Structural Biochemistry, Bacterial Gram Stain

Introduction

Gram stains’ practical functions mainly revolve around medicinal uses. The use of a Gram test identifies the source of an infection. Appropriate treatments can be determined by doctors through a mere Gram test, as it can describe the type of bacteria present in a sample. Not all bacteria fall conclusively into the Gram positive and negative category, they may stain both purple and pink. Some have waxy layers in their cell walls that are not affected by the stain.

Procedure

1. Bacterial sample is applied to a glass slide and heated so as to inactivate it and prevent it from infecting the scientist.
2. Gentian violet Iodine is applied to the sample for 60 seconds. In aqueous solutions, the gentian violet dissociates into CV+ and Cl- ions. These ions have high cell wall penetration regardless if the bacteria Gram positive or negative.
3. The CV+ ion is attracted to the negatively charged portions of the cell wall and thus stains those portions purple.
4. The iodine forms a complex with the CV+ ions and forms iodine and crystal ion complexes, in both the inner and outer membranes.
5. Slide is washed under gentle water and the Gram solution (an iodine and iodine potassium mixture) is applied to the sample, this step instigates a reaction with the gentian violet solution added in the previous step.
6. The iodine washes works to identify levels of lipopolysaccharides and peptidoglycans in the bacterial cell walls.
7. At first the sample will turn dark blue, however rinse the resulting solution with ethyl alcohol will allow the color solution to fade from certain samples. In Gram negative samples, the alcohol breaks down the crystal ion complex. On the other hand, the Gram positive samples are dehydrated from the ethanol and the crystal ion complex grows to be too big and thus trapped within the thick peptidoglycan cell walls.
8. The last set of dye, red in color will be applied and the final product can finally be analyzed.
9. Gram positive bacteria produce a purple stain and represent high levels of peptidoglycan, while Gram negative bacteria are pink and are low in peptidoglycan and lipopolysaccharide concentration.

What Gram Stain Results Mean

Gram negative bacteria produce endotoxins that are the sources of cholera and typhoid. Moreover, most Gram negative bacteria are antibiotics-resistant. Gram negative bacteria have thinner cell walls that stains pink, they also possess an additional outer lipid membrane that is separated from the periplasmic space. Gram positive bacteria possess thicker mesh like cell walls rich in peptidoglycan.

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