ND03 Introduction to Gram Staining Objective: Observing and identifying samples of bacteria based on their color and overall shape with multiple staining methods. To become familiar with an objective light microscope as well as how to load a sample for viewing. Procedure: Dry Mount: 1 Clean the slide using (70% ethanol) 2 Circle area on slide to visualize where the specimen is. 3 Place organism onto slide. If from culture use the sterile loop to spread on slide. If from plate, use sterile loop to pick colonies and mix a drop of distilled water 4 Then air dry at room temp. Until moisture has completely evaporated Wet mount: 1 Clean slide (70% ethanol) 2 circle area on slide with a wax/hydrophobic pen 3 Apply organism to slide; Place organism onto slide. If from culture use the sterile loop to spread on slide. If from plate, use sterile loop to pick colonies and mix a drop of distilled water. 4 View under microscope; Wet mount is ideal for viewing the motility of an organism. Do not dry out. 1. Clean slide (70% ethanol) 2. Apply organism to slide: -use sterile loop to put 1-3 drops/slide -spread into a thin film 3. Allow to air dry 4. Fix organism to slide by passing through flame 3 times; Do not overheat slide (takes very little heat) *series of dyes- depends on the bacteria it will either be absorbed or washed out 5. Rinse with crystal violet for 30 -60 seconds- dark purple dye 6. Rinse slide with water- (stains may wash out) 7. Cover with Gram's iodine for 30-60 seconds 8. rinse with water 9. decolorize with alcohol 10. rinse with water 11. counterstain with safranin (red/pink) for 30 seconds 12. rinse with water 13. blot dry and examine
Notes: Gram Positive bacteria = Purple · Thick peptidoglycan layer, retains crystal violet stain ? Gram Negative bacteria = Pink · Thin (single) peptidoglycan layer, damaged by alcohol rinse step and crystal violet stain washed away. . Pink color derived from Safranin (secondary counterstain) Gram (-) Rods Gram (+) Chains Gram (+) Clusters ( 10um * 10 um Negative Staining: · Commonly used to identify organisms with opaque structures · Dark background via nigrosin dye · Negatively charged, repelled from membrane Results: : organism number 1: We notice the purple background (gram-positive) Round shape cluster like phenotype, This is the staphylococcus aureus sample. Organism number 2: Pink background (gram-negative), E. coli. Rod shape Organism number 3: Dark purple stain, Rod shape. Bacillus subtilis. Organism number 4: Pink background (gram-negative). Pseudomonas aeruginosa Organism number 5 : Gram-positive; Cocci (spherical) chain; Streptococcus