Use and Care of Microscope, Aseptic Technique, Smears, Simple Stains

Aseptic Technique Principle: Proper aseptic technique prevents contamination of cultures from foreign bacteria inherent in the environment Microbiologists use aseptic technique for a variety of procedures such as transferring cultures, inoculating media, isolation of pure cultures, and for performing microbiological tests.

Aseptic Technique Flaming the loop :  Holding the loop in the flame of the Bunsen burner kills all contaminating organisms, thus sterilizing the loop.  After flaming the loop , make sure to slightly cool the loop before picking up organisms from the inoculum culture (the culture that is to be transferred.) stab loop

Aseptic Technique Flaming the Mouth of the Test Tube:  Passing the mouth of a tube through the flame of a Bunsen burner creates a convection current which forces air out of the tube.  This prevents airborne contaminants from entering the tube. The heat of the Bunsen burner also causes the air around your work area to rise, reducing the chance of airborne microorganisms contaminating your cultures. 

Smears-Clean Slides Clean both sides of glass slide with powdered soap Rinse well. Add a few drops of alcohol, Dry with Kim Wipes

Smears-Transfer of Organism Transferring bacteria from Slant: Flame Loop. Place a drop of water into the center of a clean, dry slide Re-Flame loop, allow to cool and sterilely transfer bacteria from the slant stock culture to your drop of water Spread water drop/culture mix with loop to about a quarter size circle Sterilize loop Allow to air dry

Smear-Heat Fixing Once the slide is air-dried, pass it briefly through the burner flame smear side up DO NOT COOK THE SLIDE Allow to cool before staining Purpose of heat fixing: Kills bacteria Coagulates proteins-adheres cells to slide Opens pores for easy penetration of stains

Simple Stain Microorganisms are invisible and cells are transparent under a microscope Staining makes examination easier Simple staining uses a single dye Size Shape Arrangement Method: cover previously heat fixed slide with dye. Allow to react one minute. Rinse with water. Dry with Bibulous Paper

Bacterial Stains Biological Dyes are salts- composed of two parts; cation (+) and an anion(-) If the chromophore of the stain is carried on the anion portion of the salt it is acidic These stains will react with the positive portions of the cell EXs: nigrosin, congo red, india ink If the chromophore is a cation, the dye will react with the negatively charged outer membrane of the cell and stain these components. These dyes are basic Ex-crystal violet, safranin, carbolfuschin Neutral Dyes contain color agents in both cation and anion portions of the dye and will stain all components in a cell (we will not use these in this course)

Microscopes Please refer to your lab manual (pgs 8-9) for parts and functions of the compound binocular microscopes we will use in this course Your ability to focus these microscopes independently is paramount to your success in this course

Microscopes Resolution: the ability to discern fine detail Calculating total magnification: (mag of ocular lens) x (mag of objective lens) Calculating Resolving Power: RP= wavelength of light (nm) 2 x N.A.