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
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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
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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
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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.
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____________
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.
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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.
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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.
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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
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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
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Cocci
Rod
Cocci
Spiral
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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
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Bacteria cell wall
Peptidoglycan
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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:
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Picture from http://biology.clc.uc.edu/fankhauser/Labs/Microbiology/Single_Colony_Isolation.html
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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
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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.
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Spore or
spore producer
Vegetative cell or
non-producers
Heat
+ Malachite Green
Water
Safranin
Rinse with water and look at the slide
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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
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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!
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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
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