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Microbiology > Not on Exam : Microscopy and Staining Bacteria > Flashcards

Not on Exam : Microscopy and Staining Bacteria Flashcards

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Biology 101 flashcards
1
Q

During which century were the first microscopes built?

A

1600s

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2
Q

The ability to view microscopic structures is dependent upon what four parameters?

A

1) Wavelength of radiation
2) magnification of the image
3) resolving power of the instrument
4) Contrast in the specimen

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3
Q

What is the range of wavelengths of visible light?

A

400nm to 700nm

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4
Q

What has wavelengths longer than that of visible light? How much energy do these wavelengths have as compared to visible light?

A

Infrared, microwaves, radio waves and television.

These have less energy than visible light.

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5
Q

Relationship between wavelength and energy?

A

Longer the wavelength the lower the energy Shorter the wavelength the more energy.

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6
Q

What has wavelengths shorter than that of visible light? How much energy do these wavelengths have as compared to visible light?

A

Gamma rays. These have more energy than visible light.

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7
Q

How do the wavelengths of beams of electrons compare to the wavelengths of visible light? How much energy do these wavelengths have as compared to visible light?

A

The wavelength of beams of electrons is much shorter than visible light, resulting in a significant increase in energy.

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8
Q

What is magnification?

A

Increases the apparent size of an object.

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9
Q

What causes the bending of a beam of light in a light microscope?

A

Curved glass lens. Light passing through the periphery is bent more than light passing through the center. The lens focuses on a focal point and the rays spread apart resulting in an enlarged and inverted image.

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10
Q

What causes the bending of a beam of electrons in an electron microscope?

A

Magnetic fields

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11
Q

What is a focal point?

A

Its a common point on the principal axis where all the light rays starting from the object converge.

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12
Q

What is the approximate limit of useful magnification with a light microscope? Is it possible to magnify beyond this limit?

A

The limit is approx 1,000x, yes more is possible but its empty magnification.

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13
Q

What is empty magnification?

A

Magnification beyond the point of being useful, results in faint and blurry image.

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14
Q

What is resolving power?

A

ability to distinguish objects that are closer together.

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15
Q

How is resolution measured?

A

um, objects closer together than this appear as a single object.

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16
Q

How does the resolution of a modern microscope compare to that of Leeuwenhoek’s microscopes. Does this surprise you?

A

Leeuqenhokes had a resolution of about 1um while modern microscopes have resolution of 0.2nm. Not surprising with advancements in technology.

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17
Q

What is the key determinant of a microscope’s resolving power?

A

Numerical aperture of the lens: ability of the lens to gather light.

Wavelength of electromagnetic radiation: shorter wavelengths lead to greater resolution.

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18
Q

What is contrast?

A

Refers to the differences in intensity between two objects or an object
and its background.

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19
Q

By what two means is contrast commonly increased?

A

Staining and using certain types of microscopy.

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20
Q

What is a compound microscope, and how does it differ from a simple microscope?

A

Compound microscope has a series of lenses while a simple microscope has a single magnifying lens.

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21
Q

What is an ocular lens? What is the typical magnification of an ocular lens?

A

Part of the microscope that magnifies the image produced by the microscope’s objective so that it can be seen by the human eye. Typically 10x magnification.

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22
Q

What is an objective lens? What are the typical magnifications of a light microscope’s objective lenses?

A

The lenses that directly observe the object the microscope user is examining. Typically 4x, 10x, 40x, and 100x.

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23
Q

How does one compute a microscope’s total magnification?

A

Magnification of the objective lens times the ocular lens.

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24
Q

What is oil immersion and why is it done? What effect does it have on useful magnification?

A

Done by placing oil between the slide and the highest powered objective lens. Increases resolution and reduces distortion of image.

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25
Q

Why might one use a dark-field microscope?

A

Useful for viewing pale objects.

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26
Q

How does dark-field microscopy differ from bright-field microscopy?

A

Light is stopped by directly entering the objective lens, light only scattered by specimens enters the objective lens.

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27
Q

Why might one use a phase microscope?

A

Examine living or other specimens that would be damaged by attaching to slides or staining.

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28
Q

How does phase microscopy differ from bright-field microscopy?

A

Amplifies the slight difference in refractive index between cells and tier surrounds. light is shifted “out of phase” to create better contrast.

29
Q

“in phase”

A

light has wave crests alined, waves reinforce each other.

30
Q

“out of phase”

A

light has wave crests not alined, waves interfere with each other.

31
Q

How does the light source used in fluorescent microscopy differ from that used in bright-field microscopy?

A

Uses ultraviolet light

32
Q

How does it work?

A

Fluorescent molecules absorb ultraviolet radiation and radiate the energy back as a longer visible wavelength. 3D image viewed.

33
Q

How are cells made fluorescent?

A

Some are naturally fluorescent while others can be stained with fluorescent dyes. Some fluorescent dyes are specific to certain cells.

34
Q

What is immunofluorescence?

A

Fluorescent dyes can be covalently linked to antibodies that then bind to bacterium. Used to identify pathogens and visualize cellular structures.

35
Q

How are confocal microscopy and fluorescent microscopy alike? How are they different?

A

Also uses fluorescent dyes or fluorescent antibodies. Uses ultraviolet lasers instead of ultraviolet light like in fluorescent microscopy.

36
Q

How does an image obtained through confocal microscopy compare to an image obtained through bright-field microscopy?

A

The image is three-dimensional. Only a thin plane of the sample is illuminated then multiple planes are illuminated in series.

37
Q

Compare the radiation source used in electron microscopy to that used in bright-field microscopy.

A

Bright-field is limited by the wavelength of visible light while electron microscopy resolution is much greater.

38
Q

Compare the useful magnification of electron microscopy to that of bright-field microscopy.

A

magnification typically 10,000x to 100,000x instead of 1,000x.

39
Q

Compare the resolution of electron microscopy to that of bright-field microscopy.

A

Resolution as high as 0.001nm instead of 0.2nm

40
Q

What are the two types of electron microscopes?

A

Transmission electron microscope (TEM)
Scanning electron microscope (SEM)

41
Q

Transmission electron microscope (TEM)

A

Generates beams of electrons transmitted through the specimen. Electrons pass through magnetic fields instead of glass lenses. Electrons strike fluorescent screens. Dense areas on specimen block electrons; less dense areas appear lighter. resolving power as high as 0.3nm. Monochrome 2D image produced

42
Q

How to prepare a TEM slide?

A

Sample prepared by dehydration, embedded in plastic, 100nm thickness, no live organisms.

43
Q

Scanning electron microscope (SEM)

A

Electrons focused across the surface of the sample. Surface coated with metal and electron beam knocks scardary electrons off the surface of coated specimen. magnification up to 10,000x and resolution ~20nm. Whole specimens observed resulting in realistic 3D image, monotone but man be color enhanced.

44
Q

What is the purpose of coloring microorganisms with stains?

A

Creates contrast making microorganisms and their parts more visible. Electron microscopy also requires treatment of specimens with stains or coatings.

45
Q

What is the purpose of fixation? Describe two basic means of fixation.

A

Attach the microbes more firmly to the slide. Heat or chemical.

46
Q

Steps for making smear

A

1) spread microbes in liquid on slide.
2) Air dry
3) Fix either with heat or chemical (methanol).

47
Q

What are the two components of most microbiological stains?

A

Generally salts, made of chromophore (colored portion) and an ion.

48
Q

What is a basic dye? Given an example of such a dye. What types of structures does it stain?

A

Positivity charge dye, the most common type used in microbio, stains acidic structures, binds to negatively charged molecules like DNA and proteins.

49
Q

Example of a basic dye?

A

methylene blue which contains positively charged chromophore and negatively charged chloride ion.

50
Q

What is an acidic dye? Given an example of such a dye. What types of structures does it stain?

A

Negatively charged dye, works best under acidic conditions, stain alkaline structures, binds to positively charged molecules like amino acids.

51
Q

Example acidic dye?

A

Eosin, negatively charged chromophore and positively charged sodium ions.

52
Q

What types of cellular features are stained by Sudan black? How does this compare to acidic or basic dyes?

A

Membranes. Does not form bonds with cellular chemicals functions instead functions by solubility and accumulates in the membranes.

53
Q

What is a simple stain and what is a differential stain? Why might one use a differential stain?

A

Simple stains are composed of a single dye. Differential stains use more than one dye. Results in ability to distinguish different cells, chemicals, or structures.

54
Q

Examples of differential stains:

A

gram stain
acid-fast stain
endospore stain

55
Q

Simple stain process:

A

1) prepare slides and heat fix.
2) soak the smear in dye for 30-60 seconds.
3) rinse with water
4) air dry

56
Q

What is the purpose of a Gram stain?

A

differentiates between two large groups of bacteria based on their cell wall structure.

57
Q

Describe a Gram-staining procedure, indicating the purpose of each step involved.

A

1) crystal violet, primary stain, all cells purple. 30-60s and rinse
2) Iodine, mordant, cells remain purple. 60s and rinse
3) Alcohol, decolorizer, Gram + purple, Gram - colorless. 5-10s rinse very well
4) Safranin, counterstain, Gram + stays purple, Gram - pink/red. 1-2min rinse and dry

58
Q

What would happen if any given step were omitted in a Gram-staining procedure? Go through the procedure and omit one step while leaving the others in place. Then do the same for each of the other steps.

A

Skip crystal violet= nothing is purple gram + not stained everything will end up pink.

Skip iodine= purple will rinse be cleared away from all cells with alcohol, everything pink.

Skip alcohol= Gram - does not get its color removed. Everything is purple with a pink tinge on top.

Skip Safranin= GRam + purple but Gram- stays colorless.

59
Q

What are the causes of some common difficulties with Gram stains?

A
  • Overloading your slides/ clumping resulting in uneven staining and destaining.
  • Not rinsing all the alcohol off
  • works best with younger cells, decreased cell wall integrity in older gram + cells means it will destain more easily and end up pink.
  • Destaining too little results in gram-neg cells appearing purple.
  • Destining too long results in gram-pos cells appearing pin.
60
Q

Why might one use an acid-fast stain? What types of bacteria are stained?

A

Used to stain Mycobacterium which have waxy lipid in their cell walls. Many cause diseases like tuberculosis and leprosy.

61
Q

Describe an acid-fast staining procedure, indicating the purpose of each step involved.

A

1) Flood slide with carbolfuchsin while heating. Done over steaming water and with slide covered with tissue paper.
2) remove tissue paper and remove slides.
3) decolorize with HCI/ ethanol mixture “acid alcohol”
4) counterstain with methylene blue
5) Acid fast cells stain pink, others stain blue.

62
Q

Describe the appearance of acid-fast bacteria and non-acid-fast bacteria after this staining procedure has been performed.

A

Acid fast cells stain pink while other cells stain blue. The initial stain is malachite green, water is used to destain, safranin is used as counterstain. Results in cells pink and endospores green.

63
Q

What two disease-causing bacterial genera produce endospores?

A

Bacillus and Clostridium genera

64
Q

Endospore

A

highly retractile and thick-walled structures formed inside the bacterial cells. Allows cells to go dormant and survive environmental extremes.

65
Q

Describe an endospore staining procedure, indicating the purpose of each step involved.

A

Same as acid-fast staining. Cells appear one color with endospores another color.

66
Q

Describe the appearance of a cell and of an endospore after an endospore stain has been performed.

A

green endospores inside a red cell.

67
Q

By what other name is a negative stain known? What cellular feature does it help identify?

A

Capsule stain. It helps identify the presence of negatively charged bacterial capsules. Process stains the background and leaves the cell colorless. Counterstain may be added to color the cells.

68
Q

What is the function of a flagellar stain? Can bacterial flagella be seen without it? Explain.

A

To increase the visibility of flagella by giving it color (stains) and increasing diameter (mordants). No, it is invisible to light microscopy.

Microbiology (30 decks)

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