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The third part of the experiment is endospore

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The third part of the experiment is endospore staining. After the staining by malachitegreen dye and safranin dye, the endospores ofBacillus subtiliswere marked green while thevegetative cells were stained pink. The primary stain, malachite green dye is the reason whythe endospore ofB. subtilisappeared in green after staining. When malachite green dye wasdropped on the slide, the green dye was absorbed by the bacteria. Nevertheless, the malachitegreen is a water soluble dye and unable to remain in the bacteria when the slide was rinsedwith water (Aryal, 2018). Therefore, the staining step using malachite green was carried outby placing the slide on a beaker containing boiling water. This is because the steaming stepcan soften the outer layer of endospores, allowing the dye to enter and be absorbed by theendospores. Thus, when the slide was rinsed with water, only the stain in vegetative cells
were being washed away as the attraction towards cellular materials of malachite green dye islow. Then, the bacteria was stained with safranin and it was absorbed by theB. subtilis. Thiscan be explained by the principle of positive stain. Cytoplasm of theB. subtilisis negativelycharged while safranin dye is a positive stain that contains positively charged chromophore.When safranin was added, both opposite charged ions are attracted to each other, safranin wasabsorbed by the bacteria and coloured the cytoplasm pink in colour (Aryal and Mokobi,2020). This experiment was successfully carried out as the endospores that stained in colourgreen were clearly being observed and can be easily differentiated from vegetative cells undermicroscope.Lastly, for gram staining, three different slides were used to observe the morphologyofStaphylococcus aureus, Escherichia coliand saliva smear. The main slide in thisexperiment is the glass slide that contains mixed smear ofStaphylococcus aureusandEscherichia coli. After staining of crystal violet dye and safranin,S. aureusandE. coliwereable to distinguish asS. aureuswas purple in colour whileE. coliwas coloured in pink. Gramstaining can be used to differentiate two different types of bacteria as this method depends onthe thickness of the cell wall ofS. aureusandE. coli. As mentioned in the first part of thediscussion, when primary stain, crystal violet, was added to the smear, the opposite chargedions from basic dye and cell wall of bacteria were attracted to each other. This caused the cellwall of both bacteria to absorb the stain and coloured in purple. Next, iodine that acted asmordant was added to retain the purple stain. Iodine fixed the colour by forming a crystalviolet-iodine complex to prevent the stain from being washed away. Besides the use ofmordant, thickness of the cell wall is also an important factor that affects the solubility ofcrystal violet in bacteria. In the following step, water was used to remove stain from bacteria.

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