The purpose of experiment 14 is to show how important and useful the Gram stain is in distinguishing bacteria according to their cell wall composition. The experiment likewise aims for the student to learn proper Gram staining techniques so that accurate results are achieved. Experiment 15 aims to teach the Schaeffer-Fulton method which is a spore staining technique for identifying bacteria which produce highly resistant endospores.
Materials and methods: A. Gram staining Materials: ? slides with heat-fixed smears ? Gram staining kit ? wash bottle ? bibulous paper Method: First, we applied crystal violet to the smears in the slides and let it stand for about 20 seconds, after which we briefly washed the slide with distilled water, allowing the excess to drain off. Then we covered it with Gram’s iodine solution and again, let it stand, this time for a minute before decolorizing.
We did the decolorization step by tilting the slide at a 45-degree angle and then washing the slide just until the alcohol that’s running down the slide has become colorless. Next, we again washed the slide with distilled water then covered it with safranin for 1 minute. We finished the staining procedure by washing the slide with distilled water one last time, then blotted it dry with the bibulous paper before air-drying and finally viewing it under oil immersion.
B. Spore staining: Schaeffer-Fulton Method ¬Materials: ? 24-36 hour nutrient agar culture of Bacillus megaterium ? Electric hot plate ? Small beaker ? 5% solutions of malachite green and safranin Method: We first prepared a boiling water bath, over which we later steamed (for five minutes) the slide that we covered with a piece of paper towel soaked with the malachite green stain. We then removed the slide from the water bath and allowed it to cool, after which we rinsed it with water for 30 seconds.
Next, we applied the safranin for 20 seconds, then rinsed the slide again briefly with water to remove the counterstain. We blotted it dry with the bibulous paper, then examined it with a microscope under oil immersion. Data/Results: Gram staining revealed small purple dots in slide 1 and large purple dots in slide 2, representing Staphyloccus aureus and Micrococcus luteus, respectively. Unfortunately, Serratia marcescens and Pseudomonas aeruginosa failed to grow in the culture medium so no staining results were obtained for these two species.