Lab Final Flashcards
Define aseptic technique
method of handling microbes and material in a way that minimizes contamination
Define inoculation
process of transferring a microbe from 1 medium to the next
Define inoculum
sample being transferred
Which method is used to obtain pure culture
isolation streak plate . used to sperate individual bacteria. the concentration of bacteria is so low that a single bacterium or small group of cells comes off the loop to form single isolated colonies on the surface.
Why do we not heat fix negative stain slide
we want to see the shape and size of the microbes. Heat fix it changes the shape and size of the microbes.
In negative staining why do cells remain unstained and background is colored
the cells remained unstained because the dye we use ( nigrosin) is negative charged and the cell is negative charged so it repels the color and the background is colored instead
What is the advantage of negative staining
being able to visualize delicate structures and see shape and size better
How does negative staining work
Nigrosin and eosin.
Negative charged chromophore so because cell is negative charged. repel dye and dye can’t stain cell. so dye remains outside in the background
Gram Staining: What would you observe if you decolorized your slide too much? How would your cells appear?
cells may remove the dye from both gram + and - because it will denature the peptidoglycan so both will appear pink from safranin
Gram staining: you are looking at the smear of the mixture. All o the cells, cocci and bacilli, appear deep purple. What could have gone wrong
not have decolorized long enough. If you do not do it for 15-20 sec then it may not have weakened the outer membrane of gram - enough to release the dye.
Gram staining: Explain the relationship between the observed Gram reaction and bacterial cell wall structure
gram + stain purple because of the thick peptidoglycan layer that trap the dye in the wall. Gram - stain pink because of the thin peptidoglycan wall get denatured with the alcohol so it holds the counterstain safanin
Gram stain reagents
dyes/stain: crystal violent ( primary stain)
mordant: iodine. intensity color
decolorizer: acetone-alcohol ( solvent/denaturant)
counterstain: safranin
What are the steps of gram staining
1) water and comma size of microbe
2) air dry and heat fix
3) crystal violet 1 minute
4) wash with distill water
5) iodine for 1 minute
6) decolorize till color runs off
7) safranin 1 minute
8) rinse with distill water
What is the name of the stain prodedure used in endospore stain
Schaeffer-Fulton Methods
Steps in endospore staining
Primary Stain: malachite green
Counterstain: safranin
vegative cell wall appear red
endospore appear green
Endospore: You forgot to heat-fix your slide.What would you see?
you may not see anything or minimal microbes because we heat fix it to adhere to the microbes to the slide
Endospores: List the reagents used in performing an endospore stain and describe their function
Primary Stain: malachite green. stain and penetrate spore coat
Distill water to remove excess stain
Safranin: counterstain that will stain and dye vegative cells wall red
Endospore: How did the stained slide of 24-hour culture differ from the stained slide of the 1 week old culture
stained slide of 24 hrs culture has more vegative cells and very miminal endospores while the 1 week old had very little vegative cells and more endospores. This is due to the environment stress 1 week olf cultures went throught
What is the function of blood agar
To see hemolysis type
How does alpha hemolysis appear on the
partial lysis of rbc, leads to darkening of the blood agar . possible green color
How does beta hemolysis appear
complete lysis of RBC and breakdown of hemoglobin ( colorless clearing of the blood agar)
How does gamma hyolysis appear
no hemolysis so no change
