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Microbiology > Ch. 4: Microscopy, Staining, and Classification > Flashcards

Ch. 4: Microscopy, Staining, and Classification Flashcards

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Biology 101 flashcards
1
Q

What are the 4 General Principles of Microscopy?

A

Wavelength of radiation
Magnification
Resolution
Contrast

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2
Q

How can you increase resolution?

A

Increase wavelength

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3
Q

What organisms can a compound light miscroscope (LM) see?

A 
200 nm - 10 mm
Flea
Large protozoans
RBC
Chloroplasts
Mitochondrion
Bacteria and archaea
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4
Q

What organisms can a scanning electron microscope (SEM) see?

A 
0.4 nm - 1mm
Large protozoans
RBC
Chloroplasts
Mitochondrion
Bacteria and archaea
Viruses
Ribosomes
Proteins
Diameter of DNA
Amino Acids
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5
Q

What organisms can a transmission electron miscrocope (TEM) see?

A 
RBC
Chloroplasts
Mitochonrion
Bacteria and archaea
Viruses
Ribosomes
Proteins
Diameter of DNA
Amino acids
Atoms
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6
Q

Describe light microscopes

A 
Light Microscopes – uses light passed through a specimen
Types include:
Brightfield (typically requires stain)
Darkfield (unstained live cells)
Phase-Contrast (unstained, live cells)
Differential Interference Contrast (uses two beams of light)
Flourescence (UV Light)
Confocal (Laser light)
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7
Q

Describe electron microscopes

A 
Electron Microscopes – use a beam of electrons which are either passed through or bounced off of a specimen
Types include:
Transmission (TEM)
Scanning (SEM)
Scanning Tunneling (STEM)
Atomic Force (SEM also)
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8
Q

Describe contrast

A
  • Differences in intensity between two objects, or between an object and background
  • Important in determining resolution
  • Staining increases contrast
  • Use of light that is in phase increases contrast
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9
Q

Describe simple bright-field microscopes

A
  • Contain a single magnifying lens
  • Similar to magnifying glass
  • Leeuwenhoek used simple microscope to observe microorganisms
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10
Q

Describe compound bright-field microscopes

A
  • Series of lenses for magnification
  • Light passes through specimen into objective lens
  • Oil immersion lens increases resolution
  • Have one or two ocular lenses
  • Total magnification (objective lens X ocular lens)
  • Most have condenser lens (direct light through specimen)
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11
Q

Name the 11 parts of a compound bright-field microscope

A

-(Bin)Ocular lens: remagnifies the image formed by the objective lens
-Body: transmits the image from the objective lens to the ocular lens using prisms
-Arm
-Objective lenses: primary lenses that magnify the specimen
-Stage: holds the microscope slide in position
Condenser: Focuses light through specimen
-Diaphragm: controls the amount of light entering the condenser
-Illuminator: light source
-Coarse focusing knob: moves the stage up and down to focus the image
-Fine focusing knob
-Base

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12
Q

What is a compound light microscope?

A

a microscope with a series of lenses

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13
Q

What is total magnification?

A

calculated by multiplying the objective lens

magnification by the ocular lens magnification

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14
Q

What is resolution?

A

The smallest distance that one is able to distinguish two points as being two distinct points. This distance is the resolving power

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15
Q

What is the limit of white light resolution?

A

About 0.2 microns is the limit of white light resolution, beyond that the quality of lenses is not able to improve

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16
Q

What is the lowest total magnification available using our lab microscopes?

A

40x

17
Q

What is the effect of immersion oil on resolution?

A

It lowers the angle of refraction

18
Q

Does transmission electron microscopy (TEM) go through or bounce off the specimen?

A

Through the substance

19
Q

Does scanning electron microscopy (SEM) go through or bounce off the specimen?

A

Bounces off the surface of specimen resulting in a more 3D image

20
Q

What are the steps in preparing a slide?

A
  • Clean
  • Smear
  • Fix
  • Heat-Fix
  • Stain
  • Dry
  • Observe
21
Q

What is fixation?

A

process by which internal and external structures of cells are preserved

22
Q

What is heat-fixing?

A

passing a smeared slide quickly through a flame

23
Q

What do dyes do?

A

give color to cells by chemical bonding

24
Q

What four stains do we use and what are their colors?

A

Methylene blue
Crystal violet
Safranin (red)
Carbolfuchsin (red)

25
Q

What is a mordant?

A

chemical added to make the dye bind more tightly
(increased affinity for specimen)
We use iodine

26
Q

What do differential stains do?

A

divide bacteria into separate groups based on different staining properties – different groups stain differently

27
Q

Who developed the gram stain?

A

developed by Dr. Christian Gram, 1884

28
Q

What is our staining procedure?

A

Flood Crystal Violet - 1 minute (primary stain), wash

Flood, Gram’s Iodine - 1 minute (mordant), wash

Flood, 95% ethanol - 5-10 sec (rinse to decolorize), wash

Flood, Safranin - 1 minute (secondary stain), wash, dry

29
Q

What do purple cells indicate?

A

Gram positive (G+) bacteria - trap crystal violet-iodine complex, due to thick layer of peptidoglycan in cell wall

Check for acid fast false positive!

30
Q

What do red cells indicate?

A

Gram negative (G-) bacteria - large amount of lipids in cell wall dissolved by the 95% ethanol, allowing crystal violet-iodine complex to escape leaving cells colorless. The safranin counterstain turns these cells red

31
Q

Describe acid fast staining

A

Used to stain Mycobacterium, which have high content of mycolic acid (fatty substance that holds violet stain to give a false G+)

Must use steam heat to force primary stain safranin or carbolfuchsin into cells

Acid-alcohol decolorizer that removes stain from non-acid fast cells

Counterstain with methylene blue

The acid-fast bacteria are found as red clumps of filamentous cells

32
Q

Describe negative aka capsular staining

A

reveals capsule layer around cells

Bacteria are then often stained with a simple stain (Nigrosin or India Ink)

The capsule shows as a clear halo around the bacterial cells

useful to observe Klebsiella pneumoniae & other capsule producers

33
Q

Describe endospore staining

A

Endospores do not readily take up dye, so the cells must be ‘forced’ over steam - once the stain penetrates, it is not easily decolorized

Primary = Malachite green
Counterstain with Safranin

Endospores – Green
Vegetative cells - Red

34
Q

Describe flagellar staining

A

Some bacteria have flagella, but it is not easy to stain these structures

Flagella of bacteria are coated with tannic acid or potassium alum and stained with basic fuchsin (Gray Method)

Microbiology (7 decks)

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