The Gram discoloration process was developed in the 1880s by Christian Gram ( Walker, 1998 ) and was so updated to supply a better distinction of bacteriums by Hucker in 1921 ( Murray, 2003 ) . It is used widely in both the medical and nutrient industries to assist in the designation of bacteriums by staining samples with dye. This colours the bacterium, enabling the size, form and morphology to be clearly seen when it is examined under a microscope ( Murray, 2003 ) . It besides helps to distinguish between the two chief types of bacteriums ( Gram +ve e.g. Bacillus, and Gram -ve e.g. Pseudomonas ) by the coloring material of dye that they take up and retain.
This essay will look at the Gram discoloration method and why and how it works. It will besides compare and contrast the construction of the bacterial cell walls of Gram +ve and Gram -ve bacteriums, explicating how the differing constructions enable the method to bring forth the consequences it does ; Gram +ve bacteriums demoing violet under a microscope whereas Gram -ve appear a pink coloring material.
Gram discoloration method
Measure one: Use a thin vilification of the bacteriums to a sterilized glass slide ( if excessively much is used so the consequence will be difficult to read ) .
Measure two: Heat fix the bacterium to the glass slide by go throughing it over a Bunsen burner a
twosome of times, so go forth it to chill.
Measure three: Flood the slide with 2 % Crystal Violet dye and go forth it for one minute. Then wash it exhaustively with unfertile H2O.
Measure four: Apply Gram ‘s iodine solution and go forth for one minute.
Measure five: Wash the slide with propanone for a twosome of seconds, so wash it once more
with unfertile H2O.
Measure six: Flood the slide with the counter-stain Safranin 2.5 % . Leave for 10-15
seconds, so wash it off with unfertile H2O.
Measure seven: Blot the slide gently ( so as non to upset or take the bacteriums ) with a paper towel.
Measure eight: Let the slide to dry if non exhaustively dried by the blotting. Then add a bead of Immersion oil on to the stained bacteriums.
( Jones et al, 2007 )
The slide is now ready to be examined under the microscope.
The Gram +ve and Gram -ve cell walls have a few similarities and many differences. Both have a peptidoglycan bed dwelling of n-acetylgylcosamine and n-acetylmuramic acids ( Greenwood et al, 2007 ) . In the Gram +ve bacteria this bed makes up 50-60 % of the weight of the cell wall and is really stiff. This is because of a high concentration of cross-linking of the polyose layers that make up the peptidoglycan bed. In the Gram +ve bacteria it is the outer portion of the wall, whereas in the Gram -ve cell wall it is the interior portion and lone histories for 5-10 % of the weight of the cell wall ( Boyd et al, 1991 ) . In the Gram +ve bacterium this bed makes up one of merely two beds ( the other being the cytoplasmatic membrane ) . The Gram -ve bacteria, nevertheless, has a three layered wall. These beds consist of an interior peptidoglycan bed and an outer membrane dwelling of chiefly lipolysaccharides with a infinite between them called the Periplasmic infinite which contains hydrolytic enzymes ( Boyd et al, 1991 ; Walker, 1998 ) .
The cell wall of the Gram -ve bacteria acts as a protective bed enabling it to defy the action of lysosomes and many antibiotics. Porin proteins located in the outer bed allow selective permeableness and the fond regard of certain molecules, hence halting molecules that could damage the cell from acquiring through ( Greenwood et al, 2007 ) . The following bed down, the periplasmic infinite, acts as a secondary barrier as it contains hydrolytic enzymes which can interrupt down unwanted molecules that make it through the outer membrane. In comparing the Gram +ve bacteria ‘s cell wall has small defense mechanism against such onslaughts as it does n’t incorporate that same outer membrane. It besides allows the diffusion of metabolites in and out of the cell ( Boyd et al, 1991 ) , some of which could potentially damage it. Due to these grounds the Gram discoloration method can give the cognition of whether an antibiotic is traveling to be of usage in the control of a peculiar infection.
The other differences between the two types of bacteriums are that the Gram +ve bacteriums possess teichoic and lipoteichoic acids attached to the cell wall, whereas the Gram -ve do non ( Boyd et al, 1991 ) . However the Gram -ve have flagella and pilli which the gm +ve are missing ( Walker, 1998 ) .
( Becker, 2010 )
The Gram discoloration plants by deceasing the cell ‘s cell organ and cell wall, enabling them to be clearly seen under a microscope. In the clinical intervention of many infections merely cognizing if the bacterium is Gram +ve or -ve is adequate to efficaciously handle the job. If the bacterium demo a purple/blue coloring material it is a Gram +ve bacteriums and therefore it is more likely that antibiotics such as Penicillin will efficaciously handle the infection.
The 3rd measure of the process allows Crystal Violet and I to come in the cell and organize a complex ( CV-I ) , deceasing the cell a purple/blue coloring material ( Fankhauser, 2006 ) . This is the coloring material that identifies the Gram +ve bacterium. It retains the coloring material as in the 5th measure ; the intoxicant that is used to bleach the cell dehydrates the cell wall due to the low lipid content. As the cell wall is dehydrated, the permeableness is decreased so that the CV-I composite is trapped inside the cell doing it appear purple, even after the attempted death with the counter-stain Safranin in measure six ( Walker, 1998 ) .
Gram -ve bacteria appear pink under the microscope ; this is because unlike in the Gram +ve bacterium, the CV-I composite is able to go forth the cell wall. In contrast to the Gram +ve cell wall, which is dehydrated and has its permeableness reduced, the Gram -ve cell wall has its outer membrane washed off by the intoxicant. This, in combination with the higher lipid content in the Gram -ve cell wall, means that it is non dehydrated but the permeableness of the cell wall is really increased. This increased permeableness allows the CV-I composite to be washed out of the cell and the counter-stain Safranin to be the dominant coloring material of the cell wall and cell organ when analyzing under the microscope ( Walker, 1998 ) .
There are a few things to take into history when placing bacteriums in this manner. Gram +ve bacteriums may look Gram -ve if they are taken from an ageing civilization or if they have been exposed to antibiotics. This is due to a interrupting down of the peptidoglycan bed which, as in the Gram -ve bacterium, increases the permeableness of the cell wall leting the CV-I composite to be washed off and the pink from the Safranin to be seen under the microscope. These are referred to as Gram-variable.
There are besides bacteriums that resist the Gram discoloration, either due to a waxy outer bed e.g. Mycobacteria, or the deficiency of a cell wall capable of retaining a dye e.g. Mycoplasma ( Walker, 1998 ) .
The Gram discoloration is an priceless process in the medical industry. It can supply a one trial manner of placing if an infection can be treated by certain antibiotics, enabling fast intervention of the infection. If more designation is needed it still provides a good starting point for make up one’s minding upon other trials. In the nutrient industries the Gram discoloration is used on a regular basis, frequently being the lone trial needed to place a taint in ingredients, finished merchandises or even in the mill environment itself. It is indispensable nevertheless that samples are collected aseptically and used within the shortest clip graduated table possible. Otherwise it is possible that the designation of the bacteriums could turn out false, either due to taint of the sample, or the age of the sample giving a Gram -ve consequence due to the peptidoglycan bed interrupting down. This could take to the job non being rectified or even exacerbated.