Discussion: SIMPLE STAINS I) Staphylococcus aureus The shape of Staphylococcus aureus is spherical or in other words, they were seen

een in clusters of cocci. The size of this bacteria is small. Staphylococcus aureus is arranged like a bunch of grapes.

II) Candida albicans Candida albicans is oval shaped. It is a yeast that exists in bundle or in a unicellular structure.

III) Escherichia coli Escherichia coli is a rod shaped bacterium. It is short and it has individual arrangement. It is not clustered together like Staphylococcus aureus.

IV) Bacillus subtilis Bacillus subtilis are rod shaped bacteria. Appears to have individual arrangement. Has noticeable space between each bacterium.

Simple staining is a method that is used in microscopy to enhance the visualization of microscopic images. Stains and dyes are the two major chemicals that are often used in simple staining to accentuate the structure and the morphology of the bacterial cell. In this experiment, methylene blue solution was used to stain the bacterial smears. Why is methylene blue used to stain the bacterial cells in this experiment? Since methylene blue is a basic dye, it has a high affinity for acidic substances. The presence of negatively charged molecules in the cells causes it to be attracted to the positively charged dye. Due to the staining procedure, the morphology of the bacterial cell will be clearly seen when examined under the oil immersion. From the observation, we can infer that both Escherichia coli and Bacillus subtilis are rod shaped bacteria. However, we can note the difference in their arrangment. Escherichia coli is arranged in an individual manner with noticeable space between each cell, whereas for Bacillus subtilis, each rod appears to be longer than of Escherichia coli. The bacteria Staphylococcus aureus appears oval shaped, like a bunched grape like clusters. It is also very small in size compared to the other three bacteria.

Candida albicans is a type of yeast which is found in everyone in small amount. The bacterium is oval in shape and is often the cause of yeast infection known as Candidiasis. Conclusions: I) II) The morphology of the bacteria were observed with the aid of simple stain. Simple staining technique is the best method to observe the morphology, size and the arrangement of bacteria.

DIFFERENTIAL STAINING I) Klebsiella aerogenes Klebsiella aerogenes that were observed were stained red. This indicates that it is a gram negative bacteria.

II) Escherichia coli Escherichia coli are gram negative bacteria. They were observed to be red in colour.

III) Staphylococcus aureus Staphylococcus aureus is a gram positive bacteria. They were observed to be stained blue but with slight hints of pink.

IV) Bacillus subtilis Bacillus subtilis is a gram positive bacteria. The slide observed showed that the bacteria was stained blue with very light tints of pink.

The Gram staining method is a technique that was invented by a Danish bacteriologist Hans Christian Joachim Gram in 1884 to classify bacteria into two major classes, that is gram positive bacteria and gram negative bacteria. This technique is still widely used until today. Gram positive bacteria are stained dark blue or violet when observed under the microscope whereas gram negative bacteria are stained pink in colour. Gram negative bacteria are unable to retain the crystal violet stain, thus taking in the colour of the counter stain, safranine (red). Klebsiella aerogenes and Escherichia coli are gram negative bacteria. This is because they have a thin layer of peptidoglycan that causes them to lose the blue colour of the crystal

violet dye (primary stain) when they are decolourized with alcohol(ethanol). Thus, they absorb the red colour of the safranine causing both the bacteria to appear red. However, it should be noted that gram negative bacteria are more resistant to antibiotics compared to gram positive bacteria despite their thin layer of peptidoglycan. This resistance is because the former possess a lipid rich membrane that is relatively impermeable. Staphylococcus aureus and Bacillus subtilis are gram positive bacteria. Both the bacteria have a thick layer of peptidoglycan that causes them to retain the colour of the crystal violet dye. These bacteria have a high amount of peptidoglycan in the cell wall that enables them to retain the colour of the primary stain. Conclusions : I) II) The Gram staining procedure is the simplest method to classify bacteria into gram positive and gram negative. Staphylococcus aureus and Bacillus subtilis were identified to be gram positive bacteria. Klebsiella aerogenes and Escherichia coli were identified to be gram negative bacteria.

Questions : 1) What colour would you expect Staph.aureus to be if the iodine step was omitted in the gram staining procedure? The role of iodine in gram stain is to act a mordant (a substance that sets dye). When used in the gram staining procedure, it is also known as a caustic. It functions to form large crystals with the crystal violet dye. When the crystal violet dye mixes with iodine, it will form a crystal violet iodine (CVI) complex. This complex is larger than the crystal violet molecule and is easier for the gram positive bacteria to retain the blue dye. If the iodine step was omitted in the gram staining procedure, the crystal violet dye that is used will not be able to form the CVI complex. Therefore, Staph.aureus will be decolourized by the alcohol and will appear to be red in colour (gram negative) when observed under the microscope. 2) Why would methylene blue not work just as well as safranine for counterstaining in the gram staining procedure? Initially, we use the crystal violet dye to stain the bacterial cells. The colour of this dye can vary from purple to dark blue. If methylene blue was used for counterstaining, it would be difficult to differentiate between a gram positive bacteria and a gram negative bacteria as methylene blue and the crystal violet dye have almost the same colour. Therefore, methylene blue will not work as an effective counter stain. However if safranine is used, we can clearly see the contrast between the blue colour of

the crystal violet dye and the red colour of the safranine making it easier for us to distinguish between a gram positive and a gram negative bacteria.