Lab Exam 2

Question 1

What is a culture?

Answer 1

Microbes that grow in a lab setting.

Question 2

What is an inoculum?

Answer 2

Microbes added in a growth medium

Question 3

Why do we use aseptic technique in lab?

Answer 3

To prevent cross contamination.

Question 4

Agar:

Answer 4

Solid growth media.
- Can be in a test tube: Slant (angled) or stab ( flat top)
- Can be in a petri dish

Question 5

Broth:

Answer 5

Liquid growth media.
(we use TSB: Tryptic Soy Broth)

Question 6

Types of Agar:

Answer 6

1. TSA: Tryptic Soy Agar
2. MSA: Mannitol Salt Agar
3. EMB: Eosin Methylene Blue
4. MAC: MacConkey Agar

Question 7

What were the two tools that we have used to inoculate new cultures?

Answer 7

1. Inoculating Loop
2. Cotton Swab

Question 8

Complex media:

Answer 8

Exact ingredients are unknown ex. soy (plants) , animal extracts(meat) , egg yolks, yeast. (we use in lab TSA-TSB)

Question 9

Defined media:

Answer 9

All ingredients are known in their exact amount.

Question 10

Streak plate:

Answer 10

Goal: grow a pure culture by isolating colonies
1. Spread 1 loopful of bacteria on 1/4 of plate
2. Flame loop
3. Pull bacteria from zone 1 into zone 2, covering only 1/4 of plate.
4. Flame loop
5. Pull bacteria from zone 2, into zone 3, covering only 1/4
6. Flame loop
7. Pull little bacteria from zone 3 into 4, avoid touching zone 1 and 2

Question 11

Lawn plate:

Answer 11

Goal: Heavy growth spread evenly to test the susceptibility of bacteria to many antimicrobial substances.
1. We swab the entire surface of the plate with a cotton swab, making sure there are no gaps. Antiseptic technique

Question 12

Zig zag plate:

Answer 12

Use loop to swab left and right in a few zig-zags

Question 13

Why do we flame the loop between each zone when making a streak plate?

Answer 13

To reduce the number of microbes spread in next streak. We want to reduce bacterial numbers to obtain isolated colonies.

Question 14

Effect of Temperature on Growth

• know the organisms used for this exercise

Answer 14

1. Bacillus (+)
2. Escherichia (-)
3. Staphylococcus (+)
4. Mycobacterium (acid, +)
5. Pseudomonas (-)

Question 15

Effect of Temperature on Growth

• How did we apply our microorganism to the surface of this plate?

Answer 15

Lawn plate - use cotton swab with bacteria to cover entire plate, leaving no gaps.

Question 16

Effect of Temperature on Growth

• Temperatures:

Answer 16

4 °C (Refrig. temp)
25°C (room temp)
37°C (body temp)
42°C
47°C
52°C

Question 17

Convert °C to °F:

Answer 17

°F = (°C x 1.8) + 32

°C = °F - 32 / 1.8

Question 18

Effect of Temperature on Growth:

• What temperature did all species grow best at?

Answer 18

Body temperature, 37°C.

Question 19

Effect of Temperature on Growth:

• What genus did not grow well at room temperature?

Answer 19

Mycobacterium

Question 20

Effect of Temperature on Growth:

• Why didn’t the bacteria grow at refrigerator temperature?

Answer 20

Bacteriostatic effect inhibits growth. (0-7°C)

Question 21

Evaluation of Antiseptics:

• What organisms were used?

Answer 21

1. Escherichia coli
2. Pseudomonas aeruginosa
3. Bacillus subtilis
4. Staphylococcus aureus
5. Mycobacterium smegmatis

Question 22

Evaluation of Antiseptics:

• How did we apply our microorganism to the surface of this plate?

Answer 22

Lawn plate - use cotton swab with bacteria to cover entire plate, leaving no gaps.

Question 23

Evaluation of Antiseptics:

• What were the chemicals used in lab?

Answer 23

> Iodine - halogen
3% Hydrogen peroxide - Peroxygens
2% chlorhexidine - Biguanides
70% isopropyl alcohol – alcohol
1% formaldehyde - Aldehydes
1% silver nitrate - metal

Question 24

Evaluation of Antiseptics:

• How did we evaluate whether the antiseptic was effective or not?

Answer 24

Test microbe in many antiseptics:
- Most effective = have the largest death zone
- Least effective = small to no death zone

Question 25

Evaluation of Antiseptics: - Overall data trend from project:

Answer 25

- Most resistant organism: Pseudomonas aeruginosa - Least resistant: Mycobacterium smegmatis - Most effective chemical: 1% Formaldehyde - Least effective chemical: 70% Isopropyl alcohol

Question 26

Evaluation of antibiotics: - Organisms used in this lab:

Answer 26

- Escherichia coli - Pseudomonas aeruginosa - Bacillus subtilis - Staphylococcus aureus - Mycobacterium smegmatis

Question 27

Evaluation of antibiotics: - How did we apply microorganism to this plate?

Answer 27

Lawn plate: Cotton swab with bacteria and cover entire plate, leaving no gaps.

Question 28

Evaluation of antibiotics: Antibiotics used for this lab:

Answer 28

- Chloramphenicol - protein synthesis inhibitor - Gentamicin - protein synthesis inhibitor - Penicillin - cell wall synthesis inhibitor - Colistin - cell wall synthesis inhibitor - Bacitracin - cell wall synthesis inhibitor - Ciprofloxacin – DNA synthesis inhibitors

Question 29

Evaluation of antibiotics: -How did we evaluate whether the antibiotic was effective or not?

Answer 29

Test 1 bacterium in multiple antibiotics; the Larger death zone= the antibiotic was most effective - Small death zone= the antibiotic was ineffective

Question 30

Evaluation of antibiotics: - Which term is used to describe when a bacterium is easily killed by a particular antibiotic?

Answer 30

Susceptible.

Question 31

Evaluation of antibiotics: - Overall data:

Answer 31

> Most effective: Chloramphenicol > Least effective: Colistin > Most resistant: Escherichia coli > Least resistant: Mycobacterium smegmatis

Question 32

Evaluation of antibiotics: Narrow and broad spectrum

Answer 32

Narrow: targets a single microbe group Broad: It can inhibit multiple groups of bacteria.

Question 33

Evaluation of antibiotics: Which antibiotic did not kill a few of the bacteria used in the experiment? How do we know that it did not kill? Does this antibiotic selectively kill G + or G - bacteria?

Answer 33

> Penicillin; we know it did not kill Pseudomonas aeruginosa (G-) , Mycobacterium smegmatis (acid+) or Escherichia coli (G-) because there was no death zone present. Penicillin is primarily narrow spectrum for Gram + bacteria. > Bacitracin; we know it did not kill Escherichia coli (G -) , and Pseudomonas aeruginosa ( G-) because there was no death zone present. Bacitracin is an antibiotic that primarily kills gram positive bacteria. Narrow spectrum.

Question 34

Bacterial Transformation Lab - What bacteria did we use?

Answer 34

Escherichia coli

Question 35

Bacterial Transformation Lab - What genes are found on the pGLO plasmid?

Answer 35

- bla gene: allows resistance to ampicillin - araC gene: blocks GFP gene from being able to express (glow) all the time - GFP gene: codes for fluorescence - the Ori (origin of replication)

Question 36

Bacterial Transformation Lab - Which gene, when expressed allows colonies to glow?

Answer 36

GFP ( from jellyfish )

Question 37

Bacterial Transformation Lab - What is the purpose of the LB in the plates?

Answer 37

Provide E. coli with nutrients

Question 38

Bacterial Transformation Lab - What inoculated plates or side of plates showed transformation?

Answer 38

LB/amp (+pGLO side) - growth present due to bla gene (resistance) being present within the pGLO plasmid LB/amp/arabinose plate - because growth was present and it fluoresced

Question 39

Bacterial Transformation Lab - What is needed in the media for the colonies to glow?

Answer 39

Arabinose (allows GFP gene to be expressed (glow) by blocking the araC gene from blocking GFP gene)

Question 40

Bacterial Transformation Lab - What plate shows GFP expression?

Answer 40

LB/amp/arabinose plate

Question 41

Bacterial Transformation Lab - In the LB/amp plate, what side (+ o -) should show growth?

Answer 41

positive

Question 42

Bacterial Transformation Lab - What is the name of the antibiotic resistance gene?

Answer 42

bla gene - resistant to ampicillin

Question 43

S & D media: -Define Selective media:

Answer 43

Inhibits the growth of unwanted organisms (selects what lives)

Question 44

S & D media: -Define Differential media:

Answer 44

All types of organisms grow, but they look different (color change)

Question 45

S & D media: - MSA: Mannitol Salt Agar

Answer 45

>Color red. > Selects for Gram +, selects against Gram - > Differential for mannitol fermentation *Mannitol fermentor turns yellow.

Question 46

S & D media: - EMB: Eosin Methylene Blue

Answer 46

> Color: dark purple > Selects for Gram -, selects against Gram +. > Differential for lactose fermentation > Strong fermentor: metallic green > Weak: Pink edge of colonies > non: no change just growth

Question 47

S & D media: - MAC: MacConkey Agar

Answer 47

> Color: Raspberry > Selects for Gram -, selects against Gram +. > Differential for lactose fermentation > Strong fermentor: Bright pink > Weak: Light pink (fluffy) > Non: Light brown

Question 48

S & D media: - Which plate used in lab this semester is not selective or differential?

Answer 48

- TSA: Tryptic Soy Agar is complex; exact ingredients are unknown, uses plant, animal, yeast.

Question 49

S & D media: - Gram positive organisms used:

Answer 49

- Staphylococcus aureus: is a mannitol fermenter (turns yellow) - Staphylococcus epidermidis: non fermenter ( doesn't change, stays same color)

Question 50

S & D media: - Gram negative organisms used:

Answer 50

> Escherichia coli: strong lactose fermenter > Enterobacter aerogenes : weak lactose fermenter > Proteus vulgaris: non lactose fermenter