Lab Final

Question 1

Define aseptic technique

Answer 1

method of handling microbes and material in a way that minimizes contamination

Question 2

Define inoculation

Answer 2

process of transferring a microbe from 1 medium to the next

Question 3

Define inoculum

Answer 3

sample being transferred

Question 4

Which method is used to obtain pure culture

Answer 4

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.

Question 5

Why do we not heat fix negative stain slide

Answer 5

we want to see the shape and size of the microbes. Heat fix it changes the shape and size of the microbes.

Question 6

In negative staining why do cells remain unstained and background is colored

Answer 6

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

Question 7

What is the advantage of negative staining

Answer 7

being able to visualize delicate structures and see shape and size better

Question 8

How does negative staining work

Answer 8

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

Question 9

Gram Staining: What would you observe if you decolorized your slide too much? How would your cells appear?

Answer 9

cells may remove the dye from both gram + and - because it will denature the peptidoglycan so both will appear pink from safranin

Question 10

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

Answer 10

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.

Question 11

Gram staining: Explain the relationship between the observed Gram reaction and bacterial cell wall structure

Answer 11

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

Question 12

Gram stain reagents

Answer 12

dyes/stain: crystal violent ( primary stain)

mordant: iodine. intensity color
decolorizer: acetone-alcohol ( solvent/denaturant)
counterstain: safranin

Question 13

What are the steps of gram staining

Answer 13

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

Question 14

What is the name of the stain prodedure used in endospore stain

Answer 14

Schaeffer-Fulton Methods

Question 15

Steps in endospore staining

Answer 15

Primary Stain: malachite green
Counterstain: safranin

vegative cell wall appear red
endospore appear green

Question 16

Endospore: You forgot to heat-fix your slide.What would you see?

Answer 16

you may not see anything or minimal microbes because we heat fix it to adhere to the microbes to the slide

Question 17

Endospores: List the reagents used in performing an endospore stain and describe their function

Answer 17

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

Question 18

Endospore: How did the stained slide of 24-hour culture differ from the stained slide of the 1 week old culture

Answer 18

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

Question 19

What is the function of blood agar

Answer 19

To see hemolysis type

Question 20

How does alpha hemolysis appear on the

Answer 20

partial lysis of rbc, leads to darkening of the blood agar . possible green color

Question 21

How does beta hemolysis appear

Answer 21

complete lysis of RBC and breakdown of hemoglobin ( colorless clearing of the blood agar)

Question 22

How does gamma hyolysis appear

Answer 22

no hemolysis so no change