Chapter 2 Magnification 2.1

Question 1

What is resolution vs magnification

Answer 1

1) the ability of a microscope to distinguish between two close points as separate entities , closest distance between two points that are seen as separate objects
2) how many times the image is magnified compared tit eh actual size of the specimen

Question 2

Why must you stain in cells?

Answer 2

Images tend to have a low contrast as most cells do not absorb a lot of,light in light microscopy
- cytosol is transparent like other structures , the stains increase contrast as different components will take up stains to different degrees. This now lets them be visible

Question 3

What must you do before a sample is stained

Answer 3

• place sample on slide and air dry
• heat fix by passing through flame
• now specimen will adhere to the slide and take up the stains

Question 4

What is differential staining ?

Answer 4

Where two types of organisms that would otherwise be hard to identify are distinguished . It can also be organelles within the same organism

Question 5

What stains are positively charged ?

Which are negatively charged stains, + what type of technique is it

Answer 5

Crystal violet / methylene blue are positively charged blue dyes, which are Attached to negatively charged organelles , leading to staining like this

2) nigrosin or Congo red are negatively charged and are REPELLED (not attracted ) by these negatively charged CYTOSOL. These dyes stay outside cells, leaving the organelles unstained, however this will now stand out against the background, so still visible. Thus it is a negative satin technique.

Question 6

How does gram stain technique work

Answer 6

Separates bacteria into gram positive and gram negative bacteria .

1) crystal violet (positive) is applied to a bacterial specimen , then iodine which fixes dye
2) slide washed with alcohol
3) gram positive bacteria retain crystal violet and will appear blue /purple

But

4) gram negative have thinner walls and therefore lose stain.
5) thus stained with SAFRANIN DYE, which is a COUNTERSTAIN
6) these bacteria appear red

Basically stain, fix, wash, retain. Then counterstsin and now differentiated

Question 7

What are gram positive and negative bacteria susceptible too?

Answer 7

Positive is susceptible to antibiotic penicillin, which inhibits formation of cell walls

But gram negative have much thinner walls that are not susceptible to pencil in at all

I guess this makes them positive /negative

Question 8

What is acid fast technique ?

Answer 8

Used to differentiate species of MYCOBACTERIUM from other bacteria .

• a lipid solvent is used to carry carbolfuchsin dye into the cells being studied
• washed with a dilute alcohol solution
• mycobacterium are not affected by the acid alcohol solution and retain the carbolfuchsin dye which is red
• other bacteria lose the stain as they are affected by alcohol , and thus presented with a counter stain of methylene blue .

Question 9

How to produce slides for pre preparation?

Answer 9

Fix - preserves specie,ms
Section- cut thinly
Staining - differential staining used here
Mounting - put in slide

Question 10

Scientific drawing ?

Answer 10

I tile
State magnification
Proportions

Question 11

Why is resolution lost?

Answer 11

Limited by the diffraction of light as it passes though samples .
- as structures close to each other light reflected from indicyal structure can overlap due to diffraction , thus no longer seen as separate entities .

Half the wavelength = resolution typically

Question 12

How to increase resolution ?

Answer 12

Beams of electrons have much shorter wavelength than light
- these still diffract but shorter wavelength means that indicual beams can be much closer before they overlap

So objects smaller can be seen

Question 13

Limiting factor for light microscope ? Vs EM

Answer 13

Resolution limiting factor

But electron microscope, electrons wavelength much shorter so more detail can be seen , thus produce 50k magnification without reduction being lost….

Question 14

Disadvantages of electron microcope

Answer 14

Cost preparation etc

But problem with ARTEFACTS (structures that are man made due to the hard preparation process )

Question 15

TEM vs SEM

Answer 15

TEM beam of electrons through specimen , 0.5nm resolution. However THINNSPECIMEN needed .

SEM across specimen , reoltuij not as good but 3D and thin specimen NOT NEEDED

Question 16

Fluorescent microbes?

Answer 16

Here a specimen is treated with a fluorescent dye , and illuminated with a higher light intensity

As fluorescence is the absorption and re-radiation of light , light if a longer wavelength and lower energy is emitted and used to make an image

Question 17

Laser scanning confocal microcosm

Answer 17

• Ives a single spot of focussed light across a specimen .
• causes flouresence
• emitted light filtered though pinhole aperture , so only light close to the focal plane (distance or sharpest focus ) is detected , as light from any other overt of the spice en would reduce resolution.
  Thus this radiation does not make it through the pinhole aperture

2) a laser is used instead to get higher light intensity . As very thin sections are removed and light from everywhere else is removed, HIGH RESOLUTIONS CAN BE OBSERVED

Question 18

What else is special about laser scanning confocal

Answer 18

2d image produced but 3D can be produce pd by combining different planes

• can be used on living organism , thus used for diseases of the eyes.
• shows the distribution of molecules in organisms which is good for developing drugs