الرئيسية
أحدث الصور
همس القمر2011 الخميس نوفمبر 03, 2011 7:09 amGram-Staining
IntroductionMost common bacteria are either Gram-positive or Gram-negative (based on cell wallstructure). Remember from lecture, Gram-positive cell walls consist of several layers ofpeptidoglycan (cross-linked by teichoic acid and lipoteichoic acid). Gram-negative cellwalls have one layer of peptidoglycan surrounded by a lipid-based outer membrane. Inthe 1880s, Hans Gram developed the differential method of staining that bears his name.While we still don’t know exactly why Gram-positive bacteria end up looking differentfrom Gram-negative bacteria, Gram-staining is still an important way to characterizebacteria.Gram-staining begins by getting cells to stick on a clean microscope slide. Such a prep iscalled a bacterial smear. To a bacterial smear the following chemicals are applied tomake a Gram-stain:
1. Gram’s Crystal Violet StainThe smear is flooded with Crystal Violet for 60 seconds. Crystal violet is a purplechemical that sticks to the peptidoglycan layer of the bacterial cell wall.After 60 seconds, crystal violet is rinsed off using distilled water. 2. IodineThe smear is next flooded with Iodine for 60 seconds. The iodine is called amordant—it causes crystal violet to stick to peptidoglycan like mortar causesbricks to stick together. After 60 seconds, iodine is rinsed off using distilled water.3. Acetone/Ethanol WashThe smear is next flooded with Acetone/Alcohol for JUST 5 SECONDS. TheAcetone/Alcohol washes crystal violet out of the Gram-negative cell wall. We’renot really sure why this happens, but it does. The Gram-positive cell wall retainscrystal violet as long as the acetone/alcohol wash lasts not more than a few seconds.After 5 SECONDS, the acetone/alcohol is rinsed off using distilled water. Theacetone/alcohol wash is the differential step in the Gram-stain process. That is, itis the acetone/alcohol that creates the observable difference: Gram-positive cellslook purple after this step; Gram-negative cells look clear.4 . Safranin StainThe smear is flooded with Safranin for 90 seconds to two minutes. Safranin is apink stain that sticks to cytoplasmic components of the cell. All cells becomestained with Safranin. Gram-positive cells are pink on the inside, but you can’t seethis because they are dark purple on the outside (kind of like a bon-bon). Gramnegativecells, which were cleared in the previous step, end up looking pink. After90 seconds to two minutes (the longer the better), Safranin is rinsed off withdistilled water.5. Drying the slideLM1 Ubiquity Clark College Kibota 2The completed Gram-stained slide is stuck into a book of bibulous paper andallowed to dry for a minute or two. Once dry, the slide is ready for observation.ACTIVITIESActivity 1. Cleaning a slide (each person clean two slides)1. Obtain a slide from your drawer. Make sure the slide does not have broken edges.2. Go to one of the two main lab sinks.3. Wet the slide and your fingers.4. Shake some Bon Ami cleanser on your fingers.5. Scrub the slide with your Bon Ami’d fingers to get rid of any grit on the slide.6. Remove the cleanser by rinsing with tap water.7. Soak the slide in a Coplin jar full of isopropyl alcohol (one minute). This willremove any oil that may remain on the slide.8. Remove the slide (holding it by the edges). You may allow the slide to air dry oryou may dry it with paper towel.Activity 2. Making a bacterial smear from a slant stock culture (each person)1. On a clear slide, put a SMALL drop of water (tap or distilled). The smaller thedrop, the better.2. Using aseptic technique, transfer a VERY SMALL amount of cell material from aslant stock culture to the water droplet.3. Gently mix the cells into the water droplet, then spread the water into a thin film.4. Allow the film to air dry until there is no visible liquid on the slide. It isIMPORTANT to allow complete air drying (this is why you want your waterdroplet to be small).5. Heat fix the smear by passing the slide (cell side facing the ceiling) through thecool part of the flame (the top of the flame above the blue cone) three times at adeliberate pace.a. Do not use the hot part of the flame. Do not pass the slide through the flamemore the three times. Do not let the slide linger in the flame. All of thesemistakes will cause you to cook the cells.b. Heat fixing accomplishes three important things:i. Residual water is evaporated and the cells become stuck to the slide.Without heat fixing, the cells will readily wash off the slide during thestaining process.ii. The cells are killed. After heat fixing, the slide is safe to handle.iii. The cells more readily take up stain.6. Allow the slide to cool for a minute or two.Activity 3. Making a bacterial smear from a broth stock culture (each person)1. Using aseptic technique, transfer several (four to six) loopfuls of the broth stockculture to the slide. You need to use several drops to make sure you have enoughcells on your slide to be successful.2. Spread the broth into a thin film.3. Repeat steps 4-6 from Activity 2 above.LM1 Ubiquity Clark College Kibota 3Activity 4. Gram-staining the smears1. Working on one smear at a time, perform Gram-staining using the steps listedabove.Activity 5. Microscopic observations of the Gram-stainsTo observe cell structure and stain color, YOU MUST VIEW CELLS THROUGHTHE OIL IMMERSION LENS (total magnification = 1000x). At lowermagnification, the cells will be too small to reliably determine shape, arrangement, andcolor.1. Observe the smear using the 4x, 10x, and 40x lenses. Follow the tips for focusingthat were given in Module 3: Microscope Basics.2. When in focus at high power, move the 40x lens out of the way.3. Place a drop of immersion oil on the slide in the field of view.4. Rotate the 100x (oil immersion) lens into place.a. CAUTION: The 100x lens is built to be put into oil. The 40x lens is NOT.Oil will leak into the barrel of the 40x lens and ruin it. NEVER, EVERUSE THE 40X LENS WHEN OIL IS ON THE SLIDE.5. Use the fine focus adjustment to focus the view. Be very subtle with your focusing.Even the slightest exaggeration of focusing will cause you to overshoot.Sometimes, focusing with the oil immersion lens takes a great deal of patience.Remember to jiggle the slide back and forth.6. Describe cell size, shape, arrangement, and Gram-reaction for your two smears.Questions1. What color are Gram-positive cells supposed to be after a Gram-stain? What aboutGram-negative cells?2. What characteristics can be determined in a Gram-stain?3. List all of the things that can go happen during the Gram-staining process that couldlead to an incorrect or poor result 
[justify][left]
