STAINS OF IMPORTANCE

ALT CULTURE CLUB TOPIC

BACTERIOLOGY

STAINS OF IMPORTANCE

Cells of bacteria are colorless and usually transparent and to observe their morphology in natural state is difficult to obtain with any degree of accuracy.  Men like Robert Koch and Paul Erhlich were the first ones who developed the early methods of fixing and staining bacteria.  Example of dyes used then was aniline (coal tar dye).  Affinity for nucleic material is called basic and for cytoplasmic component, acidic.
The preparation of smear, which is done by spreading the bacteria over the glass slide surface, is usually the first step in routine staining procedures.  Bacterial colony is emulsified in saline or distilled water to form a homogenous suspension.  Vigorous mixing and spreading may produce aerosols and besides, may destroy the morphology and characteristic arrangement of the cells.  The smear is air-dried and then fixed gently by passing thru a Bunsen flame or Bacticinerator with attached slide holder.  Allow the slide to cool.
There are different staining procedures but most commonly and routinely used are the Gram stain method for gram positive and gram negative bacteria and acid fast stain.  Another method is the fluorescent stain.
Gram stain reagents:
1.  Gentian violet
2.  Gram iodine
3.  Acetone-alcohol
4.  Safranin
Gram positive bacteria are stained purple
Gram negative bacteria are stained red
Acid fast stain reagents:
1.  Carbol-fuschin DSMO
2.  Acid alcohol
3.  Methylene blue
Acid fast bacilli are stained pink (against a blue background)
Other special staining procedures:
1.  Capsule stain - capsule of bacteria does not have the affinity for dyes.  India ink for Cryptococcus capsules is seen under the microscope against a black background.  In the Anthony method the cells and its background are stained but not the capsule and the surrounding envelope is seen by contrast.
2.  Staining of metachromatic granules - Methylene blue stain and Albert stain (for differential) for Corynebacterium diphtheriae.
3.  Staining of yeasts and fungi - wet mounts of yeast can be stained with methylene blue or Gram iodine; fungus spores and mycelia identification using Lacto Phenol Cotton Blue (LPCB) stain.
4.  Spore stain - Dorner method is used in which equal volume of carbol-fuschin is added to a heavy suspension of the organism and then boiled.  The spores are stained red while the rest of the bacterial cells are colorless.
5.  Conjunctival scrapings - Wright-Giemsa method is used.
6.  Fluorescent stain -  fluorescent dyes, such as auramine and rhodamine, are used.  Examination under fluorescence microscopy will show acid fast bacilli, when present, with an orange to red color.
Most commonly used stains are: Gentian or crystal violet, safranin, basic fuschin, methylene blue, Bismark brown and also malachite green.

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