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Microbiology > Lecture 3B Microscopy and Staining > Flashcards

Lecture 3B Microscopy and Staining Flashcards

1
Q

Preparation of Specimens for Light Microscopy

A
  • Smear
  • Fixing
  • Staining
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2
Q

Smear: Spread a __ of material containing microorganisms over the slide surface. Allow to __

A
  • thin film
  • air dry
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3
Q

Fixing: Process that __ microorganisms and __ them to a microscope slide. Fixing __ and __distortion of cells.

A
  • kills
  • attaches
  • preserves
  • minimizes
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4
Q

Staining: Coloring microorganisms with a dye that emphasizes certain structures. Before staining a sample, it must be __. Stains are __composed of __

A
  • fixed
  • salts
  • 2 components: positive ion (cation) and negative ion (anion)
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5
Q

The colored ion in staining is called the __

A

chromophore

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6
Q

Two types of dyes

A
  • basic dyes
  • acidic dyes
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7
Q

A type of dye
- Chromophore is in __ ions.
Most commonly used dyes.
- Bacteria are slightly __ charged at pH 7, therefore they stain with basic dyes.
- Give examples

A

BASIC DYE
- positive
- negatively
- crystal violet, methylene blue, safranin (red/pink)

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8
Q

A type of dye
- Color is in __ ions.
- Stain the background: __
- Bacteria __ stain with acidic dyes.
- Used to observe __
- Minimal distortion because heat fixing is not necessary and dye is not taken up by cells.
- Give examples

A

ACIDIC DYE
- negative
- negative staining
- do not
- cell shape, size, and capsules
Examples:
- eosin (yellow)
- nigrosin (black/dark gray)
- india ink (black/dark gray)

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9
Q

cell wall __ the acidic dye

A

repels

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10
Q

Types of staining methods

A
  • Simple Stains
  • Differential Stains
    a. Gram Stain
    b. Acid-Fast Stain (Ziehl-Nielsen Stain)
  • Special Stains
    a. Endospore Stain
    b. Capsule Stain
    c. Flagella Stain
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11
Q

A type of staining method
- Aqueous or alcohol solution of a single basic dye.
- Primary purpose is to stain the entire organism to view cell shape and basic structures.

A

SIMPLE STAINS

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12
Q

Procedure of simple staining:

A
  • stain is applied for a certain time, then washed off
  • slide is dyed and examined
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13
Q

A type of staining method
- React differently to different types of bacteria.
- Can be used to distinguish among different groups of bacteria.

A

Differential stains

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14
Q

two important differential stains used in microbiology

A
  • gram stain
  • acid fast stain (Ziehl-Nielsen Stain)
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15
Q

Who developed gram stain?

A

Hans Gram (1884), a Danish microbiologist

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16
Q

A type of staining method
- The most useful staining procedure in medical microbiology.
- culture must be young to undergo this stain
- provides useful information for disease treatment
- Distinguishes bacteria of two large and medically important groups:
__ bacteria
__ bacteria

A

Gram stain (Differential stain)

17
Q

what type of bacteria have thick cells walls

A

gram-positive bacteria

18
Q

Steps of gram stain

A
  1. Primary stain
  2. Mordant
  3. Decolorizing
  4. Counterstain
19
Q

Steps of gram stain:
1. __ stain: Cover a heat fixed smear with a basic dye (__).
2. __: After smear is rinsed with water, an __ solution is applied
3. Decolorizing:
Gram negative cells will be __.
Gram-positive cells will remain __.
4. Counterstain:
Gram-negative cells are stained __.
Gram-positive cells remain __.

A
  • primary
  • crystal violet
  • mordant
  • iodine mordant
  • decolorized
  • purple
  • pink/red
  • purple
20
Q

slide is washed with alcohol which will remove stain from gram-negative cells but not from gram-positive cells

A

decolorizing

21
Q

alcohol is rinsed off. safranin is applied, which will stain the cells that were decolorized

A

counterstaining

22
Q
  • Gram-positive cells have very __ peptidoglycan cell walls, whereas gram-negative cells have very __ cell walls. Crystal violet easily penetrates both cell types.
  • Because of its larger size, the crystal violet-iodine complex [CV-I] is not easily removed from gram- __ cells, due to their thick cell wall. The CV-I complex is readily washed out of gram-__cells with alcohol.
  • Counterstain (this refers to __ name of stain) only colors gram-__ cells.
A
  • thick
  • thin
  • positive
  • negative
  • safranin
  • negative
23
Q

Application of Gram stain
- Gram-positive cells with their very thick peptidoglycan cell walls, are __ to penicillins and cephalosporins.
- Gram-negative cells with their thin cell walls and lipopolysaccharide layer are __ to these antibiotics.

A

CHEMOTHERAPY
- susceptible
- resistant

24
Q

Limitations of Gram stain

A
  • Not all bacterial cells stain well with the Gram stain.
  • Gram-stain only works well on young bacterial cultures, that are actively growing. Therefore it is best to use cultures 18 to 24 hours old.
  • Older cultures (over 24-48 hours), are often gram-variable.
25
Q

A type of staining method
- Used to detect tuberculosis and leprosy-causing organisms of the genus __ and pathogens of the genus __.
- These bacteria have __ cell walls, which makes them difficult to stain.

A

Acid-fast Stain or Ziehl-Nielsen Stain (differential staining)
- Mycobacterium
- Nocardia
- waxy

26
Q

Acid-fast stain is a modification of a method developed in 1882 by __.

A

Paul Ehrlich

27
Q

steps of Acid-Fast Stain

A
  1. Primary stain
  2. Decolorizing
  3. Counterstain
28
Q

Steps of Acid-Fast Stain
1. Primary stain: Cover a __ with __, a red basic dye. Gently heat for several minutes to increase penetration and retention of dye. Allow to __ and __
2. Decolorizing: Slide is washed with __.
Non-acid-fast cells will be __.
Acid-fast cells will remain __.
3. Counterstain: Acid-alcohol is __. __is applied, which will stain cells that were decolorized.
Non-acid-fast cells are stained __.
Acid-fast cells remain __.

A

(1)
- heat-fixed smear
- carbolfuchsin
- cool; rinse with water
(2)
- acid-alcohol
- decolorized
- red
(3)
- rinsed off
- Methylene blue
- blue
- red

29
Q

Special Stains are Used to color and isolate specific parts of microorganisms such as: __, __, and __

A
  • endospores
  • capsules
  • flagella
30
Q

A type of staining method
- used for 2-3-day old culture; the older the better

A

Endospore stain

31
Q

Endospore stain
- Endospores are extremely __ , __ structures that are formed by some gram-positive bacteria to protect them from __
- Ordinary staining methods cannot penetrate the thick __
- The most commonly used method is __ endospore stain

A
  • resistant; dormant
  • harsh environmental conditions: heat, drought, chemicals, radiation
  • endospore wall
  • Shaeffer-Futton
32
Q

Steps for Schaeffer-Fulton Endospore Stain
1. Primary stain: __ is applied to heat fixed smear and steamed for 5-7 minutes.
2. Wash: Rinse with __ for 30 seconds.
3. Counterstain: __ will stain rest of the cell.

A
  • Malachite green (will penetrate the endospore wall)
  • water (remove green dye from the rest of the cell except for endospore)
  • safranin
33
Q

Appearance of cell with endospore

A

Pink cell with green endospore

34
Q
  • gelatinous covers on top of the cell wall, which are important virulence (disease) factors
A

capsules

35
Q

A type of staining method
- Capsules are __ to stain
- __ is used to obtain a dark background (E.g.: India ink or nigrosin).
- Cell is stained with a __ (e.g.: safranin).

A

Capsule stain (special stain)
- difficult
- negative ion
- basic dye

36
Q

Capsule appearance

A

light halo around the cell

37
Q

A type of staining method
- Flagella are appendages used for __ that are too thin to be seen easily with a light microscope.
- Staining procedures are difficult. Usually involves using a __ and coating the flagellar surface with a __
- The __ and __ of flagella can be used as diagnostic aids

A

Flagella stain
- locomotion
- mordant
- dye or metal
- number
- arrangement

38
Q

contains metal to increase the size or improve the retention of the dye

A

mordant

Microbiology (5 decks)

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