practical exercises

Gram staining technique


Bacteria vary in certain physiological and biological properties. Some staining techniques utilize these differences to stain the bacteria differently. Such staining methods are called differential staining methods, these include Gram staining and acid fast staining. Different bacteria stain differently to a common staining procedure. Gram stain was described by Danish bacteriologist Hans Christian Gram in 1884 to differentiate Streptococcus pneumoniae from Klebsiella pneumoniae in lung tissue. The original formulation comprised of aniline gentian violet, Lugol's iodine, absolute alcohol and Bismarck brown.

Principle of Gram staining technique:
There are four components of Gram stain; primary stain, mordant, decolorizer, and counterstain. When stained with a primary stain and fixed by a mordant, some bacteria are able to retain the primary while others get decolorized by a decolorizer. Those bacteria that resist decolorization and retain the primary dye are called Gram positive and those bacteria that gets decolorized and then get counterstained are called Gram negative. Various theories have been put forth to explain the differences among bacteria that are exploited by this staining technique; some are more acceptable than others.

Typically, gram positive bacteria have thicker cell walls (approximately 40 layers of peptidoglycan) compared to Gram negative bacteria, which have only 1-2 layers of peptidoglycan.

Alcohol or acetone, which is used as a decolorizer dissolves lipids in the membranes; since Gram negative bacteria have an additional membrane (apart from cytoplasmic membrane), more lipids are lost forming pores through which the dye-iodine complex escapes during decolorization. On the other hand, alcohol dehydrates the Gram positive bacteria closing the pores and shrinking the cell wall and thus prevents outflow of dye-iodine complex.

Slightly lower cytoplasmic pH (2.0) in Gram positive bacteria helps the basic dye to bind more efficiently than the Gram negative bacteria who have slightly elevated pH (3.0). This theory is probably not widely accepted.

Another theory such as presence of Magnesium ribonucleate in Gram positive bacteria and its absence in Gram negative bacteria has not received widespread acceptance.

At the end of Gram staining, Gram positive bacteria appear violet or purple whereas Gram negative bacteria appear pink.

Preparation of smear:
A grease free glass slide is taken and a circle is marked one side of the slide using a wax pencil or a glass marker pen. Marking the slide makes it convenient to identify the surface of the slide that contains the smear. On the opposite side, a small drop of sterile saline is taken. Saline may also be transferred using a bacteriological loop. A small portion of the colony is picked up using a sterile straight wire and emulsified well in the saline on the slide. The colony is spread evenly within the circled are to produce a smear. Presence of grease on the slide would prevent uniform distribution resulting in an uneven smear. Taking too much of the colony would result in excessively thick smear. Similarly, taking a very miniscule part of the colony make it very difficult to look for bacteria. Once a uniform smear is prepared, it has to be air dried. The smear must be held above the Bunsen flame at comfortable height. The smear must be not be forcibly dried by applying heat. After the smear dries, it is fixed. Smears can be fixed physically or chemically. Chemical fixatives are not useful for regular bacteriological studies. It is convenient to heat-fix the smear by gently passing the slide through the Bunsen flame once or twice. Excessive heating of the slide must be strictly avoided.

Note: You don't have to prepare smears, slides containing smear would be provided to you in the practical class.

Staining procedure:
The glass slide with smear facing upwards is placed on the staining rack. Few drops of crystal violet solution are poured over the smear taking care not to extend beyond the circled area. The stain is allowed to act for one minute. Gentian violet and Methyl violet are the other alternatives for primary stain. The slide is washed in gentle stream of running tap water. Few drops of Gram's iodine is placed over the smear and allowed to act for one minute, after which it washed in tap water. The smear is decolorized using alcohol-acetone decolorizer by holding the slide in an inclined position and pouring the decolorizer from the top end. As the decolorizer flows over the smear, it decolorizes it. When the decolorization is complete, the fluid that flows over would be colorless. The entire decolorization process should be completed within 30 seconds. Most errors in staining occur at this stage. Decolorization for excessive duration is called over-decolorization wherein Gram positive bacteria appear Gram negative. Similarly, rapid decolorization may lead to under-decolorization of smear wherein Gram negative bacteria appear Gram positive. Absolute alcohol, acetone or a mixture of alochol and acetone (1:1) are used as decolorizer. After decolorization the smear is washed and counterstained by dilute carbol fuchsin for 30 seconds. Other alternatives include safranin and neutral red. The slide is dried using blotting paperr, a drop of oil is placed on the smear and observed under 100x (oil immersion) objective.

Violet coloured, spherical shaped bacteria in singles, pairs, tetrads, short chains and irregular grape like clusters seen along with pink colored, rod shaped bacteria that are present haphazardly.

The given smear contain both the Gram positive cocci and the Gram negative bacilli.

gram stain

Note: a. Observation must be made in the following order: colour > shape > arrangement
b. Spherical shaped bacteria are referred as cocci (coccus is singular) and rod shaped bacteria as bacilli (bacillus is singular)
c. While we provide a slide containing both Gram positive cocci and Gram negative bacilli in the smear for routine practical classes, you may get slides containing either of them alone or a mixture during your examination.

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