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Aseptic Techniques Aseptic Techniques andand
Staining proceduresStaining proceduresSupplemental instruction
Picture from http://biology.clc.uc.edu/fankhauser/Labs/Microbiology/Coliform_assays/Single_Colony_Isolation.htm
•Aseptic transfer•Stanning procedure Simple, Negative, Gram, Endospore Flagella
Designed by Pyeongsug Kim ©2010 [email protected]
Fall 2011Chattahoochee Technical College
Designed by Pyeongsug Kim, ©2010 www.science-i.com
Aseptic techniquesAseptic 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….
Picture from http://www.lifelinemedical.net/manual_autoclaves.html http://biology.clc.uc.edu/fankhauser/Labs/Microbiology/Single_Colony_Isolation.html
Autoclave or steam sterilization
Flame sterilization of wire loops and needles
Flaming the mouth of test tubes and other containers
Aseptic transfer:ToolsTools
Loop
Pippet Bunsen burnerCotton swap
Spreading rod Disposal loop and needle
Inoculation needle
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Aseptic transfer:MediaMedia
______ – solid; made from brown algae.Agar______ – liquidBroth
_____________ containing the necessary nutrients are used to grow microorganisms in a laboratory Culture media
How sterilize media? Autoclaving
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Aseptic transferAseptic transfer : Transfer living microbes from one place to another aseptically, without contamination
__________ The process of introducing a microorganism or suspension of microorganism into a culture medium.
Inoculation
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____________ 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
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“….a light blue area at the top of the flame. The light blue area is the hottest area of the flame.”
Designed by Pyeongsug Kim, ©2010 www.science-i.com
Bunsen burnerBunsen burner
Picture from http://kids.britannica.com/comptons/art-53017/The-Bunsen-burner-consists-of-a-metal-burner-tube-on
Flame the wire loop until it is red hot.
Flame the loop here!
_______-contains only a single species.-Isolation technique-Streak-plate method :dilution technique, :individual colonies of microbes can be obtained.
Pure culture
Mixed culture ~ 2 or more species -We alwaysalways assumed to be a mixed culture.
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Streak-plate methodStreak-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
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Picture from http://biology.clc.uc.edu/fankhauser/Labs/Microbiology/Single_Colony_Isolation.html CTC Microbiology Lab. mannual Lab 4
Simple staining-Cell is slightly negative.- Cell will be stained.-(Basic / acidic ) stainsto 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
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Negative staining- The surrounding of the cell will be stained.- NigrosinNigrosin-(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
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Bacteria cell wallBacteria cell wall
Peptidoglycan
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Gram staining-Differential staining.-(Basic / acidic ) stains-_____________ cell wall
-Gram + ~ stainded by crystal violet Thicker 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
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Designed by Pyeongsug Kim, ©2010 www.science-i.com
Picture from http://biology.clc.uc.edu/fankhauser/Labs/Microbiology/Single_Colony_Isolation.html
EndosporeEndospore-a dormant and non-reproductive structure -allows it to survive poor environmental conditions.-resistant to heat and chemicals. Protein keratin
Mother cell holding endospore
Vegetative cell
Endospore
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“Resting bodies”
- __________ Counter staining stains vegetative cells and mother cells.
Endospore staining (Shaeffer-Fulton Method)-______________ water-soluble stains endospores. Vegetative cells and mother cells are decolorized with waterwater.
SafraninSafranin
Malachite greenMalachite green
- __________ Used to force the stain into spores if present.
Heat!Heat!
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Picture from http://pscantie.myweb.uga.edu/stain.html http://archive.microbelibrary.org/ASMOnly/Details.asp?ID=2598
Heat + Malachite Green
Water
Safranin
Vegetative cell or non-producers
Spore or spore producer
Rinse with water and look at the slideDesigned by Pyeongsug Kim, ©2010 www.science-i.com
Designed by Pyeongsug Kim, ©2010 www.science-i.com
Capsules and Slime layers Capsules and Slime layers
:employs an acidic stain(for background stain) and a basic stain(for cell
stain) to detect capsule production. -For Cell For Cell Methylene Blue (basic stain)
-For BackgroundFor Background Negative stain by NigrosineNigrosine (acidic stain)
-For capsuleFor capsule Unstained!
Capsule stainCapsule stain
Picture from http://academic.pgcc.edu/~kroberts/Lecture/Chapter%204/gramstain.html
:layers of polysaccharide or polypeptide
-Capsule ~ round or oval in shape.-Slime layer ~ irregularly shaped and loosely bound to the bacterium.
Flagella stain Flagella stain
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Picture from http://www.bd.com/ds/productCenter/SR-StainsDroppersIndicators.asp
:Thin proteinaceous structuresmotility -Leifson stain-stain coats the flagella, thereby increasing their diameter. -Allow the smear to air dry. -Do not heat fix!
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
Flagella; Allows the bacteria to move
What is the hair like structure in the left? function?
