Bacterial Morphology and Staining Techniques

Question 1

Name the morphology of this bacteria.

A spherical bacteria with a diameter of approximately 0.5 µm

Answer 1

Coccus

Question 2

Name the morphology of this bacteria.

Rod-shaped bacterial cells linked to form a chain

Answer 2

Streptobacilli

Question 3

A dye that stains only the cell leaving the background colorless.

Answer 3

Direct Stain

Question 4

Acidic stain repelled by the cell’s surface

Answer 4

Congo Red

Question 5

Mesh-like polymer present in bacteria cell walls

Answer 5

Peptidoglycan

Question 6

List the sequence for condition a gram stain.

Answer 6

1.) Crystal violet applied
2.) Iodine applied
3.) Decolorizer dehydrates Gram-positive cell walls
4.) Safranin is applied

Question 7

a cylindrical or rod-shaped bacterium 0.5 µm to 20 µm in length

Answer 7

Bacillus (bacilli, plural

Question 8

a helical or spiral-shaped bacterium approximately 15 µm in length

Answer 8

Spirillum (spirilla, plural)

Question 9

_____ are rod-shaped bacteria 0.5 - 20 µm in length.

Answer 9

Bacilli

Question 10

Only blank and blank form organized arrangements

• hint bacterial morphologies

Answer 10

Bacilli and cocci

Question 11

True/False: Spirilium remain as single cells and do not cluster.

Answer 11

True

Question 12

When a coccus bacterial cell divides but the daughter cells fail to fully separate after cell division, a pair of cocci is formed, and the pair is called a

Answer 12

diplococci.

Question 13

When cocci fail to separate and remain in groups of four, they form squares called

Answer 13

tetrads

Question 14

Cocci can also form cubical packets of eight cells, sixteen cells, and even greater numbers of cells, which are all called

Answer 14

sarcina

Question 15

_____ are bead-like chains of spherical cells.

Answer 15

Streptococci

Question 16

Simple stain

Answer 16

One dye is used to directly stain the bacterial cell or the background. Cell shape, size, and arrangement may be determined with simple staining techniques.

Question 17

Direct stain

Answer 17

A type of simple stain; only the bacterial cell becomes colored, leaving the background or external environment clear and colorless

Question 18

Negative stain

Answer 18

A type of simple stain; the cell is unstained and appears as a bright, often colorless object against a dark-stained environment.

Question 19

Differential stain

Answer 19

Two or more dyes are used. Cell morphology can be described and identification of the structural components (composition of the cell wall and other cell features like flagella, capsules, or endospores) is possible. Figure 5 shows one type of differential stain called a Gram stain.

Question 20

Negative staining results in a colorless cell on a stained background.

Answer 20

True

Question 21

Stains are salts dissolved in a liquid and are composed of positive (+) and negative (-) ions. One of the ions is colored depending on the chemical composition of the solution. The colored ions are called

Answer 21

chromophores.

Question 22

Does a basic stain have a positive or negative charged chromophore?

Answer 22

Positive

Question 23

Does an acidic stain contain positive or negative charged chromophores?

Answer 23

Negative

Question 24

List an example of a basic stain and an acidic stain.

Answer 24

Basic: Crystal violet
Acidic: Congo red

Question 25

_____ is an example of a direct stain.

a.) Crystal violet
b.) Methylene blue
c.) safranin
d.) all the above

Answer 25

d

Question 26

True/False: Gram positive have a thin peptidoglycan wall.

Answer 26

False! Thick wall

Question 27

True/False: Teichoic acids are only found in Gram-positive bacteria.

Answer 27

True

Question 28

Blank cells have a thin cell wall comprised of a single layer or sheet of peptidoglycan sandwiched between an outer and inner cell membrane.

Answer 28

gram negative

Question 29

True/False: lipopolysaccharides are found in gram positive cells only.

Answer 29

False!

The outer cell membrane also contains specialized molecules or proteins unique to Gram-negative cells called lipopolysaccharides (LPS) which are endotoxins that produce reactions such as fever or shock in Gram-negative infections

Question 30

T/F Gram-positive cells have a thick outer cell wall composed of peptidoglycan.

Answer 30

True

Question 31

True/False: Crystal violet dyes all bacterial cells, regardless of peptidoglycan thickness

Answer 31

True

Question 32

What does iodine do in a gram stain.

Answer 32

Iodine reacts with crystal violet, to form a complex that allows dyes to better adhere to the bacterial cells.

Question 33

What does decolorizer do in a gram stain.

Answer 33

Dehydrates the gram positive cell wall, trapping in dyes, the decolorizer washes dyes away from the gram negative bacteria

Question 34

What does safranin do in a gram stain?

Answer 34

No effect on gram-positive bacteria. Turns gram-negative bacteria to become pink.

Question 35

Blank washes dyes from Gram-negative bacteria.

Answer 35

Decolorizer

Question 36

What is the purpose of heat fixing? Why did the slides need to be heat fixed during the direct stain but not during the negative stain?

Answer 36

The purpose of heat fixing is to kill the bacteria on the slide and therefore preventing the bacteria from spreading. Additionally, heat fixing the slides allows the bacteria to be more readily taken up by the stain. Heat fixing is important during a direct stain because the stain binds directly to the bacteria. Heat fixing is not used during a negative stain because the background is being stained and not the actual bacteria.

Question 37

Define the three major types of bacterial shapes.

Answer 37

The three major bacterial shapes are coccus, bacillus, and spirilla. Coccus appears to be spherical shape. Bacilli have a rod-shaped appearance. Finally, spirilla bacteria have a spiral or helical shape

Question 38

Describe the difference between Gram-negative and Gram-positive bacteria. Which type of bacteria are most often treatable with antibiotics and why?

Answer 38

Gram-positive bacteria lack an outer membrane but are surrounded by a thick peptidoglycan layer. Gram-negative bacteria have an outer membrane and are surrounded by a thin peptidoglycan layer. Gram-positive bacteria are most often treatable with antibiotics because Gram-positive bacteria lack the outer membrane that often creates resistance for antibiotics. Additionally, Gram-positive bacteria have a thick peptidoglycan outer layer that readily absorb antibiotics.

Question 39

List the steps of the Gram stain technique and describe the purpose of each. Be sure to include a description of the color of the bacteria during each step.

Answer 39

The steps of the Gram stain technique are: 1.) apply the crystal violet and rinse with water after one minute 2.) add PVP iodine and rinse with water after one minute 3.) add decolorizer and rinse with water after 30 seconds 4.) counter stain with safranin and rinse with water after 30 seconds.

After the first step of adding the crystal violet, the bacteria will appear to be dark purple. Crystal violet is positively charged therefore its purpose is to adhere to both the gram positive and negative cell membranes. Next, once the iodine is added, the stain appears yellow until it is rinsed off. Once rinsed off the stain maintains the light purple appearance from the crystal violet. The purpose of adding the iodine, is to bind the iodine to the crystal violet and make a larger complex that adheres to the cell membrane. The next step is to add the decolorizer. When the decolorizer is added, the purple stain from the crystal violet disappears. The slide will appear colorless if it is Gram-negative and the slide will still have a purple appearance if it is Gram-positive. The purpose of the decolorizer is to dissolve the crystal violet. In gram negative bacteria the decolorizer will efficiently dissolve the crystal violet because the bacteria have a thin layer of peptidoglycan and are unable to retain the iodine-crystal violet complex. In gram positive bacteria, the decolorizer will not be able to dissolve the crystal violet due to the thick outer layer of peptidoglycan. Finally, the safranin is added as a counter stain. The safranin directly stains the bacteria that has been decolorized. The safranin is red in color. After the safranin is washed away and the bacteria has dried, the bacteria will appear pink or red if it is Gram-negative or the stain will remain purple if it is Gram-positive.

Question 40

What is the major advantage of a differential stain, such as the Gram stain, over a simple stain? Was this observed in your experiment?

Answer 40

A differential stain, such as a Gram stain allows for the morphology and structural components of the bacteria to be determined. In a simple stain, only the physical appearance of the bacteria may be viewed under the microscope. This was observed in the experiment. When I used a Gramstain, I could identify different types of bacteria that were Gram-positive or negative. I could also view the physical characteristics of the Gram stain bacteria under the microscope.

Question 41

_____ exist only as single cells and do not form clusters or chains.

Answer 41

Spirillum

Question 42

T/F Differential staining uses two or more dyes to stain structural components of a cell.

Answer 42

T

Question 43

The charge of _____ determines how dyes interact with bacterial cells during staining.

Answer 43

Chromophores

Question 44

_____ stains Gram-negative bacteria pink.

Answer 44

Safranin

Question 45

Gram-positive bacteria have an outer cell membrane containing endotoxins. T/F

Answer 45

False

Gram-negative bacteria have an outer cell membrane lacking endotoxins.

Question 46

Heat fixing is required for the negative staining process. T/F

Answer 46

F,
Heat fixing is not used in negative staining, only direct staining.

Question 47

Slides should be rinsed after applying _____ during the Gram staining process.

a.) crystal violet
b.) Iodine
c.) decolorizer
d.) all the above

Answer 47

d

Question 48

What color is gram negative bacteria?

Answer 48

Pink