Microbiology (Laboratory): MICROSCOPE

Base

It is the basal, horse shoe-shaped structure. It provides support to all the remaining parts of the microscope.

Pillar

A small, strong vertical projection developing from the foot or base.

Arm

It is a curved and strong structure used for handling the entire instrument.

Stage

It is a flat rectangular plate attached to the lower end of the arm. Slides or objects are placed on the stage and studied.

Aperture

A hole is present in the centre of the stage. Light rays pass through this hole.

Stage Clip

Two clips are attached on the upper part of the stage. These are used for holding the slide in position.

Body tube

It is the tubular, hollow part attached to the upper part of the arm of the microscope. It can be moved up and down with the help of adjustment knobs.

Draw tube

It is the cylindrical structure on top of the body tube that holds the ocular lens.

Revolving Nosepiece

It is a circular and revolving metallic part attached to the lower end of the body tube. It has holes in which objective lenses can be fitted.

Dust shield

It lies atop the revolving nosepiece and keeps dusts from settling in the objectives.

Coarse focus knob

It is a large-sized knob used to move the body tube up and down for bringing the object into focus.

Fine focus knob

It is a small-sized knob. It is used for fine and sharp focussing of the object. Exact focussing is done by this knob.

Ocular / Eyepiece

It is a small tube consisting of lenses that indicate the relative power of magnification.

Scanning objective

It is used for the initial location of the specimen. It has 4X magnification.

Low Power Objective

• It may also be used for the initial location of the specimen.

• It magnifies the specimens 10 X.

• It views the specimen in a larger field.

High Power Objective

It obtains greater magnification and views detailed structures of the specimen

Oil Immersion Objective

It is used for magnification of extremely small specimens, such as bacterial cells.. In using this, a drop of oil is placed on the slide for better refraction.

Illuminator/ Lamp

A steady light source (110 volts in the US) that shines up through the slide

Mirror

reflects light rays through the object. One side of the mirror is plain (used with natural light); the other is concave (used with artificial light)

a. Iris diaphragm

This part can be adjusted to vary the intensity that is projected through the slide. As there is no set rule on which setting to use for a particular power, the setting depends on the transparency of the specimen and the degree of contrast you desire in your image

b. Condenser

This is a lens that concentrates light to the specimen.

MAGNIFICATION

• is the ability to make small objects seem larger, such as making a microscopic organism visible.

RESOLUTION

• is the ability to distinguish two objects from each other.

TOTAL MAGNIFICATION

• is determined by multiplying the power of the ocular by the power of the objective in use.

The measuring unit for bacteria

micrometer

Ocular micrometer

located within the eyepiece lens, is used to determine the sizes of microorganisms that are seen under a light microscope.

Refractive index

a measure of the relative velocity at which light passes through a material.

The total magnification achieved

the product of the lens that is use

Scanning

use coarse knob

Low power

use coarse knob

High power

use fine knob

Cedar-wood oil (for an oil-immersion lens)

• Sugar solution is frequently used.

• It has the same refractive index as the glass of the cover slip so that the object is effectively immersed in it.

• The presence of the liquid increases the effective aperture of the objective, thus increasing the resolution.

Xylol cleaning method

The most widely used technique to clean the objectives of a microscope, especially the 100X objective, involves wiping out of immersion oil from the same and its surrounding mount by a piece of lens paper or a soft piece of clean cotton cloth followed by a cloth dampened with small amount of xylol.

Electron Microscope

making use of streams of electrons travelling at high speed in a vacuum

Electron Microscope image

The image produced is visible when projected into a fluorescent screen.

Principles in Electron Microscope

• Resolution strongly dependent of wavelength:

- electron microscope: about 0.2 nm

- optical microscope: about 500 nm

• Image formed due to different lateral absorption of the beam

- heavy atoms darkest

- light atoms high transmissions

• Electron microscopes often equipped with instrumentation for elemental analysis

- EDAX (Energy Dispersive Analysis of X-rays)

• Two techniques:

- Transmission Electron Microscopy (TEM)

- Scanning Electron Microscopy (SEM)

Fluorescence Microscope

a type of microscope that makes use of ultraviolet light to view the object

Fluorescence Microscope staining

a certain dyes are applied to the specimen and the specimen is exposed to the UV light and the image appears "self luminance"

Fluorescence Microscope

This is used in the examination of antigen-antibody reactions.

Principles in Fluorescence Microscope

• Fluorescence microscopy uses a much higher intensity light to illuminate the sample.

• This light excites fluorescence species in the sample, which then emit light of a longer wavelength.

• The image produced is based on the second light source or the emission wavelength of the fluorescent species - rather than from the light originally used to illuminate, and excite…

Phase Contrast Microscope

a type of microscope that makes use of special lenses, condenser and filter which permit one to observe not only the structure revealed by light passing directly through the object

Phase Contrast Microscope image

the details of object are outlined on a gray background

Phase Contrast Microscope use

used for examination of suspension of living microorganism or other transparent materials and bacteria in tissue section.

Principles in Phase contrast Microscope

• It is based on the wavelength (nature) of light rays and the fact that light rays can be in phase or out of phase.

• Different shades of grey are distinguished to our eyes due to differences in amplitude of light rays.

• PCM converts invisible small phase changes caused by the cell component into visible intensity changes.

Darkfield Microscope

• Make use of a special condenser which produces a hollow cone of visible light such that it will pass only through the object on focus and none of the light goes directly up to the objective, the field will appear dark and the object on focus appear bright, this is used in examination of unstained microorganism suspended in fluid.

Principles in Darkfield Microscope

1. Uses a light microscope with an extra opaque disc underneath the condenser lens, or a special condenser having a central blacked-out area, due to which the light coming from the source cannot directly enter into the objective.

2. The path of the light is directed in such a way that it can pass through the outer edge of the condenser at a wide-angle and strike the sample at an oblique angle.

3. Only the light scattered by the sample reaches the objective lens for visualization.

4. All other light that passes through the specimen will miss the objective, thus the specimen is brightly illuminated on a dark background.

Inverted Microscope

is a microscope with its light source and condenser on the top, above the stage pointing down, while the objectives and turret are below the stage pointing up.

Inverted Microscope

• It was invented in 1850 by J. Lawrence Smith,

• A faculty member of Tulane University (then named the Medical College of Louisiana).

Principles in Inverted Microscope

• The working principle of the inverted microscope is basically the same as that of a light microscope.

• In an inverted microscope, the source for transmitted light and the condenser are placed on the top of the stage as shown in the above picture, pointing down toward the stage.

• The objectives are located below the stage pointing up. The specimens or cells are observed through the bottom of the cell culture vessel.

• To meet the criteria for successfully inverted microscopy, the bottom of the culture vessel must have the highest optical features, which are given for the ibidi Polymer Coverslip and the ibidi Glass Coverslip.

1850 - J. Lawrence Smith

Who invented the inverted microscope?

Care and Maintenance of the Microscope

• Good preventive maintenance and care includes:

• Regular cleaning of oculars and objectives

• Avoid damaging oculars and other optics with eye make-up or other debris

• Careful handling to avoid abrupt motions

• Protect from direct sunlight, high temperature, humidity, dust and vibration

• Use appropriate materials to clean the lenses

Microscope Care

• Always carry with 2 hands

• Never touch the lenses with your fingers.

• Only use lens paper for cleaning

• Keep objects clear of desk and cords

• When you are finished with your "microscope", rotate the nosepiece so that it's on the low power objective, roll the stage down to lowest level, rubber band the cord, then replace the dust cover.

• Electron Microscope

• Fluorescence Microscope

• Phase Contrast Microscope

• Inverted Microscope

• Darkfield Microscope

What are the types of microscope?

Using High Power

• Your slide MUST be focused on low power before attempting this step

• Click the nosepiece to the longest objective

• Do NOT use the Coarse Focusing Knob, this could crack the slide or the lens

• Use the Fine Focus Knob to bring the slide into focus

Robert Hooke 1665

An English physicist looked at a sliver of cork through a microscope lens and noticed some "pores" or "cells" in it.

Mechanical Parts

For adjustments and support

Optical Parts

Enlarge the specimen; These are the set of lenses or lens system

Illuminating Parts

Provide the light

16th Century

Having been constructed in the ________, microscopes have revolutionized science with their ability to magnify small objects and produce images with definitive structures that are identifiable.

MICROSCOPE

• are instruments that are used in science laboratories, to visualize very minute objects such as cells, tissues, microorganisms, giving a contrasting image that is magnified.

• are made up of lenses for magnification, each with their own magnification powers.

• depending on the type of lens, it will magnify the specimen according to its focal strength.

• offer the ability to see great detail of objects that are hundreds of times too small to see with your naked eye.

• are generally made up of structural parts for holding and supporting the microscope and its components and the optical parts which are used for magnification and viewing of the specimen images

Mechanical Part

Base

Mechanical Part

Pillar

Mechanical Part

Arm

Mechanical Part

Stage

Mechanical Part

Aperture

Mechanical Part

Stage Clip

Mechanical Part

Body tube

Mechanical Part

Draw tube

Mechanical Part

Revolving Nosepiece

Mechanical Part

Dust shield

Mechanical Part

Coarse focus knob

Mechanical Part

Fine focus knob

Optical Part

Ocular / Eyepiece

Optical Part

Scanning objective

Optical Part

Low Power Objective

Optical Part

High Power Objective

Optical Part

Oil Immersion Objective

Illuminating Part

Illuminator/ Lamp

Illuminating Part

Mirror

Illuminating Part

a. Iris diaphragm

Illuminating Part

b. Condenser