Laboratory Experiments in Microbiology-Exercise 7

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Lab portion Gram Staining
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1

What is the Gram stain useful for?

It is a differential stain that allows you to classify bacteria as either gram negative or gram positive.

2

Who discovered the gram staining technique?

Hans Christian Gram

3

What are the gram staining techniques?

1.Apply primary stain- (crystal violet) all bacteria are stained purple by this basic dye
2. Apply mordant Gram's iodine- the iodine combines with the crystal violet in the cell to form a crystal violet-iodine (CV-1 complex)
3. Apply decolorizing agent (ethanol). The primary stain is washed out of some bacteria (decolorized) and some are unaffected.
4. Apply secondary stain or counterstain (safranin) this basic dye stains the decolorized bacteria red

4

What is the most important determining factor in the procedure of Gram staining?

bacteria differ in their rate of decolorization

5

The bacteria that decolorize easily are called?

gram-negative

6

The bacteria that decolorize slowly and retain stain are?

Gram-positive

7

Why do bacteria stain differently?

Chemical and physical differences in their cell walls

8

Bacterial cell walls are complex lattice structures composed of layers of ______.

peptidoglycans

9

Gram-positive cell walls contain multiple layers of peptidoglycans making them __________.

thick

10

Gram-negative bacterial cell walls contain a__________ layer of peptidoglycans surrounded by an outer layer of lipoproteins, phospholipids and lipopolysaccharides.

thin

11

Crystal violet is picked up by the __________.

cell

12

Iodine react with the dye in the ___________to form CV-1 that is ________ than the crystal violet that entered the cell.

cytoplasm
larger

13

The alcohol decolorizing agent ________________ the cell wall of the ___________ cells.

dehydrates
gram-positive bacteria

14

True or false. The CV-I cannot be washed out of the gram-positive bacteria.

true

15

Safranin stains the decolorized bacteria __________.

red

16

Gram stain is most consistent when used on young cultures less than _______________.

24 hours old

17

Why are cells less than 24 hours better?

because when the bacteria dies their cell walls degrade and may not retain the primary stain- which would give inaccurate results.

18

What are the gram staining reagents?

Crystal violet
Gram's iodine
Ethanol
Safranin

19

What do you rinse the stain with?

distilled water

20

Preparation of Gram staining:

prepare slide with circle on back of each slide
label the slides for each culture
smear with the inoculated loop
air dry smear
fix the slides

21

Gram staining procedure:

1. starting with fixed smear cover the smear with crystal violet for 1 minute
2. gently wash off crystal violet with water until it runs clear
3.cover the smear with Gram's iodine for 30 seconds
4.gently wash smear with water
5. decolorize with ethanol
6.gently wash off ethanol
7.cover the smear with safranin for 1 minute
8. wash the smear with water
9. blot it dry

22

What are some common sources of Gram staining error?

a. loop too hot
b. excessive heat during heat fixing
c. the decolorizing agent (ethanol) left on too long
d. the culture was too old
e. the smear was too thick

23

What color will a gram-negative cell turn?

pink

24

A gram positive cell?

purple

25

Which organism is a coccus?

staphylococcus

26

Bacteria: Staphylococcus epidermidis
name the morphology and arrangement and gram stain result

coccus
staphylo
purple

27

Bacillus subtilis

rod
single
purple

28

Escherichia coli

single cell
bacillus
pink

29

Can iodine be added before primary stain in gram stain?

yes

30

Considering you can't identify bacteria from a Gram stain, why might a physician perform a Gram stain on a sample before prescribing an antibiotic?

To identify which type of antibiotics to use. There are antibiotics that are gram specific and there are broad spectrum antibiotics that are effective against both gram negative and gram positive


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