Lab Final Review - Week #2 Flashcards

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1
Q

What is a stain (chemically)?

A

an organic compound made up of a benzene ring plus a chromophore and an auxochrome group

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2
Q

The ability of a stain to bind to macromolecular componenets like proteins or nucleic acids depends on?

A

The electrical charge found on the chromogen portion and the electrical charge of the organisms being stained

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3
Q

Acidic stains

A

Anionic; negative chromogen -> binds to positively charge components like proteins

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4
Q

Basic stains

A

Cationic; positive chromogen -> binds to negatively charged components like nucleic acids

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5
Q

What is a chromogen?

A

It is the colored compound in a stain

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6
Q

What time of stain is usually used for bacterial staining? Why?

A

Basic stains, because of the negative charge on bacterial surfaces

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7
Q

Simple staining

A

Use of a single stain, which produces a distinctive contrast between the organism and the background. It is useful in the visualization of the morphilogical shape an arrangement of an organism

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8
Q

Differential staining

A

Use of two contrasting stain; at least 4 chemical reagents are needed to accomplish this. It is useful in separating microorganisms into groups & visualization of structures

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9
Q

What are the 4 primary rules in preparing bacterial smears for staining?

A
  1. clean slides
  2. label slides
  3. smear bacteria
  4. heat fixation
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10
Q

How would you prepare a bacterial smear using a broth culture?

A

Use one or two loopfuls and spread to about the size of a dime

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11
Q

How would you prepare a bacterial smear using cultures from a solid medium?

A

dilute the culture by placing one or two loopfuls of water on the slide in which the cell swill be emulsified. Spread to the size of a nickel

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12
Q

Why is heat fixation of the prepared slide so important?

A

So that the bacterial smear does not wash away during the staining procedure. Heat fixating coagulates the bacteria and fixes them to the glass surface

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13
Q

How could a smear that is too thick yield a false result?

A

Because of the thickness it could prevent stain to be washed away that otherwise would be

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14
Q

What are the 3 most commonly used basic stains?

A
  1. methylene blue 2. crystal violet 3. carbol fuchsin
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15
Q

How are simple stains clinically relevant?

A

Although they are not differential or diagnostic, simple stains are a quick procedure that allow for determining whether the sample has the presence of a foreign bacterial pathogen

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16
Q

What type of stain is used for negative staining?

A

Acidic stain ( such as India ink or nigrosin)

17
Q

What are the 2 practical applications of negative staining?

A
  1. because heat fixation is not used, the cells maintain their natural size and shape
  2. it is possible to observe bacteria that are difficult to stain
18
Q

What is the principle application of negative staining?

A

To determine if an organism possesses a capsule (it can also show spore formation)

19
Q

What is a capsule?

A

A gelatinous outer layer that makes the microorganism more virulent

20
Q

The technique of negative staining if often used to identify what important infectious agent?

A

Cryptococcus neoformans

21
Q

What is the most important differential stain used in bacteriology?

A

The Gram stain

22
Q

Gram stain allows for the division of bacterial cells into what two groups?

A

Gram-positive and gram-negative, this reaction and differentiation is based on the chemical composition of their cell walls.

23
Q

What type of cell wall do gram-positive cells have?

A

They have a thick peptidoglycan layer

24
Q

What type of cell wall do gram-negative cells have?

A

They have a very thin peptidoglycan layer and are surrounded by outer lipid layers

25
Q

What is peptidoglycan composed of?

A

It is a polysaccharide composed of two chemical subunits: N-acetylglucosamine and N-acetylmuramic acid. Peptidoglycan chains are cross linked by means of the enzyme transpeptidase

26
Q

What are the 4 reagent steps of a gram stain?

A
  1. primary stain
  2. mordant
  3. decolorizing agents.
  4. counterstain
27
Q

What is the primary stain? What does it do?

A

Crystal Violet, it stains all cells purple

28
Q

What is the mordant? What does it do?

A

Gram’s Iodine, it increases the cell’s affinity for the stain by binding to the stain forming an insoluble complex. Now, all cells appear purple-black

29
Q

What is the decolorizing agent? What does it do?

A

Ethyl Alcohol 95%, it serves a dual function: 1. protein dehydrating agent 2. lipid solvent. Because of this it ultimately decolarizes gram negative cells

30
Q

What is the counterstain? What does it do?

A

Safranin, it stains pink those cell walls which have previously been decolorized

31
Q

What is the most critical phase of the gram stain procedure and why?

A

The decolorizing step because over-decolorization will result in loss of the primary stain even in the gram positive stain, causing them to stain and appear as negative. And under-decolorization will not remove all stain from the gram negative organisms causing them to appear gram postive

32
Q

Why is it important to rinse the slide with water between each step in the gram staining procedure?

A

Because it removes excess reagent, thereby preparing the slide for the subsequent reagent

33
Q

Why is best to gram stain cultures that are less than 24 hours old?

A

Because as cultures age they lose their ability to retain the primary stain, causing them to appear gram-variable.

34
Q

Why is gram staining clinically important? 2 reasons

A
  1. it is much more rapid than culturing a specimen

2. it allows for the correct type of antibiotic to be used

35
Q

How much diluent and how much bacteria ought to be used when making a smear from an agar plate?

A

2 loopfuls of water and a small amount (tip of loop or needle) of bacteria

36
Q

What cell component is bound by negative stain?

A

none

37
Q

What color would a gram negative bacteria appear if stained with methylene blue?

A

purple

38
Q

What color would a gram negative bacteria appear if stained by nigrosin?

A

it would not be colored at all

39
Q

What color would a gram negative bacteria appear after gram-staining?

A

pink