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THE GRAM STAIN TECHNIQUE 6 49 the Gram 6 Stain Technique 43038_CH06_0049.qxd 1/3/07 3:44 PM Page 49 THE GRAM STAIN TECHNIQUE 6 49 The Gram 6 Stain Technique n 1884 Christian Gram, a Danish physician, discovered that certain bacteria, after being stained with crystal violet and I iodine, would lose their color on subsequent treatment with alcohol while other bacteria would retain the color. Bacteria could thus be separated into two groups on the basis of their reaction to this procedure. Those retaining the stain are known as gram-positive bacteria, while those losing it are gram-negative bacteria. The Gram stain technique is perhaps the most important differ- PURPOSE: to differentiate between gram-positive and ential staining procedure in microbiology because the great majority gram-negative bacteria; to of bacteria are either gram-positive or gram-negative. (Spirochetes determine cell size, shape, and mycobacteria are notable exceptions—they do not respond to the and arrangement. procedure.) The differential staining has been elucidated through electron micros- copy. The crystal violet stain and iodine form a complex that exits easily from gram-negative bacteria but not from gram-positive bacteria when alcohol is applied. Also, large amounts of peptidoglycan in the cell walls of gram-positive bacteria trap the complex. Gram-negative bacteria have less peptidoglycan and thus fail to trap the complex. The Gram stain technique is a four-part procedure in which two dyes, a mordant, and a decolorizing agent are used. It is important to note that the terms “gram-positive” and “gram-negative” do not refer to electrical charges but are merely used for convenience to indicate the retention (positive) or loss (negative) of the crystal violet-iodine complex. S pecial Materials • Cultures of selected bacterial species • Crystal violet • Gram's iodine solution • 95% ethyl alcohol • Safranin 43038_CH06_0049.qxd 1/12/07 11:29 AM Page 50 50 6 THE GRAM STAIN TECHNIQUE P rocedure 1. Prepare air-dried, heat-fixed smears of selected bacterial organisms on a Quick Procedure clean slide, as outlined in Exercise 4A. Generally, three smears will fit on Gram Stain a slide, with room for a label. 1. Stain heat-fixed slide with 2. Flood the smears with crystal violet (the primary stain), and allow the crystal violet for 1 min; stain to remain 1 minute as indicated in Figure 6.1A. Wash with water (Fig- wash. ure 6.1B). It is not necessary to blot the slide. 2. Flood with Gram’s iodine for 1 min; wash. 3. Flood the smears with Gram’s iodine solution for 1 minute (Figure 6.1C). Wash with water, but do not blot (Figure 6.1D). The iodine acts as a mor- 3. Decolorize for two dant; that is, it increases the affinity of dye to the cell by forming a complex 15-sec periods with alcohol; wash. with the stain. 4. Stain with safranin for 4. Decolorize the smears with 95% ethyl alcohol as follows: Pour a few 30 sec; wash; dry; drops of alcohol on the smears (Figure 6.1E), and rock the slide back and observe. forth for 15 seconds. Allow the alcohol to drip off, then repeat this decol- orization procedure for another 15-second period. (As a general rule, decolorization should continue until the ethyl alcohol running off is no longer purple. However, this may be difficult to discern when performing To avoid spilling stain on the procedure for the first time.) Wash the slide with a gentle stream of your fingers, place a heavy water but do not blot (Figure 6.1F). It should be pointed out that excessive line of wax pencil between the smear and the area decolorization may cause gram-positive bacteria to lose their color where you will later place (appear gram-negative), and that insufficient decolorization may allow the slide label. ! gram-negative bacteria to retain a purple color (appear gram-positive). At Gram's Crystal iodine Water violet A Apply crystal violet to the B Rinse the smear. C Cover the smear with Gram's bacterial smear for 1 minute. iodine for 1 minute. Water Alcohol Water D Rinse the smear. E Decolorize the smear for 15 F Rinse the smear. seconds, two times. FIGURE 6.1 The Gram stain technique. 43038_CH06_0049.qxd 1/3/07 3:44 PM Page 51 THE GRAM STAIN TECHNIQUE 6 51 Water Safranin G Counterstain the smear with safranin H Rinse the smear. for 30 seconds. I Blot the smear dry with bibulous paper. FIGURE 6.1 The Gram stain technique (continued). this point, gram-positive bacteria retain the crystal violet iodine com- plex while gram-negative bacteria lose the complex and would appear transparent if observed with the microscope (Figure 6.2). 5. Flood the smears with safranin (the counterstain), and allow the stain to remain for 30 seconds (Figure 6.1G). Wash gently with water (Figure 6.1H), and blot the slide to dry it (Figure 6.1I ). Safranin, a red dye, will stain bacteria that lost the crystal violet-iodine complex during decolorization (i.e., the gram-negative bacteria). It will have no effect on gram-positive bacteria. 6. Examine the smears under the lower power lens to orient yourself, Stained slides are viewed then move to the 40X lens and finally the oil immersion lens. Scan the slide without a coverslip. thoroughly to locate areas where the bacteria are widely separated. Gram- positive bacteria will appear blue to purple, while gram-negative bacteria will be orange to red. If some of each color are observed, scan the slide a few moments more and decide which is the predominant color. Be sure to ! confine your work to areas containing thin smears of bacteria, since thicker areas may resist decolorization due to the heavy concentration of bacteria. Prepare representations of the organisms in the Results section. Include the complete binomial name of the organism, the Gram reaction, and the magnification. If the slide will be retained, the oil may be removed by gently blotting the slide. Label the slide with your name, the organisms’ names, the date, and the designation “Gram stain.” 43038_CH06_0049.qxd 1/3/07 3:44 PM Page 52 52 6 THE GRAM STAIN TECHNIQUE Gram- positive Gram- negative A Application of B Application of C Alcohol wash D Application of crystal violet iodine (decolorization) safranin (primary stain) (mordant) (counterstain) FIGURE 6.2 The color changes in gram-positive and gram-negative bacteria during the Gram stain procedure. 7. It is valuable to prepare Gram stains of mixtures of gram-positive and gram-negative bacteria to test your ability with this technique. If not pro- vided, to prepare a mixture, add a loopful of bacteria to a loopful of water on a slide. Now flame the loop, obtain a sample of a second organism, and mix the two organisms together in the water. Additional practice may be obtained by Gram staining samples of dental plaque obtained from the tooth-gum junction. In this case, a mixture of forms and Gram reactions will be observed. A sample from a soil suspension or hay infusion may also be used. The instructor may provide unknown bacteria for determination of the Gram reaction. Record all drawings in the Results section. Q uestions 1. Why is the Gram stain technique more valuable than the simple stain technique in the diagnostic laboratory? Under what circumstances might the simple stain be preferable to the Gram stain technique? 2. Suppose a student reached for the alcohol bottle for the decolorization step and instead took the bottle of distilled water. What would be the color of a gram-positive and a gram-negative bacterium at the conclusion of the pro- cedure? Explain thoroughly. 3. How might the Gram stain technique be of value in establishing the purity of a culture of bacteria? 4. Illustrate the result if congo red, an acidic dye, was substituted for safranin in the Gram stain technique. 5. Describe the procedures needed to properly see Gram-stained organisms with the oil-immersion lens. 43038_CH06_0049.qxd 1/3/07 3:44 PM Page 53 THE GRAM STAIN TECHNIQUE 6 53 Name Date Section Exercise6 Results The Gram Stain Technique Gram-Stained Bacterial Smears Organism: ______________________ ______________________ ______________________ Gram reaction: ______________________ ______________________ ______________________ Magnif.: ______________________ ______________________ ______________________ Organism: ______________________ ______________________ ______________________ Gram reaction: ______________________ ______________________ ______________________ Magnif.: ______________________ ______________________ ______________________ 43038_CH06_0049.qxd 1/3/07 3:44 PM Page 54 54 6 THE GRAM STAIN TECHNIQUE Organism: ______________________ ______________________ ______________________ Gram reaction: ______________________ ______________________ ______________________ Magnif.: ______________________ ______________________ ______________________ Organism: ______________________ ______________________ ______________________ Gram reaction: ______________________ ______________________ ______________________ Magnif.: ______________________ ______________________ ______________________ Organism: ______________________ ______________________ ______________________ Gram reaction: ______________________ ______________________ ______________________ Magnif.: ______________________ ______________________ ______________________ Observations and Conclusions: .
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