front 1 simple stains | back 1 staining procedure that only uses one stain and all bacteria are stained similarly used for cell morphology, size, arrangement |
front 2 differential stains (gram) | back 2 first step in identifying bacteria uses two contrasting stains seperated by a declorizing agent |
front 3 Acid fast | back 3 used to keep stain in place when washed with acid |
front 4 Spore stain | back 4 used to observe endospores, capsules and flagella |
front 5 types of staining techniques | back 5 aa |
front 6 smears | back 6 speading a small amount of bacteria broth culture or solid inoculum mixed with a drop of distilled water on a clean slide allowing to dry |
front 7 heat fixing a sample | back 7 passed over incommator several times or placed over a slide warmer |
front 8 chemically fix the sample | back 8 cover smear with 95% methanol for 1 min |
front 9 what is the purpose of heating/ fixing the smear? | back 9 to kill the bacteria and preserves with minimal distortion/shrinkage |
front 10 simple stains | back 10 bacteria cell surfaces negatively charged and basic dyes are used as stains use of a single basic dye is called a simple stain doesnt tell you if its gram positive or negative |
front 11 common basic dyes | back 11 1. methylene blue 2. crystal violet 3. safranin 4. malachite green |
front 12 simple staining steps | back 12 1. use a clothespin to hold slide 2. cover slide with methylene blue and leave for 30-60 sec 3. wash off excess stain with distilled water 4. blot smear with paper towel and air dry |
front 13 which bacteria is a rod? | back 13 bacillus megaterium |
front 14 which bacterium is larger | back 14 bacillus megaterium |
front 15 what value is a simple stain? | back 15 enhances the contrast between the bacterium and its surrounding material and permits greater clarity of detail |
front 16 a | back 16 B |
front 17 another method of fixing a smear? | back 17 chemically fixing |
front 18 how does alcohol chemically fix the bacteria? | back 18 1. removes water (imp for mounting the cell) 2. denatures proteins (metabolism of the cell is stopped and cell dies) 3.it dissolves and removes lipids (cell membrane of bacteria is harmed by alcohol) |
front 19 negative staining | back 19 does not stain bacteria stains background |
front 20 dyes used for negative staining | back 20 india ink nigrosin |
front 21 steps in negative staining | back 21 1. add drop of nigrosin on clean slide 2.aseptically add organism 3.place second clean slide on the surface of the first slide and draw it back into the drop 4.when drop flows across the width of spreader slide 5. push spreader slide to other end 6. air dry |
front 22 which cell is a rod? | back 22 bacillus subtilis |
front 23 why is the size more accurate in a negative stain than in a direct stain? | back 23 No heat fixing or chemicals are used so there is no distortion of the cell |
front 24 what microscopic technique gives the field similar appearance to that seen in a negative stain | back 24 dark field microscope |
front 25 could any dye be used in place of nigrosin for negative staining? | back 25 no only a cell that does not penetrate the cell, india ink works |
front 26 why can carbolfuchsin be used as a direct stain and as a negative stain? | back 26 ph is basic |
front 27 Acidic congo red does not conatin suspended particiles like nigrosin but can give the appearance of a negative stain. what is the basis for this stain? | back 27 b/c it it acidic ( negative charge ) stain cannot bind to negatively charged bacteria |
front 28 Gram staining steps | back 28 1. apply primary stain (crystal violet) 30 sec bacteria stained purple (gram plus and minus are blue) 2. apply mordant (gram iodine) 10 sec (gram plus and minus blue) 3. apply decolorizing agent (ethanol)10-20 sec (gram plus blue gram minus colorless) 4. apply secondary stain or counter stain (safranin) 30sec the dye stains decolorized bacteria red (gram plus blue glam minus red |
front 29 Colors Crystal violet Safranin | back 29 crystal violet-purple safranin-red |
front 30 use of mordant | back 30 grams iodine binds to the crystal violet preventing the compound from being clear thru the cell wall of the gram positive bacteria |
front 31 gram positive cell wall | back 31 thick layer of peptidoglycan |
front 32 gram negative cell wall | back 32 thin peptidoglycan layer covered by a lipopolysaccharide layer |
front 33 no data | back 33 gram negative bacteria |
front 34 no data | back 34 gram positive bacteria |
front 35 no data | back 35 mycobacteria |
front 36 no data | back 36 fungi |
front 37 acid fast staining | back 37 differential stain |
front 38 principal behind this staining | back 38 carbolfuschin binds to mycolic acids on the outside of the acid-fast bacteria which is not washed away with the acid-alcohol mixture that decolorizes all other bacteria |
front 39 dyes used for acid fast staining | back 39 carbofuschin and methylen blue |
front 40 acid fast steps | back 40 1. prepare fixed smear of culture 2. cover the smear with carbolfuchsin for 5 min 3. wash with distilled water 4. wash with decolorizer (acid alcohol) for 1 min 5. wash water 6. counterstain with methylene blue for 1 min 7. examine slide under microscope |
front 41 Endospores | back 41 "resting bodies" do not metabolize resistant to heat chemicals and harsh environments, are formed when nutrients are not available generally impermeable to stains so heat must be applied |
front 42 capsule | back 42 increase virulence(disease causing ability) decrease being phagocytiezed |
front 43 Flagella | back 43 motility location and presence are helpful in identifying bacteria |
front 44 Steps in staining endospores | back 44 1. make smear let air dry, and heat fix 2. tear small piece of paper towel and place over smear 3.cover smear and paper with malachite green and steam the slife for 5 mins (keep wet) 4.remove paper and throw away, wash smear with h20 5.counterstain with safranin for 30 sec 6.wash with h20 30 sec 7.examine under microscope |