Transcript 슬라이드 1
Aseptic Techniques and Staining procedures •Aseptic transfer •Stanning procedure Simple, Negative, Gram, Endospore Flagella Supplemental instruction Fall 2011 Chattahoochee Technical College Picture from http://biology.clc.uc.edu/fankhauser/Labs/Microbiology/Coliform_assays/Single_Colony_Isolation.htm Designed by Pyeongsug Kim ©2010 [email protected] Aseptic techniques :the process of growing pure (uncontaminated) cultures, and is essential for proper characterization of a bacterium :use of sterile media and equipment. For examples…. Autoclave or steam sterilization Flame sterilization of wire loops and needles Flaming the mouth of test tubes and other containers Picture from http://www.lifelinemedical.net/manual_autoclaves.html http://biology.clc.uc.edu/fankhauser/Labs/Microbiology/Single_Colony_Isolation.html Aseptic transfer:Tools Spreading rod Loop Disposal loop and needle Inoculation needle Pippet Cotton swap Designed by Pyeongsug Kim, ©2010 www.science-i.com Bunsen burner Aseptic transfer:Media _____________ Culture media containing the necessary nutrients are used to grow microorganisms in a laboratory ______ Agar – solid; made from brown algae. ______ Broth – liquid How sterilize media? Autoclaving Designed by Pyeongsug Kim, ©2010 www.science-i.com Aseptic transfer : Transfer living microbes from one place to another aseptically, without contamination Inoculation __________ The process of introducing a microorganism or suspension of microorganism into a culture medium. Designed by Pyeongsug Kim, ©2010 www.science-i.com ____________ are used to Wire loops transfer microorganisms from liquid media to liquid or solid media. : streak in solid media _________ Needles are used for transferring microorganisms to deep tubes. used to dip into agar. Designed by Pyeongsug Kim, ©2010 www.science-i.com Bunsen burner “….a light blue area at the top of the flame. The light blue area is the hottest area of the flame.” Flame the loop here! Flame the wire loop until it is red hot. Picture from http://kids.britannica.com/comptons/art-53017/The-Bunsen-burner-consists-of-a-metal-burner-tube-on Designed by Pyeongsug Kim, ©2010 www.science-i.com Mixed culture ~ 2 or more species -We always assumed to be a mixed culture. Pure culture _______ -contains only a single species. -Isolation technique -Streak-plate method :dilution technique, :individual colonies of microbes can be obtained. Designed by Pyeongsug Kim, ©2010 www.science-i.com Streak-plate method - Isolation - decrease cell density leading to individual cells being deposited separately on agar. -In each quadrant, you should…. Flam the loop! Why??? Sterilize loop facilitating to decrease cell density Picture from http://biology.clc.uc.edu/fankhauser/Labs/Microbiology/Single_Colony_Isolation.html CTC Microbiology Lab. mannual Lab 4 Designed by Pyeongsug Kim, ©2010 www.science-i.com Simple staining -Cell is slightly negative. - Cell will be stained. -(Basic / acidic ) stains to stain the CELL, -methylene blue, crystal violet, and safranin. -Heat - fixed. Kill and adhere bacteria to the slide. -Morphology shape(cocci, rod), size, and arrangements Designed by Pyeongsug Kim, ©2010 www.science-i.com Cocci Rod Cocci Spiral Designed by Pyeongsug Kim, ©2010 www.science-i.com Negative staining - The surrounding of the cell will be stained. - Nigrosin -(Basic / acidic ) stains NOT stain the CELL, -Do NOT heat fix! -Advantages minimal cell shrinkage so it is proper for the bacteria to withstanding heat-fixing. determine the accurate the cell size. -Morphology shape(cocci, rod), size, and arrangements Designed by Pyeongsug Kim, ©2010 www.science-i.com Bacteria cell wall Peptidoglycan Designed by Pyeongsug Kim, ©2010 www.science-i.com Gram staining -Differential staining. -(Basic / acidic ) stains -_____________ Peptidoglycan cell wall -Gram + ~ stainded by crystal violet Thicker peptidoglycan -Gram - ~ stainded by safranin Thin peptidoglycan -Culture 24 hour-old or less Best for staining -Heat - fixed. -Morphology shape(cocci, rod), size, and arrangements - crystal violet: Designed by Pyeongsug Kim, ©2010 www.science-i.com Picture from http://biology.clc.uc.edu/fankhauser/Labs/Microbiology/Single_Colony_Isolation.html Designed by Pyeongsug Kim, ©2010 www.science-i.com Endospore “Resting bodies” -a dormant and non-reproductive structure -allows it to survive poor environmental conditions. -resistant to heat and chemicals. Protein keratin Vegetative cell Mother cell holding endospore Endospore Designed by Pyeongsug Kim, ©2010 www.science-i.com Endospore staining (Shaeffer-Fulton Method) Malachite green -______________ water-soluble stains endospores. Vegetative cells and mother cells are decolorized with water. - __________ Heat! Used to force the stain into spores if present. - __________ Safranin Counter staining stains vegetative cells and mother cells. Picture from http://pscantie.myweb.uga.edu/stain.html http://archive.microbelibrary.org/ASMOnly/Details.asp?ID=2598 Designed by Pyeongsug Kim, ©2010 www.science-i.com Spore or spore producer Vegetative cell or non-producers Heat + Malachite Green Water Safranin Rinse with water and look at the slide Designed by Pyeongsug Kim, ©2010 www.science-i.com Capsules and Slime layers :layers of polysaccharide or polypeptide -Capsule ~ round or oval in shape. -Slime layer ~ irregularly shaped and loosely bound to the bacterium. Capsule stain :employs an acidic stain(for background stain) and a basic stain(for cell stain) to detect capsule production. -For Cell Methylene Blue (basic stain) -For Background Negative stain by Nigrosine (acidic stain) -For capsule Unstained! Picture from http://academic.pgcc.edu/~kroberts/Lecture/Chapter%204/gramstain.html Designed by Pyeongsug Kim, ©2010 www.science-i.com Flagella stain :Thin proteinaceous structures motility -Leifson stain -stain coats the flagella, thereby increasing their diameter. -Allow the smear to air dry. -Do not heat fix! Picture from http://www.bd.com/ds/productCenter/SR-StainsDroppersIndicators.asp Designed by Pyeongsug Kim, ©2010 www.science-i.com What is the surrounding the cell (white) part? Chemical compound? Capsule-polysaccahride What is the function of structure in the bacteria? Resistance of chemcals, heat What is the hair like structure in the left? function? Flagella; Allows the bacteria to move Designed by Pyeongsug Kim, ©2009