Supplemental instruction Chattahoochee Technical College Aseptic Techniques and Staining procedures Supplemental instruction Aseptic transfer Stanning procedure Simple, Negative, Gram, Endospore Flagella 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 webmaster@science-i.com

Designed by Pyeongsug Kim, ©2010 www.science-i.com 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 Designed by Pyeongsug Kim, ©2010 www.science-i.com

Designed by Pyeongsug Kim, ©2010 www.science-i.com Aseptic transfer:Tools Spreading rod Disposal loop and needle Loop Inoculation needle Pippet Cotton swap Bunsen burner Designed by Pyeongsug Kim, ©2010 www.science-i.com

Designed by Pyeongsug Kim, ©2010 www.science-i.com Aseptic transfer:Media _____________ containing the necessary nutrients are used to grow microorganisms in a laboratory Culture media ______ – solid; made from brown algae. Agar ______ – liquid Broth How sterilize media? Autoclaving Designed by Pyeongsug Kim, ©2010 www.science-i.com

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

Designed by Pyeongsug Kim, ©2010 www.science-i.com ____________ are used to transfer microorganisms from liquid media to liquid or solid media. : streak in solid media Wire loops _________ are used for transferring microorganisms to deep tubes. used to dip into agar. Needles Designed by Pyeongsug Kim, ©2010 www.science-i.com

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

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

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

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

Designed by Pyeongsug Kim, ©2010 www.science-i.com Cocci Cocci Rod Spiral Designed by Pyeongsug Kim, ©2010 www.science-i.com

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

Designed by Pyeongsug Kim, ©2010 www.science-i.com Bacteria cell wall Peptidoglycan Designed by Pyeongsug Kim, ©2010 www.science-i.com

Designed by Pyeongsug Kim, ©2010 www.science-i.com Gram staining -Differential staining. -(Basic / acidic ) stains -_____________ 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: Peptidoglycan Designed by Pyeongsug Kim, ©2010 www.science-i.com

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

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

Designed by Pyeongsug Kim, ©2010 www.science-i.com Endospore staining (Shaeffer-Fulton Method) ______________ water-soluble stains endospores. Vegetative cells and mother cells are decolorized with water. Malachite green - __________ Used to force the stain into spores if present. Heat! - __________ Counter staining stains vegetative cells and mother cells. Safranin 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

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

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

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

Designed by Pyeongsug Kim, ©2009 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