Simple Stains & Gram stain

Making a smear 1. clean the slide - Take slide out of beaker, and wash with detergent - Rinse really well, and clean with alcohol. - Once slide is clean, label one side so you know the top from the bottom. 2. Flame loop to sterilize (aseptic technique) 3. Once loop is cool, place under tap water and place loopful of water on slide. 4. Reflame loop before taking a little bit of culture and smear it into tap water. Should be about the size of a quarter. 5. Let air dry

Heat fixing Once smear has air dried, inspect it to see if it cloudy; if you can not see through it you have too many cells. Using a bunsen burner or fireboy, pass the smear through the flame, being careful not to burn yourself or the smear. Check the temperature of the slide on the back of your hand, it should just be warm to the touch.

What does heat fixing do? 1. Kills the bacteria 2. Coagulates the proteins of the cells and adheres them to the slide. 3. Opens the pores of the cells, so they can accept the dye.

Staining Microorganisms are almost invisible by the naked eye, and even under a microscope are almost transparent. Staining the cells, allows for examination of bacteria. Allows the researcher to: 1. look at shape 2. look at size 3. look at arrangement

What types of dyes? Simple staining only uses on type of stain, so all bacteria will be the same color. Dyes are salts made up of two parts: cation (+) and anion (-) Acidic dyes- if the color portion is carried in the (-) portion of the dye; will react with (+) portion of the cell (cytoplasm). Basic dyes- if the color portion is carried in the (+) portion of the dye; will react with (-) portion of cell (outer membrane). Used for identification since bacteria carry a net negative charge on the surface. What we will use in today’s lab.

Steps to Staining 1. Place newly made smear on rack on sink. 2. Flood slide with dye- methylene blue 3. Let it react for 1 minute, then gently rinse with tap water until water that runs off is clear. 4. Gently dry slide with bibulous paper; pat and move the slide until it is dry.– DO NOT USE PAPER TOWELS 5. Make labeled drawings of each organism– arrangement and shape.

Ocular piece Binocular tube Revolving nose-piece Objective lenses- 4x- scanning 10x-low power 40x- high power dry 100x- high power oil-immersion Condenser Stage adjustment knobs Lamp intensity dial Focusing knobs- course and fine Eye piece scale Interpupillary scale Base Power button Arm

Microscopes Antoin Van Leeuwenhoek Compound light microscope- two lenses Total magnification= eyepiece (10x) X objective lens Resolution-ability to discern fine detail. High energy light plus high gathering light lenses= sharp resolution Resolving Power (RP)= λ (nm)/ (2)(N.A.)

Arrangements and Shapes Rod (bacillus)- rod-shaped, can be single or in chains. Cocci- circular, can be single, clusters, chains, or pairs. Vibrio- comma shaped. Spirochetes- spiral shaped.

Gram + Gram - Developed by Christian Gram He noticed that some bacteria retained dye while others washed out. Differential stain- shows differences among bacteria. Gram positive bacteria- have thick peptidogylcan layer with teichoic acid. Gram negative bacteria- have thin peptidoglycan layer in cell wall that is covered with lipopolysaccharide layer (LPS).

Steps to Gram-Staining 1. Prepare a smear and heat-fix- put both cultures on same slide so you can compare. 2. Flood both smears with crystal violet (primary dye), react for 1 min. 3. Rinse slide until water runs off clear. DO NOT DRY, shake off excess water. 4. Flood both smears with Iodine (mordant), react for 1 min, then pour off Iodine. 5. Flood both smears with decolorizer for 4-10 sec, as soon as stain stops coming off STOP. 6. Immediately rinse with water, shake off excess. 7. Counterstain with Safranin for 1 min, rinse with water then blot dry. 8. Examine under 100x lens.