1. 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
Designed by Pyeongsug Kim ©2010

2. 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
Designed by Pyeongsug Kim, ©

3. 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, ©

4. 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, ©

5. 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, ©

6. 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, ©

7. 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
Designed by Pyeongsug Kim, ©

8. 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, ©

9. 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 CTC Microbiology Lab. mannual Lab 4
Designed by Pyeongsug Kim, ©

10. 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, ©

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

12. 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, ©

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

14. 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, ©

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

16. 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, ©

17. 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
Designed by Pyeongsug Kim, ©

18. 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, ©

19. 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
Designed by Pyeongsug Kim, ©

20. 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
Designed by Pyeongsug Kim, ©

21. 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