ex 5,6,7

Question 1

what does a gram - cell wall look like?

what color is gram - after a gram stain

Answer 1

cell wall has an outer membrane and a thin peptidoglycan layer

gram - is red/pink after a gram stain

Question 2

what does a gram + cell wall look like?

what color is gram + after a gram stain?

Answer 2

cell wall has a thick peptidoglycan layer

purple after a gram stain

Question 3

what is a gram stain

Answer 3

widely used differential staining method

differentiates bacteria as gram + or gram - according to their type of cell wall

Question 4

steps of gram stain

Answer 4

1) step: primary stain
reagent: crystal violet
time: 1 min

2) step: mordant
reagent: iodine
time: 1 min

3) step: decolorizer
reagent: 95% ethanol
time: 15 sec

4) step: counterstain
reagent: safranin
time: 2 min

Question 5

colors of gram + and gram - after each step of a gram stain

Answer 5

1) primary stain
gram + : purple
gram - : purple

2) mordant
gram + : purple
gram - : purple

3) decolorizer
gram + : purple
gram - : colorless

4) counterstain
gram + : purple
gram - : red/pink

Question 6

If crystal violet is added second,

Answer 6

gram + will appear purple

Question 7

If iodine is applied first,

Answer 7

a gram + cell will not have gain any color

Question 8

Without the mordant being added after the crystal violet has already been applied, the CV-I complex will not form, and Gram-positive cells will not retain the purple stain when the decolorization is performed. Thus, if the

Answer 8

Thus, if the
iodine is added first and crystal violet second, Gram-positive cells will end up red/pink after steps
three and four are performed

Question 9

colors of gram + and gram - at the end of a gram stain

Answer 9

gram + purple

gram - red pink

Question 10

acid-fast stain stains

what molecule?

know ex

disease causing species

diseases

Answer 10

molecule: mycolic acid; waxy lipids

example: mycobacterium and norcardia

disease causing species: Mycobacterium tuberculosis and Mycobacterium leprae

diseases: Tuberculosis (TB) and leprosy

Question 11

endospore stain

be able to identify

know names of stains/reagents used

examples

Answer 11

identify: dead bacteria: red/pink endospore:

primary stain: malachite green

decolorizer: water

counterstain: safranin

ex: Clostridium & Bacilis

Question 12

capsule stain

identify

how stain is performed

Answer 12

identify:
capsule appears as a halo surrounding the purple bacterial cells

protocol:
1) add 1-2 drops of india ink to end of slide

2) aseptically mix in 2 loopfuls of Enterobacter aerogenes from a broth culture using a loop

3) get a clean slide and hold at an angle over the 1st slide so the edge touched the india ink. Push the 2nd slide to spread the bacteria and ink across the surface

4) allow smear to air dry.

5) brief heat fix (1 time)

6) put slide on rack over a staining tub and apply
crystal violet. let stain for 1 min

7) VERY GENTLY rinse with water

8) CAREFULLY blot slide with bubulous paper or allow to air dry. view at 1000X TM

Question 13

purpose of flagella

Answer 13

chemotaxis: movement of bacteria in response to chemical signals

Question 14

parts of flagella

Answer 14

filament: the tail-like projection

hook: connects the filament to the basal body

basal body: the motor

Question 15

flagella arrangement
monotrichous

Answer 15

one flagellum

Question 16

flagella arrangement
bitrichous

Answer 16

two flagella on one side of the cell

Question 17

flagella arrangement
amphitrichous

Answer 17

two flagella one on each side of the cell

Question 18

flagella arrangement
lophotrichous

Answer 18

bundle of flagella on one side of the cell

Question 19

flagella arrangement
peritrichous

Answer 19

flagella all over the slide

Question 20

Why is iodine important in the Gram stain? What does the iodine do?

Answer 20

Iodine is important because it is added as a moderant to form the crystal violet-iodine complex which causes the gram positive stain to be a darker purple.

Question 21

A. What color would Gram-positive cells appear if step 3 was omitted when performing a Gram stain (if you did step 1, step 2, step 4)?

Answer 21

A. purple

Question 22

B. What color would Gram-negative cells appear if step 3 was omitted when performing a Gram stain (if you did step 1, step 2, step 4)?

Answer 22

B. purple

Question 23

Suppose you do a Gram stain on a pure culture of Gram-positive cocci. When you view the slide, you see some purple spheres and red spheres adjacent to one another. What should you conclude and why?

Answer 23

The pure culture could be an old bacterial culture because older cells do not retain the crystal violet as well as the younger cell. This causes the stain to be pink and purple. Another reason could be the species of the bacteria do not stain according to their cell wall. The bacteria could be gram positive according to their cell wall but appear mostly red/pink after the gram stain.

Question 24

A. In the endospore stain, what color do the endospores stain?

B. What is the name of the primary stain?

Answer 24

A. green

B. malachite green

Question 25

Why might a doctor perform a Gram stain on a clinical sample before prescribing antibiotics to a patient for a bacterial infection?

Answer 25

To determine whether the bacteria is gram negative or gram positive and prescribe the right antibiotics and not prescribe the opposite which would not be effective.

Question 26

Streptococcus pneumoniae that are capable of causing pneumonia are encapsulated bacteria (meaning they have a capsule). Describe what you would expect to see under the microscope after performing a capsule stain with india ink (acidic dye) and safranin (basic dye.

Answer 26

A black background with a clear capsule surrounding a pink/red cluster of cocci bacteria.

Question 27

Identify two diseases that are caused by acid-fast bacteria.

Answer 27

Tuberculosis and Leprosy

Question 28

A. What color would Gram-negative cells appear if step 2 was performed before step 1 in a Gram stain (if you did step 2, step 1, step 3, step 4)?

Answer 28

A. red/pink

Question 29

B. What color would Gram-positive cells appear if step 2 was performed before step 1 in a Gram stain (if you did step 2, step 1, step 3, step 4)?

Answer 29

B. red/pink

Question 30

A. What color would Gram-negative cells appear if step 4 was omitted (if you did step 1-3, but not step 4)?

Answer 30

A. colorless

Question 31

B. What color would Gram-positive cells appear if step 4 was omitted (if you did step 1-3, but not step 4)?

Answer 31

B. purple

Question 32

What is the most time-sensitive step in the Gram stain? What may happen if you let this step go too long?

Answer 32

Step 3 is the most time sensitive step because if the 95% Ethanol is rinsed too long then the stain could be over-decolorized.

Question 33

Provide the 3 basic parts of a bacterial flagellum. Briefly describe what each part does.

Answer 33

The filament is the tail like projection you can see. The hook connects the filament to the basal body. The basal body is the motor.

Question 34

What color does an acid-fast cell stain?

Answer 34

red

Question 35

Gram-positive cultures that are older than 24hrs can appear as Gram negative sometimes. Why does this happen?

Answer 35

Their peptidoglycan weakens and can no longer resist the decolorizer.

Question 36

If a bacterium can form an endospore, why would it? What is the purpose of an endospore?

Answer 36

A bacterium forms an endospore to survive. The purpose of an endospore is to not let the bacteria be destroyed. Endospores are very resistant heat, desiccation, radiation, antimicrobial compounds, and stains.