ex 3 & 4

Question 1

solid media comes in 3 major forms

Answer 1

agar plates

agar slants

agar deeps

Question 2

advantage of slants

Answer 2

provide a solid surface for bacteria to grow, allowing for isolation

Question 3

deeps are useful for

Answer 3

growing bacteria that require less oxygen than what is found in the standard atmosphere and for showing motility

Question 4

an inoculating loop is used for

Answer 4

transferring bacteria to sterile broth, plates, and slants

Question 5

an inoculating needle is used for

Answer 5

stabbing deeps

Question 6

slide smears are used in

Answer 6

direct (positive) staining

Question 7

differential staining techniques are used in

Answer 7

gram stain

endospore

acid-fast

but NOT for negative staining

Question 8

aseptic technique

Answer 8

hold inoculating loop/needle, heat whole length of the wire

wait for it to cool

place cooled tube in broth culture

Question 9

how to inoculate broth media

Answer 9

place loop in culture media then place in broth media

Question 10

how to inoculate slant media

Answer 10

aseptically begin at the bottom of the slant and zigzag to the top

Question 11

how to inoculate deep media

Answer 11

aseptically stick needle in culture media

Question 12

how to inoculate TSI media

Answer 12

aseptically stab center through the center of the medium to the bottom of the tube and then streaking the surface of the agar slant

Question 13

how to label agar

Answer 13

initials

date

lab instructor

section number

Question 14

slide smear

Answer 14

label slide

sterilize loop

for the B. subtilis on solid media place 1-2 loopfuls of distilled water in the center of one circle,

reflame loop

cool loop

scrape a small amount of culture from the surface of the agar

sterilize loop

let smear air dry

once dried heat fix 2-3 times

now smear is ready staining

Question 15

simple stain

Answer 15

slide smear

place slide smear on the rack above the drainage tub

cover smear with metheylene blue for 1 min

rinse with diluted water

blot dry with bibulous paper

observe under microscope 100x immersion oil

Question 16

negative stain (capsule stain)

Answer 16

dye (india ink) is added first to the end of the slide

dye is spread across the slide

Question 17

Can you determine whether or not a bacterial culture grown in broth is a pure culture by
visually inspecting it without a microscope? In an agar deep? On a slant? On a plate?
How can you tell

Answer 17

you cannot determine
this is why we use staining techniques to see if it is pure or not

Question 18

What is the purpose of flaming the loop before obtaining inoculum?

Answer 18

sterilize the loop and not contaminate the inoculum

Question 19

What is the purpose of flaming the loop after you have used it to transfer bacteria?

Answer 19

kill any bacteria that is left on the loop

Question 20

Why must a loop be cooled before you touch it to a culture?

Answer 20

if it is not cooled it will the bacteria

Question 21

How can you tell if media is sterile before you use it for an experiment?

Answer 21

if there are any abnormalities in growth and smell

Question 22

What is the purpose of heat fixing the smear prior to direct staining?

Answer 22

heat fixing is to make the cells stick to the slide and kills the bacteria

Question 23

What would happen if you heat fixed a smear before it air-dried? What would you
expect to see when viewing the slide using the microscope

Answer 23

if you heat fix before it dried the smear will boil and the cellular morphology will be lost

you will not be able to see anything

Question 24

Why must a loop be cooled before you touch it to a culture?

Answer 24

A loop must be cooled before you touch it to a culture because if it is not it will kill the bacteria.

Question 25

what do you use to inoculate a slant tube

Answer 25

To inoculate the tube you would need to use an inoculating loop.

Question 26

Can you determine whether or not a bacterial culture grown in broth is a pure culture by visually inspecting it without a microscope? In an agar deep? On a slant? On a plate? How can you tell?

Answer 26

No you will not be able to tell whether or not a bacterial culture grown in broth, agar deep, agar slant. You can tell by using staining techniques to see if the bacterial culture is a pure culture. you can SOMETIMES tell on a plate

Question 27

What is the purpose of flaming the loop after you have used it to transfer bacteria?

Answer 27

The purpose of flaming the loop after is to kill any bacteria left on the loop.

Question 28

What 2 scientific terms describe the cellular morphology and the arrangement of Staphylococcus epidermidis?

Answer 28

Two scientific terms to describe the cellular morphology and the arrangement are the cells are gram-positive and cocci. staphylo-clusters

Question 29

1. Explain how you would prepare a bacterial smear using a broth inoculum (you do not have to explain all steps of aseptic technique, but say "using aseptic techniques.....etc

Answer 29

1. To prepare a bacterial smear using a broth inoculum you would need to turn on the bunsen burner and use aseptic techniques on the inoculating loop allow it to cool and hold it with your dominant hand. With your non dominant hand you will be holding the broth culture and open it with your dominant hand. Then you will heat the mouth of the tube a couple of times then insert the incoluating loop into the tube. Then you will need to heat the mouth of the tube a couple of times and close the tube. Place the test tube back into the rack. Grab the sterile media tube, open it, burn the mouth a couple of times, insert the inoculating loop with the broth inoculum into the sterile media, heat the mouth of the tube a couple of times, and close the tube. Use aseptic technique on the inoculating loop.

Question 30

2. Then explain the steps you would do to perform a simple positive stain using your smear from above (use methylene blue).

Answer 30

2. Grab a glass slide and label it with two circles and label the bacterial culture next to the circle. Turn on the bunsen burner. Sterilize the incoluting loop and allow it to cool down. Grab the sterile media tube with the broth inoculum tube with your non dominant hand. Open the tube and sterilize it over the bunsen burner a couple of times. Put the incoluting loop in the tube and place loopfuls onto the slide. Sterilize the tube again over the flame and close the tube. Place the tube back on the tube rack. Allow the smear to air dry completely. Once air dried heat fix the smear and place it on top of the drainage tub. Cover the smear with methylene blue for one minute. Rinse off the methylene blue with distilled water. Blot the slide dry with bibulous paper. Observe under the microscope.

Question 31

3. What is the difference in this process if your original inoculum provided by Dr. K was a plate?

Answer 31

3. The difference would be to label an agar plate and label it with the bacterial species. Repeat the sterilization process with the inoculating loop and lift the agar plate cover and streak the surface in a zig-zag pattern instead of using a tube. To stain a solid media you would need to place 1-2 loopfuls of distilled water in the inoculating loop. Sterilize the inoculating loop and allow it to cool. Once it has cooled scrape a small amount of the culture from the surface and spread it into the water on the slide. Sterilize the loop. Allow to air dry and the rest of the process is the same.

Question 32

What would happen if you heat fixed a smear before it air-dried? What would you expect to see when viewing the slide using the microscope?

Answer 32

If you heat fixed a smear before it air-dried the smear will boil and the cellular morphology will be lost. You would not be able to see anything using a microscope because the smear would be lost.

Question 33

What would happen if you didn’t heat-fix a smear before performing the direct stain? What would you expect to see when viewing the slide using the microscope? (You saw this in the virtual lab)

Answer 33

If a smear is not properly heat fixed before performing the direct stain then the cells would be rinsed off. You will not be able to see anything because the cells would be rinsed off.

Question 34

What scientific term describes the cellular morphology of Bacillus subtilis? Escherichia coli?

Answer 34

The scientific term that described the cellular morphology of Bacillus subtilis are bacilli which means the cells are rod-shaped. The scientific term that describes the cellular morphology of Escherichia coli are bacilli which means the cells are rod-shaped.

Question 35

Which of the following answers is the BEST order for inoculating sterile broth from a broth culture? 1. Flame the top of the test tube 2. Flame the inoculating loop 3. Remove test tube cap 4. Replace test tube cap 5. Dip inoculating loop in culture 6. Dip inoculating loop in sterile broth

Answer 35

2, 3, 1, 5, 1, 4, 3, 1, 6, 1, 4, 2

Question 36

What is the purpose of flaming the loop before obtaining inoculum?

Answer 36

The purpose of flaming the loop is to sterilize the loop and not contaminate the inoculum.

Question 37

How can you tell if media is sterile before you use it for an experiment?

Answer 37

You can tell is a media is sterile by seeing if there are any abnormalities in growth and smell after incubating the media overnight