LABORATORY INSTRUMENTS

Microscope

Micro = Small, Scope = to view.

It magnifies the image of the object to be visualized

through it. Normally, the laboratory microscopes provide magnification of 40x (scanner), 100x (low power), 400x (high power) and 1000x (oil immersion). The totalmagnification is obtained by multiplying the magnificationof the objective with that of the eyepiece.

Parts of the Microscope

It has three sets of parts. They are the:

1. Stand,

2. Mechanical adjustments, and

3. Optics or the lenses.

Stand

It consists of:

1. The tube—supports objectives and eyepiece.

2. The body—gives support to the tube.

3. The arm—gives correct height and angulation to thebody and the tube.

4. The stage with a pair of spring clips or a mechanical stage.

5. The substage holds the condenser lens with its iris diaphragm and a holder for light filters and stops.

6. The foot on which other parts rest, can be in tripod or

horseshoe shape.

Mechanical Adjustments

Focusing Adjustments

These are coarse and fine adjustments.

Coarse Adjustment

Controlled by a pair of large knobs, one on each side of the body. On rotating this, the tube moves with its lenses.

Some microscopes have this attached to the stage; so that instead of the tube, the stage moves up and down. Coarse adjustment is enough for low power lenses.

Fine Adjustment

Necessary for high power and oil immersion lenses. This is

usually controlled by two smaller knobs on each side of the

body. They may be graduated to indicate the movement in

microns.

Draw Tube

It is used to adjust the distance between the objective lensand the eyepiece lens.

Inclination

The arm can be tilted upon the foot by a hinge.

Condenser Adjustments

Focusing of condenser is done by rotating a knob present on one side below the stage.

Aperture Adjustment

It is done by the iris diaphragm (made up of leaves).

Centering of Condenser

It is done to bring the light beam accurately through the instrument. In some microscopes, it is permanently fixed.

Mechanical Stage

It has knobs for moving the slide across or along the stage.

Monocular, Binocular and Digital Microscopes

Monocular—has only one eyepiece .

Binocular—has 2 eyepieces, the only advantage it offers is

that it causes less strain on the eyes .

 Nowadays digital microscopes are available, here digital image is

projected onto a digital display device

Microscope Optics

Objective

On objective quality, depends, the quality of the image.These are usually made up of more than one lens. On each objective is engraved the magnification power.

Numerical Aperture

Numerical aperture (NA) of the objective is important, for on this, depends, among other things, the amount of lightwhich the lens passes and the detail which it can make visible, on which it is said to resolve.

Oil Immersion Objectives

They are used to avoid bending of light beam (with higher magnification). The oil used should have the same optical properties as glass, e.g. cedar wood oil. Liquid paraffin can also be used.

Objective Aberrations

With increasing magnification certain optical aberrations

creep in:

1. Spherical aberration—edge of the lens gives slightly higher magnification than its center.

2. Chromatic aberration—blue light is magnified slightly more than red.These aberrations can be avoided by using a series of lenses made of special glass, carefully calculated and designed.

Objective Qualities

1. Achromatic—are the usual average quality lenses and are good enough for routine laboratory work.

2. Fluorite (Fi)—are highly corrected and expensive, have a wider field and are good for searching blood films.

                                                   microscobic objectives

3. Apochromatic (Apo)—are very highly corrected and costly and are only of value in special work.

Spring-loaded Objectives

The high power objectives (40X and 100X) of most modern micobjective will be pushed in rather than pushed through a specimen, if such an objective is accidentally pressedroscopes are spring loaded, i.e. the front mount of the against a specimen when focusing .

Working of Oil Immersion Objectives

A beam of light passing from air into glass is bent; and while passing from glass to air, it is bent back again.The bending effect and its limitations can be avoided by replacing the air between the specimen and lens with an oil which has optical properties similar to that of glass,

i.e. immersion oil. When an appropriate oil is used, the light passes in a straight line from glass through the oil and back to glass as though it were passing through glass all the way. Whenever possible, the immersionoil recommended by the manufacturer of a microscope

should be used 

Eyepiece

The most commonly used eyepiece is known as Huygens eyepiece which has 2 lenses mounted at a correct distanceapart, with a circular diaphragm between, which give asharp edge to the image. These are available in different magnifications. Lesser the magnification, brighter and

sharper is the image. For routine work, a 10X Huygens is good enough. The 15X eyepieces are also available, as are wide field ones.

Condenser and Iris

Condenser is a large lens mounted below the stage, with an iris and diaphragm. There may be 2 or more lenses. Itsfunction is to deliver the light beam to the objective at a sufficiently wide angle.

The Mirror

It is placed below the condenser and iris, it can be turned inany direction. It reflects the light beam from the source to the iris and condenser. It usually has two mirrors mounted

back to back, one flat and the other concave. Flat mirror is used in the presence of condenser and the concave without the condenser.

Light Source

Daylight

Use of direct sunlight is bad for the microscope and the eye. It is best to use reflected sunlight of a dull whitebackground. It is not sufficient for oil immersion lens and it is not available during evening or night.

Electric Light

A 60 watt frosted electric lamp placed 18" away from themicroscope is sufficient for most routine work. Manymicroscopes are now provided with built-in sources of illuminations. In the absence of electricity, a battery lamp or an oil lamp can be utilized. The light from these artificialsources is rather yellow but may be used. Best, however,are halogen lamps.

Special Applications of the Microscope

Phase Contrast Illumination

This is needed to visualize transparent microorganisms suspended in a fluid. Ray of light travels in a wave form in a straight line. Two such rays traveling together are said to

be in phase, and they produce a brighter illumination. If,however, these rays are out of step with each other, theyare said to be out of phase. They interfere and produce less

bright illumination. Phase contrast microscopy makes use of this property of rays to help or hinder each other and therebyresulting increased contrast in the microscopic image.

The desired effect is brought about by placing anannulus in the condenser and a phase plate in theobjective. A circle is engraved in the phase plate which matches the ring of beam coming through the condenserand annulus. This circle makes the wave take a longer or

a shorter step, so becoming out of phase with those aves which pass through the rest of the plate.Supposing that the specimen is suspension free fluid,the only light that reaches the eye is that which goes from the annulus through the phase plate. Whereas presence of organisms would diffract and scatter the light. The lightpassing through the fluid gets out of phase with the light thathas the organisms stand out in contrast to their background.

Equipment Needed

An annulus, a phase plate and a telescope that is needed

for adjusting the rings of both annulus and the phase plate.

Method

1. Focus the specimen with the right objective afterilluminating the microscope.

2. Place the matching annulus at its position.

3. Remove the eyepiece and put the telescope in its place, adjust it till the two rings, one bright and one dark arein focus.

4. Adjust condenser screws till the bright annulus ring fits exactly into the darker ring of the phase plate.

5. Remove the telescope, replace the eyepiece, focus and examine the specimen.

Importance

This method is made use of for examining live organisms,

for examaple,

a. Cholera vibrios

b. Amebae

c. Trypanosomes

d. Trichomonas, and

e. Other flagellates.

It can also be used for platelet counting and for examining routine urine specimens.

Demerits

a. A halo is seen around each particle, it gives a falseappearance of its structure.

b. In addition, some resolution power is lost but this is more than compensated for by the increased contrastthat is produced.

Dark Ground Illumination

This method too, is used for visualizing organisms

suspended in fluid, both the structure and the motility

of the organisms can be seen. In this method, the lightenters the special condenser which has a central blackedout area so that light cannot pass directly through it to enter the objective. Instead the light is reflected to pass through the outer rim of the condenser at a wide angle which illuminates the microorganisms by a ring of light surrounding them In this method, the light that is seen comes only from the microorganisms themselves and not from the light source. Hence, the organisms are brightly illuminatedagainst a dark background. Though useful, this method is rather cumbersome.

Equipment Needed

1. An oil immersion dark ground condenser with the centering screws.

2. A funnel stop for insertion in 100X objective to reduce its NA and exclude light coming directly from thesource.

3. A brightly illuminated microscope lamp.

4. Scratchless slides not more than 1 mm thick

.Method

1. Fit the dark ground condenser and raise it to stage level.

2. Place the coverslipped specimen on the thin polished glass slide. Both, the coverslip and the slide should be absolutely clean.

3. Place a drop of immersion oil between the condenser and the slide.

4. Adjust light source and the mirror properly.

5. Focus 10X objective and observe.

6. Focus condenser up or low, so that the ring ultimately becomes just a spot of light. Focus this spot right in thecenter.

7. Use 40X objective; if needed, use the 100X oil immersion by inserting the funnel stop into it.

Demerits

1. Focusing and/or centering of condenser is difficult asis the alignment of the lamp.

2. Difficulties may arise under the following circumstances:

• Smear traces on the slide or coverslip

• If the specimen is dense

• A bubble is present in the immersion oil

• Insufficient oil contact below or above the slide.

Importance

1. This method is of particular importance for the

examination of Treponema group of organisms.

2. It can also be of use for microfilariae, for the sheath

of the pathogenic forms can be clearly seen which

otherwise needs to be stained.

3. For examining the rapid movement of Vibrio cholerae.

4. In addition, this method can be used for:

• Leptospira

• Borrelia, and

• Spirillum species.

• The ideal objective for dark ground illumination is the 50X fluorite as this lens gives a clear, sharp

and a well-illuminated image.

Fluorescence Microscopy

This method entails the illumination of particles/microorganisms(previously stained with a fluorescent dye) withultraviolet (UV) light into visible light (yellow or orange),by lengthening their wavelength. This procedure is madeuse of for visualizing, besides other things, mycobacteria

glowing against a black background.All other wavelengths emitted by the lamp except the

ultraviolet (UV) are to be filtered off (by using appropriate optical filters) and no harmful rays of UV light should reach the observer’s eye (by using an immersion dark ground condenser as described for previous method). Again,another filter is used to remove all unwanted fluorescent light by placing a secondary or a barrier filter above the

                                                                      Components of fluorescence system                                

Equipment Needed

1. A fluorescent lamp (mercury vapor or quartz iodine, the

latter is better, being cheaper, lighter and easier to use).

2. A blue (primary or exciting) filter, generally a BG 12.

3. A yellow (secondary or barrier) filter.

4. An immersion dark ground condenser.

5. A nonfluorescent immersion oil, e.g. liquid paraffin.

Importance

1. For identifying mycobacteria.

2. It is used extensively in fluorescent antibody techniques used in parasitology and bacteriology.

3. It is also used widely in histopathology of kidney, skin, etc. where immune/autoimmune basis of disease isexpected. In fact, anything can be confirmed withhigh degree of sensitivity and specificity, if antibodiesagainst it (later tagged with a fluorescent dye) can beproduced.

4. Used widely in cytogenetics.

Electron Microscope

Basic Principle

The resolution of the light microscope has been shown to be limited by the NA and the wavelength of light employed. As the degree of correction in glass lenses is very high, the main limitation is imposed by the light(e.g. half wavelength of light), giving a normal resolution of approximately 250 nm; and when UV light is used,a resolution of about 100 nm. By the substitution of an electron beam for light rays, a much greater degree of resolution can be obtained; since at an acceleration of

50,000 volts, electrons have a wavelength of only 0.001 nm;therefore, a theoretical resolving power of 0.0005 nm couldbe attained, which would enable molecules to be seen.Unfortunately, the degree of correction that is currently

feasible with transmission electron microscope (TEM)lenses will permit a resolution of only 0.25 nm, but this is still a thousand times greater than that possible with thelight microscope. A further difficulty with the TEM is that,since electrons have poor penetrating power, the sections

to be examined must be very thin, less than 50 nm thick.

This necessitates the use of special hard embedding media(plastics) and special ultra-microtomes to cut such thinsections. Steel knives cannot be used to cut these sections;either glass or diamond knives are used.