Microbiology 101 - Lab 4A, 2/15/10 Finish broth cultures **Handle tubes gently so you will not disturb the growth** Procedure: Observe growth in culture tubes and describe as turbidity, pellicle, or sediment type growth. Write results in notebook. Why did you do the sham-inoculation? Size, Shape, and Motility of Microbes Objective: Be able to microscopically examine material in a liquid medium and stained smear. Observe the size difference between prokaryotic and eukaryotic cells and between stained and unstained cells. Terms: Motility: the ability of an organism to move by itself from one location to another. Brownian motion: Quivering at the same or close location caused by random movement. Wet Mount Cell types and characteristics: Saccaromyces cerevisiae: brewer's yeast, eukaryote, 5-10 Ám RBC's (red blood cells): eukaryotic, lack nuclei, biconcave discs, ~7.5 Ám in diameter Staphylococcus epidermidis: bacteria, cocci, non-motile, 0.8 - 1 Ám in diameter Proteus vulgaris: bacteria, rod-shaped, motile 2 - 3 Ám long, 0.6 Ám width. Procedure: Using a sterile loop, place a couple of loopfuls of liquid culture in the middle of a clean glass slide. Carefully place a cover slip over the drop of culture. View under microscope at 10x, 40x, and 100x focusing first at the edge of the cover slip (cells may be easier to see if illumination is decreased). When finished place slides in bleach water and return to slide boxes. Discard cover slips in the sharps container. Direct Stain / Indirect Stain Objective: To learn the principles and procedures for different staining methods. Principles: Staining increases the contrast between the cells and their surroundings. Staining makes focusing with a microscope easier and allows observation of bacterial morphology, internal structures, etc. Types of stains: Simple stain (basic or acidic stain): one step is needed. All the cells have the same color after staining. Differential stain: detect differences in microbes based on chemical properties (e.g. gram stain, acid-fast stain) Structural stains: special stains for detecting certain structures (e.g. flagella, endospores, cell wall, etc) General concepts of staining: Dyes: Dyes are salts in which one ion is colored. Basic stain: positive ion is colored. Used for direct staining. Acidic stain: negative ion is colored. Used for indirect staining. At neutral pH, bacterial cells have a slight negative charge. A change in pH can change the surface charge and cause changes in staining characteristics. Staining with basic stains: Colored positive ions of basic stain are attracted to negatively charged cells. This is direct stain because the cells are directly stained. Staining with acidic stains: Colored negative ion repelled from negatively charged cell surface. Staining results in a colorless cell on a stained background. This is called an indirect stain because cells themselves are not stained, but the contrast between the cells and the colored surrounding is increased indirectly. Examples of several common stains: Basic stains: Methylene blue Crystal violet Safranin Carbol fuchsin Acidic stains: Eosin Nigrosine Comparison between direct and indirect stain: Indirect Direct Dye used Acidic dye (-) Basic dye (+) Staining results Colorless cells and colored background Colored cells & colorless background Heat fixing No Yes Rinsing after staining No Yes Adv vs. Disadv Clearer picture of cell size and shape Distortion of true cell size Procedures: Direct Staining: Mark your slide to indicate which side your smear is on Using your loop make a thin smear of bacterial mixture on a slide Air dry 5-10 minutes and heat fix (heat fixing attaches cells to the slide). Stain with methylene blue (~60 seconds) Rinse with water Blot slide (GENTLY) with paper towel Observe cells under microscope from low power up to oil immersion Slides can be saved for future reference or soak in bleach water Indirect Staining: Add one drop of nigrosine stain to one end of a slide. Transfer two loops of the bacterial mixture to the drop of stain. Add the nigrosine to the slide before adding the culture to prevent contamination of the bottle of stain. Use a second slide to spread the stain over the surface. (The purpose of this is to create a different thickness of the stain along the slide.) Air dry completely. Observe under microscope using low power, high dry, and oil immersion. Slides can be saved for future reference or soak in bleach water. **Notes: Klebsiella pneumoniae - rod shaped, Micrococcus luteus ? cocci There will be a lab report on wet mount, direct stain, and indirect stain. The report will be due on 5A (February 22). Make notes and observations of your slides and procedures (draw each slide). PAGE 3
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