Staining- Stainingo Staining = coloring key structures of a microbe using a dyeo Smear = thin film containing a solution of microbes fixed and attached to a slide Fixing the microbes usually kills them ☹ - Staining Basicso Simple Stain Uses one basic stain, so the cells are all the same color  Increased contrast between cells and backgroundo Steps for a Simple Stain Smear -> air dry -> fixing (using heat) -> stain -> rinse -> dry -> examineo Basic Dyes Positive charge  Bonds to negatively charged parts of the cello Acidic Dyes  Negative charge Bonds to positively charged parts of the cello Mordant Maintains the integrity of the stain Enlarges specimen- Shapes and Arrangements of Bacteriao Shape #1: Cocci (Coccus)  Can be singular, pairs, chains, tetrads, and clusters Spherical  Example: Streptococcus (chain of spherical-shaped bacteria)o Shape #2: Bacilli (Bacillus) Rod-shaped- Can also be coccoid shaped - Curved rods = vibrios Singular or short chains  Example: Bacillus anthracis o Shape #3: Spirilla (Spirillum) Spiral-shaped (like a corkscrew pasta noodle �) Flexible helix = spirochete  Example: Heliobacter pylorio Arrangements  One plane of division = pairs and chains- Pairs = diplo (e.g. diplococci) - Chains = stepto (e.g. streptococci) Two-three planes of division = tetrads and cubical packets  Random planes of division = clusters - Clusters = staphylo (e.g. staphylococcus)- Gram Staino Differential staino Gram positive bacteria stains purple Bacillus, Streptococcus, Staphylococcus, Mycobacteriumo Gram negative bacteria stains pink/red Escherichia, Heliobacter, Pseudomonas, Salmonellao Differences in color is a result of differences in the structure of the cell walls- Gram Stain Process o Step 1: Primary Stain Crystal violet  Stains both positive and negative bacteria Gram Positive BacteriaGram Negative Bacteriao Step 2: Mordant Gram’s Iodine Forms large crystals of dye Gram Positive BacteriaGram Negative Bacteriao Step 3: Decolorizer  Alcohol/alcohol acetone Removes crystal violet from Gram negative bacteriaGram Positive BacteriaGram Negative Bacteriao Step 4: Counterstain Safranin (red) Stains Gram negative bacteriaGram Positive BacteriaGram Negative