USE of MICROSCOPE and

EXAMINATION of LIVING MICROORGANISMS

Light microscopy uses visible or ultraviolet light to illuminate an object.

The light passes through several glass lenses that alter the path of the light and produce a magnified image of the object.

Observing and Drawing Objects:

Because the light rays from an object cross before

reaching your eye, the image you see through our

light microscopes will be inverted and upside

down.

Sitting on the stage Viewed through the lens

Magnification Magnification: the increase of an object's apparent size.

Total magnification is the product of the magnifying powers of the individual lenses. The magnifying capability of a

microscope is the product of the individual magnifying powers

of the two lenses;

1- Ocular lens (eyepiece) : The lens nearest the eye to magnify

object 10 times (10X)

2- Objective lens: The lens nearest the specimen to magnify

object 4, 10, 40, and 100 times (4X, 10X, 40X, 100X)

Total magnification = ocular x objective

Resolution: is the degree to which the detail in the specimen

is retained in the magnified image. The ability to see in detail is

essential lest everything appears as an unresolved blur.

Magnifying object by using microscope is useful only if detail

can be accurately preserved and observed.

Resolving Power; is the closest spacing between two points at which the points

can still be seen clearly as separate entities. The smaller the

value for resolving power, the smaller the object that can be

seen distinctly.

Resolving Power;

Wavelenght of the light used/2*Numerical aperture (NA), where NA is N*sin.

N: refractive index

: angle between the most divergent light ry gathered by the lense and the centered of the lens

Types of Microscope

Compound Microscope

These are light illuminated.

The image seen with this type of microscope is two dimensional.

This microscope is the most commonly used.

You can view individual cells, even living ones.

It has high magnification. However, it has a low resolution.

Dissection or Stereoscope

is light illuminated.

The image that appears is three dimensional.

It is used for dissection to get a better look at the larger

specimen.

You cannot see individual cells because it has a low

magnification. earth worm

Confocal Microscope

This microscope uses a laser light.

This light is used because of the wavelength.

Laser light scan across the specimen with the aid of scanning

mirrors.

Then image is then placed on a digital computer screen for

analyzing.

Scanning Electron Microscope (SEM)

SEM use electron illumination. The image is seen in 3-D.

It has high magnification and high resolution.

The specimen is coated in gold and the electrons bounce off to

give you and exterior view of

the specimen.

The pictures are in black and white.

Eyes of Mosquito

Transmission Electron Microscope (TEM)

TEM is electron illuminated.

This gives a 2-D view.

Thin slices of specimen are obtained.

The electron beams pass through this.

It has high magnification and high resolution.

Plant cell

STAINING TECHNIQUES

Wet mount, hanging drop, Brownian movement

Simple stain:

- Direct Staining

- Negative Staining

Differential stain

Structural or special stainining

Simple staining is used to observe cell morphology, size and

arragement

Experimental Section

1. Wet Mount

2. Simple staining with cyrstal violet

3. Negative staining