lab 4 gram stain/endospore

Question 1

Gram ___________ bacteria have a cell wall that’s composed of between 60 - 90% ____________

Answer 1

B. Positive ; peptidoglycan

-thick call wall

Question 2

what is gram neg

Answer 2

-thinner cell wall also mase of peptidogylcan which is surrounded by an outer membrane with high lipid contect

Question 3

what is the primmary stain for gram

Answer 3

crystal violet

Question 4

what is the mordant for gram and what does it do

Answer 4

iodine, causes crystal violet to form large crystals within the cell wall

Question 5

what is the decolorizer for this lab

Answer 5

alcohol/acetone solution

Question 6

what does the decolorizer do

Answer 6

dissolves the cyrstal violet and at the same timw dehydrates the thick cell wall of the gram pos (which makes it difficult for the stain to leave the cell wall)

Question 7

what color is gram pos

Answer 7

purple

Question 8

what color is gram neg

Answer 8

clear bc of the decolorizer reaction

Question 9

what is the counterstain for gram

Answer 9

safranin

Question 10

what makes gram pos pink ?

Answer 10

safranin

Question 11

explain the procedure…

Answer 11

1. start with bacteria smear
   2 cover the smear with crystal violet stain for 1 min
   3 gently and thoroughly rinse the slide with water
   4 cover the smear with gram iodine for 1 min
   5 gently and thoroughly rinse the slide with water
   6 add decolorizer dropwise until runnoff is clear (20-30)
   7 gently and thoroughly rinse the slide with water
   8 cover the smear with a safranin stain for 1 min
   9 gently and thorughly rinse.. done

Question 12

what was the result of e coli for this lab…

Answer 12

gram neg
color-pink
shape- rod
arrangemnet- none

Question 13

what was the result of staphyloccus aureus

Answer 13

gram-pos
color-purple
shape- sphiricle
arramgment- cluster

Question 14

How many steps are in the gram stain procedure

Answer 14

4

Question 15

What is the first step in the gram stain procedure?

Answer 15

The heat-fixed smear is flooded with primary stain crystal violet
[for 1 minute]

Question 16

What is the second step in the gram stain procedure?

Answer 16

adding mordant [gram’s iodine]
[for 1 minute]

Question 17

What is the third step in the gram stain procedure

Answer 17

[Remember to hold slide at an angle when washing]
(Wash the slide with water [for 5 seconds])

Then wash with the decolorizer 95% ethanol until no more crystal violet washes off the slide.

(Wash the slide with water [for 5 seconds])

Question 18

What is the fourth step in the gram stain procedure?

Answer 18

Counter-stain the bacteria with safrinin [for 1 minute]

(Then wash the slide with water [for 5 seconds]

Question 19

Why do we use mordant in the gram stain procedures?

Answer 19

It fixes the primary stain [Crystal Violet] to the cell wall

Question 20

True OR False?
Safrinin does not affect the color of the gram positive cells

Answer 20

True

Question 20

Why do we use decolorizer [95% ethanol]?

Answer 20

It dissolves the outer membrane in gram negative organisms [which removes the Crystal Violet stain]

Question 21

The ____________ stains cells that have become colorless [from the decolorization step]

Answer 21

safrinin

Question 22

True OR False?
It’s best to perform the gram stain on young, actively growing cultures.

Answer 22

True,
because older bacterial cultures do NOT show typical gram stain results

Question 23

What bacteria did we use for the gram-stain procedure?

Answer 23

1) Staphylococcus aureus [Staph. A}
2) Escherichia Coli [E Coli]

Question 24

What are endospores

Answer 24

dehydrated cells with thick cell walls survive extream heat, lack water

Question 25

what is the main stain for endospore

Answer 25

malachite green

Question 26

what is the counterstain stain for endospore

Answer 26

safranin

Question 27

what is the color of the endospore

Answer 27

green and the surrounding vegetative cell pink

Question 28

The spore stain is a ______________________ stain

Answer 28

differential stain

Question 29

Endospores are produced by

Answer 29

Bacillus and Clostridium

Question 30

Endospores are resistant to

Answer 30

desiccation, heat, chemicals (disinfectants, acids, and staining reagents)

Question 31

Bacteria produce endospores in response to

Answer 31

hostile environments conditions (heat, cold, radiation,etc.)

Question 32

Endospores are a

Answer 32

Protective mechanism

Question 33

In the proper environment, an endospore will germinate to produce a new ____________ cell, which can grow & reproduce

Answer 33

vegetative

Question 34

The endospore first develops within the mother vegetative cell when nutrients such as ___________ & ____________ become limiting

Answer 34

carbon & nitrogen

Question 35

________________ are not metabolically active and are resistant.

Answer 35

spores

Question 36

Any __________ cells will decolorize with water

Answer 36

vegetative cells

Question 37

Step 1 of Schaffer-Spore stain

Answer 37

Prepare a heat-fixed smear of Bacillus Subtilis

Question 38

Step 2 of Schaffer-Spore stain

Answer 38

Place slide on top of beaker of steaming water

Question 39

Step 3 of Schaffer-Spore stain

Answer 39

Saturate smear with malachite green & steam for 3 minutes

Question 40

Step 4 of Schaffer-Spore stain

Answer 40

Remove from water bath, set on staining tray, and cool it

Question 41

Step 5 of Schaffer-Spore stain

Answer 41

Gently rinse with squeeze bottle

Question 42

Step 6 of Schaffer-Spore stain

Answer 42

Counterstain with safrinin [for 30 seconds]

Question 43

Step 7 of Schaffer-Spore stain

Answer 43

Step 7 of Schaffer-Spore stain

Question 44

Step 8 of Schaffer-Spore stain

Answer 44

Examine slide under oil immersion lens

Question 45

It takes _____________ hours to incubate and see endospores

Answer 45

72 hours

Question 46

m. smegmatis + s. aureus results (acid fast stain)

Answer 46

shape-rod color-pink acid fast- positive