Lab 2: Staining Bacterial Cells *Burners: Be careful! Pull back long hair… rubber bands on computer Remove dangling scarves Put away everything but essential! Be careful with dangling sleeves….

Why stain bacteria? Enables us to see them Aids with classification Hay infusion: lots of bacteria, hard to see since they were not stained (clear MOs in clear liquid) Aids with classification No limbs or leaves to help us identify bacteria! Use different results from staining to help ID

Smear Prep, Heat Fixing Draw a LONG OVAL on your slide with a Gram stain pen, label with name of culture Fill oval with bacteria using aseptic techniques (later) BEFORE staining: Slide must be air dried only THEN heat fix: pass slide through a flame: this keeps cells from being washed off and preserves their arrangement and morphology Name

Types of stains Simple stains: use ONE dye and ALL cells are stained the SAME color Simple direct stain: cell is stained Bacteria have a negative charge and dye has a positive charge; therefore, color is attracted to the cell (eg., Methylene Blue—actually a chloride salt) Negative stain: stains the surroundings Dye has a negative charge; therefore, color is repelled by the cell

Types of Stains (con’t) Differential stains: more than one dye used and certain properties of bacteria allow them to stain different colors Gram stain (today’s example) Primary stain: Crystal Violet All bacteria stain purple Mordant: Gram’s Iodine Acts to hold crystal violet in cell walls of Gram + cells Decolorizer: Ethanol Removes color from Gram – cells but Gram + cells remain purple Counterstain: Safranin Gram – cells stain red/pink and Gram + remain purple

Why do Gram + and Gram – cells react differently to decolorizing? Cell wall contains a thick layer of peptidoglycan and teichoic acid Thick peptidoglycan layer holds crystal violet Gram – Cell wall contains a thin layer of peptidoglycan and NO teichoic acid Also contains a complex outer layer composed of lipopolysaccharide (LPS) Layers not thick enough to hold purple dye

Cell walls

Morphology coccus (cocci) = spheres bacillus (bacilli) = rods spirillum (spirilla) = spirals Arrangement: (subjective!) staphylo- = in bunches like grapes (Staphylococcus) strepto- = in chains (Streptococcus)

Always use ASEPTIC TECHNIQUE Flaming loop, flaming lip of tube, not touching or breathing on plates, loops, or inside of tubes, keeping caps on… Lysol bench before and after use … All transfers done to prevent unwanted microbes getting in, only the one you put there is there Very Important, you will use all semester We will demo aseptic technique for you

Lab Flow DEMOS of Techniques Draw a large/Long oval in the center of 3 slides Label slides: Mixed culture: tells you which side of slide contains bacteria Apply loopfuls of culture until have a thin covering filling oval on all slides Allow slides to air dry completely, only then heat fix all the slides While waiting for slides to dry, view Simple stain (Methylene Blue) of Mixed culture Demo Gram Stain Choose 1 slide to Gram Stain View and draw on 1000x total magnification Have a TA look at your Gram stain technique Repeat Gram staining using the TAs suggestions until you run out of time. Discard all slides in the lysol bin. Get TA initials on your Gram stain (if its correct)