Microbiology- Chapter 3 (The Methods For Studying Microorganism)

Front 1

What are the 5 I's in studying microorganisms?

Back 1

1. The Five I's
• Inoculation
• Incubation
• Isolation
• Inspection
• Identification

Front 2

Explain Inoculation.

Back 2

• Introducing a sample (the inoculum) into a container with a nutrient medium
• The medium contains appropriate nutrients that sustain the growth of microorganisms
• Some microbes have to be inoculated into a living organism
*some microorganisms can not live in artificial mediums, they do not multiple or grow
*Example: viruses

Front 3

Explain Isolation.

Back 3

• Isolation: Separating one species from another
• Obtaining Pure Culture
• Cultures composed of cells arising from a single cell- Pure Cultures
*individual cells multiple and produce a clone which eventually becomes visible within the naked eye
*pure culture is a culture that rises from a single cell
*calling it a clone can also be correct

Front 4

What are the methods to obtain a pure culture?

Back 4

•Streak Plate method
•Dilution method
*could not obtain pure culture before cause it wasn't the right medium
*one colony is one pure culture
*dilution method requires you to dilute samples in such a way that it will inoculate into others it will produce a colony

Front 5

What are the measures to take when working with microbiological medial?

Back 5

• Need to be sterilized
• Prevent contamination

Front 6

Name the physical types of media?

Back 6

-Liquid
-Solid (layer)

Front 7

Name the chemical compositions of media.

Back 7

-Synthetic
- Nonsythetic (Complex)

Front 8

Name the functional types of media.

Back 8

• 3.Functional Type
- General type (not specific)
- Enrichment (specific)
-Selective
-Differential

Front 9

What is synthetic media?

Back 9

• Known chemical composition (NaNO3 - 3g/k glucose 2g/l…)

Front 10

What is nonsynthetic media?

Back 10

• Contains chemically undefined components (pepton, beef extract…)

Front 11

What does enrichment media do?

Back 11

Supports the growth of a specific group of microorganism (Ex. N2-Fixing)

Front 12

What is selective media?

Back 12

Favor specific microorganisms and inhibits the others (methylene blue inhibits the growth of Gram+ bacteria)

Front 13

What is differential media?

Back 13

Contain substances that permit detection of microorganisms with specific metabolic activity (blood agar)

Front 14

Explain Incubation.

Back 14

Microbiological media are placed in temperature controlled chambers (incubators)
temp- 20-40C
Pathogenic- 37C ( Human body temp!)

Front 15

Who invented the compound microscope?

Back 15

• Robert Hooke

Front 16

How does a microscope work?

Back 16

• The specimen is magnified with the objective lens (real image)
• This image is magnified by ocular lens (virtual image)
• An enlarged and inverted image is received by retina

Front 17

What are the basic features of microscopy?

Back 17

• Magnification
• Resolution
• Contrast

Front 18

What is magnification?

Back 18

results of Light Refraction

Front 19

What are the color coded objective lens and their magnifications?

Back 19

Smallest objective lens = 4x (40x) (red)
10x (100x) (yellow)
40x (400x) (blue)
100 (1000x) (green/black)

Front 20

Why use oil immersion?

Back 20

Use with higher powder objectives.

Immersion oil has the same refraction index as the glass.
Refractive index is a measure of relative velocity at which light passes through a material.

*refraction index of immersion oil is the same as glass

Front 21

What is resolution?

Back 21

Resolution (resolving power) is the ability of a lens to distinguish two adjacent points as two separate objects. In light microscopes resolution is 0.2 μm (limit - 2000x)

Front 22

Which images can be resolved?

Back 22

Anything larger than 0.2 um (bacteria)

Front 23

How does the resolution depend on the wavelength?

Back 23

• Resolving distance = wavelength of light /2x NA
•The shorter the wavelength - the greater the resolution

Front 24

Explain Contrast in terms of microscopy.

Back 24

• Specimen must contrast with their medium
• Change the refractive index of specimen
•Stain the specimen
•Refractive index - a measure of light bending ability

Front 25

What are the types of light microscopes?

Back 25

1.Bright field
2.Phase contrast
3.Fluorescent
4.Dark field
5.Differential
6.Interference
7.Confocal

Front 26

What are the types of electron beam microscopes?

Back 26

1.Transmission
2. Scanning

Front 27

What are the parts of light microscopy?

Back 27

• Illuminator
-so there is a light
• Condenser
-makes the light smaller
• Objective Lens
• Ocular Lens (eyepiece)

Front 28

Explain dark field microscopes?

Back 28

• Best for observing pale objects
• Only those light rays scattered by specimen enter objective lens
• Specimen appears light against dark background
• Increases contrast and enables observation of more details

*advantages: increases contrast, can see more detail in cells that normally not seen in bright-field microscopy

Front 29

Explain Fluorescent Microscopy.

Back 29

*Fluorescent is the ability of certain substances to absorb short wavelength of light and emit light at a longer wavelength *makes it glow*

*in a spectrum, dyes absorb a short wavelength and illuminate length a different wavelength: longer

Front 30

Explain Immunofluorescence as a diagnostic procedure.

Back 30

• Antibody produced against a specific bacterium
• Conjugate antibody and fluorochrome
• Treat the unknown bacterium
• If suspected bacteria present they will bind tagged antibodies
• Ultraviolet (or near) light is used as a light source

Front 31

Explain phase microscopes?

Back 31

Provides better contrast and more details in the cell.
The light rays that hit the specimen travel a different path than the rays, which do not hit the specimen.

*rays are in phases (all in the same phase), sometimes it's possible for rays to be out of phase
*the light rays get deviated by specimen is 1/4 wavelength out of phase -> phase plate deviates the rate even further -> deviated ray is now 1/2 wavelength out of phase

Front 32

Explain Differential Interference Microscopy (Nomarsky) .

Back 32

• uses two beams of light
• Produces contrast from refractive index gradients
• Higher resolution
• 3-D images

Front 33

Explain Confocal Microscopy.

Back 33

• Also uses fluorescent dyes
• Use UV lasers to illuminate fluorescent chemicals in a single plane of the specimen that is not thicker than 1.0 μm
• Resolution increased by up to 40% because emitted light passes through pinhole aperture
• Computer constructed 3-D images

Front 34

Name some advantages of using EM?

Back 34

• Resolving distance = Wavelength of light 2

Wavelength of visible light = 4000A
- Resolution light microscopy : 2000A (2 μm)

• E.M. uses an electron beam as a source of illumination (100,000 times shorter wavelength than visible light)
-Resolution (EM): 2 A
- Objects are magnified 10,000X to 100,000X

Front 35

Explain transmission electron microscope.

Back 35

• Image formed by the electrons transmitted through a specimen
• A specimen is a thin section of material (fixed, embedded, and sliced - never alive)
• TEM - objects smaller than 0.2 mm

Front 36

What is the preparation of a specimen for TEM image?

Back 36

• Fixation
• Dehydration
• Embedding with plastic
• Sectioning
• Staining

Front 37

Explain Scanning Electron microscope.

Back 37

• Used to study the surface of the cell/tissue
• The beam scans the surface
• Image is formed by the electrons reflected from the surface
• 3-D view

Front 38

What is the preparation of specimens for optical microscopes?

Back 38

• Wet Mount (living) preparation
• Unstained
• Stained (methylene blue)

• Heat fixed smear
• Thin film of material containing microorganisms is spread over the surface of the slide
Air dried
Heat fix (kill and fix bacteria to the slide)

Front 39

Name the two differential stains?

Back 39

-Gram Stain
- Acid-Fast Stain

Front 40

What are the special stains?

Back 40

- Negative (Capsule) Stain
- Flagellar Stain
- Fluorescent Stains
-Endospore Stain