Special stains

Question 1

what species produce endospores?

Answer 1

Bacillus and Clostridium

Question 2

why are endospores produced?

Answer 2

-they are produce when environments become unfavorable
-depletion of nutrients, change in temperature, lack of water
-when the environment becomes favorable again they will germinate back into vegetative cells

Question 3

Endospores

Answer 3

-are dormant forms of the bacteria resistant to heat, ultraviolet radiation, toxins, chemical disinfectants, freezing, and desiccation

Question 4

In normal environmental conditions how do bacteria exists?

Answer 4

-in a vegetative state where they are able to reproduce and grow

Question 5

What stain is used to identify endospores?

Answer 5

Schaeffer- Fulton stain

Question 6

What does the Schaeffer- Fulton stain identify?

Answer 6

vegetative and endospore forms of Bacillus and Clostridium

Question 7

Steps in Endospore stain (Schaeffer-Fulton)

Answer 7

1. Place malachite green stain on the slide while holding over steam for 5 minutes (heat is the mordant)
2. decolorize the stain with water (vegetative cells: white, endospores: green)
   3.Flood the smear with the counterstain safranin for 1 minute, then rinse with water
   (vegetative cell: red, endospore: green)

Question 8

What is the mordant in the Schaffer Fulton stain

Answer 8

heat

Question 9

what is the primary stain in Schaffer- Fulton stain

Answer 9

malachite green

Question 10

What is the counterstain in Schaeffer-Fulton stain

Answer 10

safranin

Question 11

What species contain mycolic acid

Answer 11

mycobacterium

Question 12

what does mycolic acid do?

Answer 12

-is a waxy outer layer on cells that block primary stains from being absorbed

Question 13

What can certain species of Mycobacterium cause?

Answer 13

-Leprosy or tuberculosis

Question 14

What does acid-fast staining (Ziehl-Neelsen) identify?

Answer 14

-bacteria with mycolic acid

Question 15

Steps in Acid-fast staining (Ziehl-Neelson)

Answer 15

1.Prepare a mixture of S.epidermis and Mycobacterium thin smear
2.Place carbolfuchsin stain on the slide while holding over steam for 5 minutes (heat is the mordant, which softens the waxy outer layer, allows for penetration)
-all bacteria appear magenta
3.Decolorize with acid alcohol dropwise until smear is decolored, rinse with water (removes stain from non-acid fast cells)
4.counterstain with methylene blue, flood the slide for 2 minutes, the rinse off with water
results: mycobacterium (magenta)
s. epidermis: blue

Question 16

How to create a thin smear?

Answer 16

1. create smear of bacteria on slide
2. air dry
3. heat fix

Question 17

What is methylene blue

Answer 17

a positively charged, basic stain and will bind to the surface of non-acid fast cells

Question 18

What is glycocalyx

Answer 18

-is a gelatinous sticky substance surrounding the outside of the cell
-made up of proteins and polysaccharides

Question 19

capsule

Answer 19

-a glycocalyx that is firmly attached to the cell surface
-this can prevent bacteria from being recognized by the host
-can also camouflage by containing chemicals that are found in the host cell

Question 20

Negative capsule stain

Answer 20

1. use a primary dye such as crystal violet to stain bacteria
2. stain the background with an acidic negatively charged dye like Nigrosin or India Ink
   Background: India Ink or Nigrosin (pink)
   Bacteria: Crystal violet (purple)
   capsule: clear, colorless, white halo

Question 21

What is the mordant in the Acid-fast staining (Ziehl-Neelson)

Answer 21

heat

Question 22

what is the primary stain in Acid-fast staining (Ziehl-Neelson)

Answer 22

carbolfuchsin

Question 23

what is the secondary stain in Acid-fast staining (Ziehl-Neelson)

Answer 23

methylene blue

Question 24

crystal violet

Answer 24

-is positively charged/basic and will stain the negatively charged features on the bacteria besides the capsule

Question 25

Name two negatively charged dyes

Answer 25

Nigrosin and India Ink

Question 26

How do negatively charged dyes work?

Answer 26

they repel the cell/capsule that is also negatively charged

Question 27

Flagellar staining

Answer 27

- primary stain and mordant are mixed and used as the primary stain -this combination allows the dye to be less soluble and coat the surface of the flagella -this increases the diameter/size and colorize it so easier to see

Question 28

Monotrichous

Answer 28

-one flagella only on one side ex: vibrio cholerae

Question 29

Amphitrichous

Answer 29

-having a single flagellum on each of the two opposite ends ex: spirillum volutans

Question 30

Lophotrichous

Answer 30

multiple flagella at one end or end point ex: helicobacter pylori

Question 31

Peritrichous

Answer 31

having flagella all over, covering the entire surface ex: Proteus vulgaris

Question 32

Vegetative cell

Answer 32

-is the parent cell of an endospore. the vegetative cell breaks open to release the endospores into the environment

Question 33

Why can you not see flagella on smears stained with usual technique?

Answer 33

-The flagella are to thin to see without a compound microscope, their width is less than the resolution of microscope

Question 34

Why is heat used to stain bacterial endospores?

Answer 34

-heat drives the stain into the cell

Question 35

what are some characteristics of endospores?

Answer 35

-endospores are resilient forms of a bacterial cell that forms within the parental cell. They have an impervious cell wall and are resistant to heat/cold, pathogens, toxic changes and desiccation

Question 36

What is the difference between central and terminal endospores?

Answer 36

central endospores are located within the middle of the vegetative cell. And terminal endospores are located at the end of vegetative cells.

Question 37

What is meant by the term vegatative cell?

Answer 37

-is the parent cell of an endospore, it breaks open to release the endospore into the environment

Question 38

How does the spore stain resemble the acid-fast stain

Answer 38

-both the endospore and acid-fast stain require the use of heat to drive the primary stain through the cell wall

Question 39

What pathogen can be presumptively identified using the acid fast stain?

Answer 39

mycobacterium species

Question 40

Why can you not see flagella on smears stained within the usual staining technique?

Answer 40

-the flagella are too thin to see with a compound microscope

Question 41

What is the principle behind the negative staining technique?

Answer 41

-negative stains, stain the entire background surround the cells. A counter stain allows the cells to be visualized and the capsule is left unstained. This leaves a halo like capsule visible between negative and counter stains

Question 42

What structures may be observed with a negative staining technique

Answer 42

capsules

Question 43

what stains endospores?

Answer 43

malachite green

Question 44

what stains vegetative cells in the endospore stain?

Answer 44

safranin

Question 45

what stains the background in the negative stain?

Answer 45

India Ink or nigrosin

Question 46

What is the primary stain for acid fast?

Answer 46

carbol fuchsin