Gram staining - micro lab PDF

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Jasmanjeet Singh BIO 3300 Prof. Joseph Marlon 15 February 2018 Lab Report #1: Gram Staining Procedure and analysis Introduction : The Gram stain is a differential stain commonly used in the microbiology. Gram stains differentiate bacteria on the basis of their cell wall structure. Some bacteria is considered Grampositive and gram-negative, but they have different cell wall structures. Gram-negative cell walls are more complex. The Gram staining method is the most important procedure in microbiology was developed by a Danish physician. Gram-positive bacteria is characterized by having a thick layer of peptidoglycan, called a capsule. It makes up 90% of the cell wall of bacteria that are cocci shaped, such as streptococcus and staphylococcus. When stained, this type of bacteria will become purple. Gram-negative bacteria has a thin layer of peptidoglycan, and is called a slime layer. In contrast to gram-positive, it only makes up 10% of a cell wall, but has a high lipid content. –stains red/pink. When stained with the safranin, the gram-negative bacteria will be stained pink.

Objectives:

In this procedure, the goal is to differentiate between the two major categories of bacteria: Grampositive and Gram-negative. As well as to understand how the Gram stain reaction affects Grampositive and Gram-negative bacteria based on the biochemical and structural differences of their cell walls. PROCEDURE: 1. Using a sterile inoculating loop, add 1 drop of sterile water to the slide. Prepare a mixed smear of Escherichia coli (G- rod) and Staphylococcus epidermidis (G+ coccus). 2. Air dry and Heat fix. 3. Cover the smear with Crystal Violet (primary stain) for 1 min. 4. Gently wash off the slide with water. 5. Add Gram’s Iodine (mordant) for 1 min. 6. Wash with water. 7. Decolorize with 95% ethanol. This is the "tricky" step. Stop decolorizing with alcohol as soon as the purple color has stopped leaching off the slide (time will vary depending on a thickness of smear). Immediately wash with water. Be sure to dispose of all ethanol waste in the appropriately labeled waste container. 8. Cover the smear with Safranin for 30 seconds. 9. Wash both the top & the bottom of the slide with water. 10. Blot the slide with bibulous paper. 11. Using the 10X objective lens, focus first on the line and then on the smear. Follow the focusing procedure in Lab #1. Use the focusing procedure in Lab #2 to view the smear using the 100X (oil immersion lens). Materials: - Glass Slide - Escherichia coli in broth culture -Escherichia coli in agar culture - Bacillus sp. in broth culture - Bacillus sp. in agar culture - Staphylococcus aureus in broth culture - Actinomycetes sp. in broth culture - Actinomycetes sp. in agar culture - Kimwipe - Bunsen burner - Dropper - Distilled water - Inoculation loop Discussion/ conclusion:

They were four reagents that were in the gram staining experiment. Mordant attach the crystal violet tightly to the wall of gram-positive cells. Primary stain initially stains the cells wall of both gram-positive and gram-negative bacteria. Decolonize removes the crystal violet from the gram-negative cells walls. Lastly was counterstain stains cells that are point are colorless (gram- negative cells). In this game stain experiment, the cell wall is the most involved because of this the site where gram-positive cells retain crystal violet and where gram-negative are decolorize by a mixture of isopropyl alcohol and acetone. Young culture has an importance in the gram stain experiment because old cells tend to stain gram-negative due to the cell wall deterioration regardless of their origin. Gram staining is important in differentiation gram positive and gram negative bacteria in which the gram positive and gram negative bacteria in which the gram-positive bacteria stained purple color while gram-negative organism. stained pink. Escherichia coli is Gram-negative while Bacillus sp., Staphylococcus aureus, and actinomycetes are Gram-positive bacteria....