol fixation is recommended for fixing the smear when looking for gram negative intracellular diplococci.

3. It is more bactericidal than heat so it is preferable to fix the smear prepared from sputum.(M. Tuberculosis)

OTHER CHEMICAL FIXATIVES

1. Potassium permanganate. (anthrax bacilli)

2. Formaldehyde vapour (Mycobacterium species)

GRAM STAIN

DISCOVERED BY
Hans Christian Joachim Gram was a Danish physician and bacteriologist who developed in 1884 the most widely used method of staining bacterial cells for microscopic study.

PRINCIPLE

Crystal violet (gentian violet) serves as the primary stain, binding to the bacterial cell wall after treatment with a weak solution of iodine which serves as the mordant for binding the dye. Some bacterial species, because of the chemical nature of their cell wall, have ability to retain the dye iodine complex even after treatment with an organic decolouriser such as mixture of equal parts of 95% ethyl alcohol & acetone. Dye retaining bacteria stain dark-purple while bacteria which are decolorized take the counter stain (safranin) and appear pink.

PROCEDURE

1. Lay the fixed slide on a staining rack. Cover the smear completely with the gentian violet.

    Keep it for I minute.

2. Pour off the stain and rinse with running tap water.

3.  Flood the slide with Gram’s iodine solution and wait for 2-3 minute.

4. Gently drain off the iodine solution and rinse with running tap water. 

5. Decolorize rapidly (few seconds) with acetone/alcohol by holding the smear between thumb & forefinger.

6. Wash the slide under running tap water &drain.

7. Pour safranin solution on the slide and keep for 30-60 seconds.

8. Wash off the stain with tap water.

9. Wipe the back of the slide clean, and place in a draining rack for the smear to air-dry.

10. Examine the smear microscopically with oil immersion objective.


RESULTS

Gram positive bacteria            - Dark purple

Yeast cells                                   - Dark purple

Gram negative bacteria           - Pale to dark red

Nuclei of pus cells                    - Red

Epithelial cells                           - Pale red 




MECHANISM OR THEORIES:

        LIPID CONTENT THEORY

        MAGNESIUM   RIBONUCLEATE THEORY

         ISOELECTRIC POINT THEORY


1.  Lipid content theory - Gram positive organisms contain more protein while gram negative contain more lipid. While decolorizing, the lipid get dissolved, thus leaking of dye-iodine complex out of the cell occurs. Thus gram negative take the counter stain. In gram positive organisms, the protein in cell wall gets dehydrated, shrinking the pore size. Thus do not get decolorized & take primary stain.

2. Magnesium ribonucleate theory – Present in Gram Positive Bacteria and there is formation of magnesium  ribonucleate – dye – iodine complex which is insoluble in alcohol used as a decolorizer.

3.   pH theory – As compared to Gram Negative Bacteria, Gram Positive Bacteria have more acidic protoplasm and the primary stain used is basic in nature. Hence, Gram Positive Bacteria retain primary stain more strongly than Gram Negative Bacteria. Also the iodine makes the cytoplasm more acidic and acts as a mordant, fixes the stain in bacterial cell.

Cell wall permeability – The cell wall of Gram Positive Bacteria contain more mucopeptide because of which it is thicker & stronger, hence dye iodine complex does not come out of cell easily.

USES OF GRAM STAIN

Gram stain is useful for presumptive diagnosis of:

Meningitis from CSF sample - pneumococci, streptococci, meningococci, haemophilus etc.

Diphtheria from throat swab – by demonstration of Chinese letter pattern of corynebacterium diphtheriae with clubbed ends.

Upper respiratory tract infection from sputum specimens

Urinary tract infection

Dysentery

Cellulites, osteomyelitis, abscess etc.

Gas gangrene

Opportunistic diarrhea in patients on prolonged antibiotic therapy, and various other conditions.

NOTE:

Different primary stains that can be used in gram stain are methyl violet, gentian violet and crystal violet.

Different counter stains that can be used in gram stain are safranine, neutral red, dilute carbol fuchsin and basic fuchsin.

Different decolourisers that can be used in gram stain are acetone, alcohol or a mixture of acetone and alcohol in 1:1 ratio.

Gram’s iodine acts as a mordant in gram stain. A mordant is a substance which helps in fixation and/or penetration of the dye inside the cell. 

Non toxic stain during which cells retain their viability.

TYPES OF STAIN

Simple Stains

Definition: Watery solution of a single basic dye is used to stain the smear.

Example: Methylene blue or basic fuchsine

Use: They just demonstrate the presence of bacteria in the smear

Disadvantage: Since only one stain is used, it is not possible to say whether the organism is gram positive or gram negative.

Negative Stains

Definition: Bacteria are mixed with dyes. The background gets stained leaving the bacteria contrastingly colourless.

Example: Indian ink, Nigrosin

Use: For demonstration of bacterial capsule.

Silver Impregnation Method

Definition: Bacterial cells and appendages that are too thin and delicate cannot be seen under ordinary microscope. These delicate structures are thickened by impregnation of silver on the surface to make them visible under light microscope.

Use: To demonstrate spirochaetes and bacterial flagella.


Differential Stains

Definition: They impart different colours to different bacteria or their structures.

Example: Gram stain and ZN stain.

Use: Gram stain differentiates bacteria into gram positive and gram negative. Acid fast stain (ZN stain) differentiates bacteria into acid fast and non acid fast.

Special Stains

Definition: They are devised for some special purpose, to identify some particular bacteria or its component.

Example: Albert’s stain, Neisser’s stain and Ponder’s stain for Corynebacterium diphtheriae.

Use: They demonstrate metachromatic granules of corynebacterium diphtheriae.