Microorganisms through a Microscope Flashcards

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1
Q

Differentiate b/w an acidic dye and a basic dye, and give some examples of the commonly used dyes

(Why do basic dyes stain bacterial cells? Why don’t acidic dyes stain bacterial cells?)

A
  1. Acidic Dye: Positive ions – Includes eosin, acid fuchsin, & nigrosin.
    - - Not commonly used because bacteria are slightly negatively charged at pH 7, and acidic dyes are not attracted to most types of bacteria
    - - The stain colors the background instead
  2. Basic Dye: Negative ions – Includes crystal violet, methylene blue, malachite green & safranin
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2
Q

Name the 3 kinds of staining techniques

A
  1. Simple
  2. Differential
  3. Special
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3
Q

Explain the simple staining technique. What’s the primary purpose of simple stain?

A
  1. Simple staining technique uses an aqueous or alcohol solution of a single basic dye
  2. The primary purpose: to highlight the entire microorganism so that cellular shapes and basic structures are visible
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4
Q

What is the chemical called when added to intensify a staining solution?

A

Mordant

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5
Q

What are the two common differential stain techniques?

A
  1. Gram Stain

2. Acid-fast Stain

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6
Q

Explain the Gram Staining procedures in 4 major steps

A
  1. Application of crystal violet (purple dye)
    - - After a short time, wash off the purple dye with water
  2. Application of iodine (mordant)
    - - After a short time, wash off the iodine with water
  3. Alcohol or alcohol-acetone solution wash (decolorization)
    - - After a short time, rinse off the alcohol
  4. Application of safranin (counterstain, red dye)
    - - After a short time, wash off safranin with water and blot dry before examination
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7
Q

What color does Gram (+) bacteria appear? And Gram (-)?

A

Gram (+): Blue/Purple

Gram (-): Red/Pink

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8
Q

Why can Gram (+) bacteria trap crystal violet stain, but not Gram (-)? Hint: cell wall structure / composition

A
  1. Gram (+) bacteria: Thicker Peptidoglycan cell wall inhibits the Crystal violet-Iodine (CV-I) complex to be washed off by alcohol
    - - Peptidoglycan: disaccharides + aa’s
  2. Gram (-) bacteria: Thin Peptidoglycan and they contain a layer of lipopolysaccharide (LPS), which is disrupted by alcohol wash, so the CV-I complex is washed out through the thin layer of peptidoglycan, appearing colorless until counterstained with safranin.
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9
Q

Are Gram (+) or Gram (-) bacteria easily killed by Penicillin and Cephalosporin? Why?

A
  1. Gram (+) bacteria are killed easily by PCN and Cephalosporin
  2. Gram (-) bacteria are more resistant to these antibiotics because they can’t penetrate the LPS layer.
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10
Q

Which genus of bacteria usually use the acid-fast staining technique? And give examples of the pathogens.

A
  1. Mycobacterium, including Mycobacterium tuberculosis and Mycobacterium leprae.
  2. Nocardia
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11
Q

Explain the acid-fast staining procedures in 3 major steps

A
  1. Application of red dye carbolfuchsin, and heat for several mins
    – Cool and Wash off stain with water
  2. Application of acid-alcohol (decolorization)
    Note: red stain is removed is NOT acid-fast (colorless)
  3. Application of methylene blue (counterstain)
    – Appear blue if not acid-fast
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12
Q

How does acid-fast stain work?

A

Acid-fast stain is more soluble in the cell wall lipids (waxy material) than in the acid-alcohol, therefore, retaining the red color

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13
Q

When is the special staining used? Give some examples

A

To color and isolate specific parts of microorganisms, such as Endospores, Flagella, and Capsules.

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14
Q

Explain the Capsule Stain procedure in 2 major steps (capsule stain is also called negative stain)

A
  1. Mix the bacteria in color solution: India ink or nigrosin
    - - Providing a dark background
  2. Stain the bacteria with a Simple Stain, ex. safranin
    - - The Capsule doesn’t accept the dye, but the bacterial cell does. Thus, unstained halos appear microscopically
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15
Q

Explain the Endo(spore) Staining procedure in 3 major steps. What color do we see under the microscope after the staining?

A

Schaeffer-Fulton endospore stain:

  1. Application of Malachite green and steam for 5 mins
    - - Heat helps the stain penetrate the endospore wall
  2. Wash with water for 30 secs
  3. Application of safranin (counterstain)
    - - Stain bacterial cells other than the endospores.

Note: the endorspores appear green w/i red or pink cells

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16
Q

Explain the Flagella Staining and why is it needed?

A
  1. Application of a mordant and carbolfuchsin
    - - To build up the diameters of the flagella until they become visible
  2. Bacterial flagella are generally too small to be see without staining
17
Q

How is total magnification of an object calculated?

A

Magnification of the objective lens X Magnification of the ocular lens

18
Q

When/why is immersion oil used?

A

It is used with the oil immersion lens to reduce light loss b/w the slide and the lens

19
Q

An electron microscope differs from a light microscope in that ___ focused by ___ is used instead of light, and the image is viewed not through the ocular lenses but on ___.

A
  1. Objects smaller than about 0.2 microns
  2. A beam of electrons (travel in waves)
  3. Electromagnetic condenser lenses
    (Note: images are always black and white)
20
Q

The max. magnification of a compound microscope is ___; that of an electron microscope, ___.
The max. resolution of a compound microscope is ___; that of an electron microscope, ___.
One advantage of a scanning electron microscope over a transmission electron microscope is ___.

A
  1. 2000X, 10,000X
21
Q

Endospores can be seen as refractile structures in unstained cells and as colorless areas in Gram-stained cells. Why is it necessary to do an endospore stain to verify the presence of endospores?

A

Because they can’t be differentiated from inclusions of stored material w/o a special stain