Gram stain
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Experiment 1 Gram stain Yuyan Wang Methods for staining bacteria Bacterial stain Living bacteria stain Killed bacteria stain Positive staining Negative staining Common stain Single-dye stain Special stain Complex stain Acid fast stain Endospore stain Capsule stain Cell wall stain Gram stain MEDICAL MICROBIOLOGY EXPERIMENT 1 Wang Yuyan Demonstation Escherichia coli (Gram stain, oil 100×) 大肠埃希菌（革兰染色，油镜 100×) MEDICAL MICROBIOLOGY EXPERIMENT 1 Wang Yuyan Streptococcus (Gram stain, oil 100×) 链球菌（革兰染色，油镜 100×) MEDICAL MICROBIOLOGY EXPERIMENT 1 Wang Yuyan Staphylococcus (Gram stain, oil 100×) 葡萄球菌（革兰染色，油镜 100×) MEDICAL MICROBIOLOGY EXPERIMENT 1 Wang Yuyan Neisseria meningitidis (cerebrospinal fluid，methylene blue stain, oil 100×) 脑膜炎双球菌（脑脊液，美兰染色，油镜 100×) MEDICAL MICROBIOLOGY EXPERIMENT 1 Wang Yuyan Capsule of pneumococcus （capsule stain, oil 100×) 肺炎球菌的荚膜（荚膜染色，油镜 100×) MEDICAL MICROBIOLOGY EXPERIMENT 1 Wang Yuyan Spore of Clostridium tetani （spore stain, oil 100×) 破伤风梭菌（芽胞染色，油镜 100×) MEDICAL MICROBIOLOGY EXPERIMENT 1 Wang Yuyan Flagella of Bacillus proteus（Flagella stain, oil 100×) 变形杆菌的鞭毛（鞭毛染色，油镜 100×) MEDICAL MICROBIOLOGY EXPERIMENT 1 Wang Yuyan Gram stain Divides bacteria into 2 groups Gram--positive Gram Thick peptidoglycan ® 90% peptidoglycan ® Teichoic acids ® Not many polysaccharides ® MEDICAL MICROBIOLOGY Gram--negative Gram ® Thin peptidoglycan ® 5-10% peptidoglycan ® No teichoic acids ® Outer membrane has lipids, polysaccharides EXPERIMENT 1 Wang Yuyan application ® Identification: Color, shape, arrangement, endospore ® Diagnosis toxin, endotoxin ® Treatment antibiotic selection: G+, G- MEDICAL MICROBIOLOGY EXPERIMENT 1 Wang Yuyan Effects of reagents on bacterial cells primary stain: crystal violet, causes both gram-positive and gram-negative organisms to become purple. Mordant: Gram’s iodine combine with crystal violet to form a relatively insoluble compound in the bacteria. Decolorization: 95% ethanol, the gramnegative bacteria are leached colorless. Counterstain: safranin, adds a pink color to the decolorized gram-negative bacteria without affecting the color of the purple grampositive bacteria MEDICAL MICROBIOLOGY EXPERIMENT 1 Wang Yuyan Materials l 4 students sharing ® ® Cleaning paper for lens (the blue box) 1 bottle of immersion oil (cedar oil） and aether (cleaning solution) 1 bottle of saline Drying paper Marker l 2 students sharing ® ® ® ® Bacteria Strains: Staphylococcus albus and Escherichia coli (cultures on slants). ® Gram stain kit:1 ® Bunsen burner ® Dipper l Everyone: ® Microscope slides: 1-2 ® Inoculating loops:1 ® Microscope (in the storages under the bench) MEDICAL MICROBIOLOGY EXPERIMENT 1 Wang Yuyan Gram stain ® 1. 2. 3. Procedure Prepare bacterial smears Gram stain Observation by microscope MEDICAL MICROBIOLOGY EXPERIMENT 1 Wang Yuyan Preparation of smear ® Take a clean slide ® Maker the slide ® Flame an inoculating loop, let it cool, and transfer two loopfuls of water to the center of the target circle. Strain 1 strain 1 + 2 Strain 2 Your slide may be dived into three areas for staining MEDICAL MICROBIOLOGY EXPERIMENT 1 Wang Yuyan Pick up organisms from a tube slant Pick up a very small amount of the organisms CAUTION ! Be sure to cool the loop completely before inserting it into a medium. A loop that is too hot will spatter the medium and move bacteria into the air. MEDICAL MICROBIOLOGY EXPERIMENT 1 Wang Yuyan Making bacterial smears Be sure to flame the inoculating loop before returned it to rack. MEDICAL MICROBIOLOGY EXPERIMENT 1 Wang Yuyan MEDICAL MICROBIOLOGY EXPERIMENT 1 Wang Yuyan Gram stain 1. Cover the smear with crystal violet for 10 seconds. 2. Briefly wash off the stain. Drain off excess water. 3. Cover the smear with Gram’s iodine solution for 10 seconds. 4. Pour off the Gram’s iodine and washed by water. 5 Flood the smear with 95% ethyl alcohol for 10 to 20 seconds. This step is critical. Thick smears will require more time than thin ones. Decolorization has occurred when the solvent flows colorlessly from the slide. 6. Stop action of the alcohol by rinsing the slide with water for a few seconds. 7. Cover the smear with safranin for 10 seconds. 8. Wash gently for a few seconds, blot dry with bibulous paper, and air-dry. 9. Examine the slide under oil immersion. MEDICAL MICROBIOLOGY EXPERIMENT 1 Wang Yuyan 10 10 seconds 10 MEDICAL MICROBIOLOGY EXPERIMENT 1 Wang Yuyan MEDICAL MICROBIOLOGY EXPERIMENT 1 Wang Yuyan MEDICAL MICROBIOLOGY EXPERIMENT 1 Wang Yuyan MEDICAL MICROBIOLOGY EXPERIMENT 1 Wang Yuyan Oil Immersion ® Turn on microscope Adjust brightness Insert slide Add drop of oil on TOP OF COVERSLIP Rotate nosepiece until 100X objectives straddle slide Rotate oil immersion lens (100x) into oil Use coarse adjustment to lower stage until specimen appears Use fine adjustment knob to sharp focus Observe and draw ® Clean objectives with lens paper when finished! ® ® ® ® ® ® ® ® MEDICAL MICROBIOLOGY EXPERIMENT 1 Wang Yuyan MEDICAL MICROBIOLOGY EXPERIMENT 1 Wang Yuyan Report Requires 1. Objective 2. Principles 3. Materials 4. Procedure 5. Results (draw what you observed by the microscope, or describe the phenomenon or changes emerged in your experiment ) 6. Discussion (the notes in procedures or analysis of the results) MEDICAL MICROBIOLOGY EXPERIMENT 1 Wang Yuyan Questions ® Which step is the most crucial or most likely to cause poor results in the Gram stain? Why? ® Which phase of the bacteria ther have a typical shape and color by Gram staining, lag, logarithmic, stationary, decline phase ? why? ® What part of the bacterial cell is most involved with Gram staining, and why? ® How to protect yourself against lab-infection in the medical microbiology lab (BSL-1,2). MEDICAL MICROBIOLOGY EXPERIMENT 1 Wang Yuyan
