BIO 171- Microbiology

Main reason for using wet mount is to be able to view motility of organism. Do NOT let dry out- reapply distilled water if needed to keep organism viable. Allows to see flagella- some are not motile and stay in a fixed location. Can be a key to diagnosing pathogen. Gram Staining: 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: purple -thick peptidoglycan layer; keeps crystal violet color GRAM NEGATIVE: pink -thin peptidoglycan layer and is damaged by alcohol rinse & crystal violet rinsed away; pink color is from counterstain (Safranin) Once stained you can identify based on color/shape: Gram positive: Purple- bacteria in chains/clusters (cocci shaped) Gram negative: Pink- bacteria in rod shaped Gram staining isn’t always guaranteed to identify samples ACID FAST STAINING: -strong resistance to decolorization -very few structures are acid fast -commonly used to identify mycobacterium (TB) -carbolfuchsin dye retained (red); bacteria will remain red on blue background NEGATIVE STAINING: -used for organisms with opaque structures -dark background (Nigrosin) both dye and cell membrane negatively charged so dye is repelled - will be able to see the clusters and shapes of bacteria WET LAB: 1. put on PPE ( gloves, eye wear, lab coat) 2. put samples in tube and ran the top of tubes through flame to sterilize 3. cleaned slides with chem wipe 4. draw circle on slide with wax pen to keep bacteria in circle 5. flame top of tube again 6. put loop in tube and swirl to collect sample 7. spread thin layer inside circle on slide (wax keeps liquid inside) 8. let air dry 9. heat fix by running slide over flame 3 times – should be no liquid lef 10. repeat with remaining 4 samples *sterile loop should be disposed of in biohazard waste bin as well as anything else and put in autoclave to be sterilized before disposal. APPLYING STAIN TO EACH SAMPLE: Supplies needed: 5 specimens, tray to dye slides in, distilled water, chem wipes, clips, timer, crystal violet, alcohol, iodine, safranin 1. Cover entire circle with crystal violet dye for 1 min (all slides done simultaneously) 2. rinse off with water until no more color comes off 3. cover slides with iodine- apply with eye dropper don’t touch specimen (sit for 1 min) 4. rinse thoroughly with water 5. decolorization with alcohol can see some losing dye while others retain it 6. rinse thoroughly with water 7. counterstain with safranin thoroughly let sit for 45 seconds 8. final rinse –thoroughly 9. blot dry with wipe and then let air dry *make sure to throw wipes and everything in biohazard waste bin 10. gently use wipe to make sure slide is dry –remark if wax pen came off EXAMINE SLIDES UNDER MICROSCOPE: -40x magnification; stage lowered all the way -open clips and place slide on stage to secure -illumination set at 50% -diaphragm open all the way -no longer any liquid- take off eye wear -Focus microscope: 1. raise sample to come into focus using coarse adjustment; make sure sample is in light field 2. dark image in focus use fine focus to make image clear; move stage if needed Results: Organism 1: (Gram +); Purple color; round cluster- Staphylococcus Aureus Organism 2: (Gram -); Pink color; rod shaped; E-Coli Organism 3: (Gram +); dark purple; rod shaped- Bacillus Subtilis Organism 4: (Gram -); pink color; rod shaped; Pseudomonas Aeruginosa Organism 5: (Gram +); purple color; spherical shaped w/chain structure; Steptococ