ELN Lab 6 5-21.docx

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Department
Microbiology and Cell Science
Course Code
MCB 3020L
Professor
ashgar

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Chloe Evetts (8990-4674) Cameron Jacobs May 21, 2013 Gram Stain, Bacterial Shapes and Arrangement Purpose: The purpose of this experiment is to identify Gram negative and Gram positive specimens on a slide. Because the two types of bacteria have different characteristics to their cell walls, the stains react differently with the specimens. Gram positive bacteria has a membrane surrounded by a thick cell wall of peptidoglycan. Gram negative bacteria has an inner membrane covered by a thin layer of peptidoglycan and then another outer membrane. The difference in thickness of the peptidoglycan layers contributes the different results of the two types of bacteria after staining. Gram positive bacteria hold the original purple stain because of its thick cell wall. Gram negative bacteria, however, will shed this stain once the alcohol is applied and then will easily take up the new pink stain. When viewing our resulting slide, gram positive will appear purple, and gram negative bacteria will appear pink. This procedure is very important to the medical community and to our society because bacteria are sensitive to certain types of antibiotics, based on if they are gram positive or negative. It is helpful to know what type of bacteria is present before prescribing antibiotics. Hypothesis: Gram staining is an effective method for identifying bacteria because the different types of bacteria (gram negative, and gram positive) respond differently to staining. By the end of this experiment, we should be able to successfully identify the gram negative and gram positive bacteria within the sample. Procedure: 1. Light Bunsen burner to create an aseptic environment, and flame inoculating loop to sterilize it. Cool loop. 2. Gram stain can be performed from liquid culture, from cells grown on an agar plate, or from an environmental sample like your cheek swab. Select sample to choose from. Follow below procedures to successfully obtain a sample. o Cells from liquid culture: Vortex culture. Use your sterilized loop or a pipette and add a drop of culture (10-20 μl) to your slide. Spread around and make a thin layer of bacteria cells on a clean glass slide from a fresh liquid culture o Cells from agar plate/colony: add a drop of water (10-20 μl) with a pipette or sterilized loop to the glass slide. Use sterilized loop and touch lightly the surface of a single isolated colony on an agar plate and mix thoroughly with the drop of water. o Cheek swab: to examine the cells and bacteria inside of your cheek or plaque from your teeth use a pipette tip and rub it on the inside of your cheek or along the gum line of your teeth. Mix the content with a drop of water on your slide 3. Allow the bacterial suspension to air dry completely. 4. Hold slide with cloth pin and use heat fixation to heat-fix the bacteria to the slide. Do so by gliding the slide through the Bunsen burner. Place slide on staining rack following the heat fixation. 5. Gently flood the smears with crystal violet (primary stain) and let stand for 1 minute. Tilt slide and let crystal violet run off. 6. Gently flood the smears with Gram’s iodine (mordant) and let stand for 1 minute. o 7. Hold the slide with the clothespin, tilted 90 and add decolorizer drop by drop to the slides until the decolorizer runs clear and no color comes of the slide (about 10-30 sec). 8. Rinse the smears in a gentle and indirect stream of tap water for 2 seconds. 9. Gently flood the smears with safranin (counterstain) and let stand for 30 to 60 seconds. 10. Rinse the smears in a gentle and indirect stream of tap water for 2 seconds. 11. Air dry and blot (DO NOT RUB) the smear dry with bibulous paper. 12. Examine dry slide under 100x oil immersion without a cover slip. 13. Gram-positive bacteria appear blue or violet and gram-negative bacteria appear pinkish red. Results: The results of this gram stain were clusters of cocci gram positive bacteria. Discussion/Conclusion: By effectively completing the gram stain procedure, I was able to identify whether the bacteria present in my unknown was either gram positive or gram negative. The bacteria present showed up purple (due to the crystal violet stain). This means that it is gram positive. The morphology is cocci, and
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