How To Outline A Circle About 1 Cm In Diameter On The Center Of A Clean Glass Slide.

Microscopy

(This passage was adjusted from Microbiology: A Laboratory Manual, fifth edition, Cappuccino, J.S. and Sherman, N., Benjamin/Cummings Science Publishing.)

Purposes

1.To become familiar with the history and diversity of microscope instruments.

ii.To understand the components, use, and care of the compound brightfield microscope.

3.To larn the correct utilise of the microscope for observation and measurement of microorganisms.

INTRODUCTION

Microbiology, the branch of science that has so vastly extended and expanded our knowledge of the living world, owes its being to Antony van Leeuwenhoek. In 1673, with the help of a crude microscope consisting of a biconcave lens enclosed in ii metallic plates, Leeuwenhoek introduced the globe to the existence of microbial forms of life. Over the years, microscopes have evolved from the unproblematic, single-lens instrument of Leeuwenhoek, with a magnification of 300, to the present-day electron microscopes capable of magnifications greater than 250,000. Microscopes are designated as either light microscopes or electron microscopes. The erstwhile use visible light or ultraviolet rays to illuminate specimens. They include brightfield, darkfield, phase-contrast, and fluorescent instruments. Fluorescent micro-scopes use ultraviolet radiations whose wavelengths are shorter than those of visible light and are not directly perceptible to the human eye. Electron microscopes use elec-tron beams instead of light rays, and magnets instead of lenses to observe submicro-scopic particles.

Essential Features of Various Microscopes

Brightfield Microscope

This instrument contains two lens systems for magnifying specimens: the ocular lens in the eyepiece and the objective lens located in the nose-piece. The specimen is illuminated by a beam of tungsten light focused on it by a sub-stage lens called a condenser, and the result is that the specimen appears dark against a bright background. A major limitation of this system is the absenteeism of contrast between the specimen and the surrounding medium, which makes it hard to observe living cells. Therefore, most brightfield observations are performed on nonviable, stained preparations.

Darkfield Microscope

This is like to the ordinary lite microscope; however, the condenser system is modified so that the specimen is not illuminated straight. The con-denser directs the light obliquely so that the calorie-free is deflected or scattered from the spec-imen, which then appears vivid confronting a dark background. Living specimens may be observed more than readily with darkfield than with brightfield microscopy.

Stage-Contrast Microscope

Observation of microorganisms in an unstained state is possible with this microscope. Its optics include special objectives and a condenser that make visible cellular components that differ only slightly in their refractive indexes. As light is transmitted through a specimen with a refractive index different from that of the surrounding medium, a portion of the low-cal is refracted (bent) due to slight varia-tions in density and thickness of the cellular components. The special optics convert the deviation between transmitted calorie-free and refracted rays, resulting in a significant vari-ation in the intensity of light and thereby producing a discernible image of the struc-ture under report. The image appears nighttime against a light groundwork.

Fluorescent Microscope

This microscope is used almost oftentimes to visualize speci-mens that are chemically tagged with a fluorescent dye. The source of illumination is an ultraviolet (UV) calorie-free obtained from a high-pressure mercury lamp or hydrogen quartz lamp. The ocular lens is fitted with a filter that permits the longer ultraviolet wavelengths to pass, while the shorter wavelengths are blocked or eliminated. Ultraviolet radiation are absorbed by the fluorescent label and the energy is re-emitted in the course of a different wavelength in the visible light range. The fluorescent dyes absorb at wavelengths between 230 and 350 nanometers (nm) and emit orange, yellow, or greenish light. This microscope is used primarily for the detection of antigen-antibody reactions. Antibodies are conjugated with a fluorescent dye that becomes excited in the presence of ultraviolet light, and the fluorescent portion of the dye becomes visible against a black background.

Electron Microscope

This instrument provides a revolutionary method of microscopy, with magnifications up to one meg. This permits visualization of submicroscopic cel-lular particles besides equally viral agents. In the electron microscope, the specimen is illu-minated by a axle of electrons rather than light, and the focusing is carried out by elec-tromagnets instead of a set of optics. These components are sealed in a tube in which a consummate vacuum is established. Transmission electron microscopes crave speci-mens that are thinly prepared, fixed, and dehydrated for the electron beam to pass freely through them. As the electrons pass through the specimen, images are formed by direct-ing the electrons onto photographic film, thus making internal cellular structures visi-ble. Scanning electron microscopes are used for visualizing surface characteristics rather than intracellular structures A narrow beam of electrons scans back and forth, producing a three-dimensional image as the electrons are reflected off the specimen's surface.

While scientists accept a diversity of optical instruments with which to perform routine laboratory procedures and sophisticated inquiry, the compound brightfield micro-scope is the "workhorse" and is commonly found in all biological laboratories. Although y'all should exist familiar with the basic principles of microscopy, you probably accept not been exposed to this diverse array of complex and expensive equipment. Therefore, only the compound brightfield microscope will be discussed in depth and used to examine specimens.

USE OF THE MICROSCOPE

PURPOSES

To become familiar with the:

1.Theoretical principles of brightfield microscopy.

2.Component parts of the compound micro-scope.

iii.Use and care of the compound microscope.

4.Practical use of the chemical compound microscope for visualization of cellular morphology from stained slide preparations.

PRINCIPLE

Microbiology is a science that studies living organisms that are besides modest to be seen with the naked eye. Needless to say, such a report must involve the utilize of a good compound microscope. Although at that place are many types and variations, they all fundamentally consist of a 2-lens system, a variable simply controllable light source, and mechanical adjustable parts for determining focal length between the lenses and specimen.

Components of the Microscope

Phase

A fixed platform with an opening in the middle allows for the passage of light from an illu-minating source below to the lens system in a higher place the phase. This platform provides a surface for the placement of a slide with its specimen over the central opening. In addition to the fixed stage, almost microscopes have a mechanical phase that can be moved vertically or horizontally past means of adjustment controls. Less sophisticated micro-scopes have clips on the stock-still stage, and the slide must be positioned manually over the central opening.

Illumination

The light source is positioned in the base of the instrument. Some microscopes are equipped with a congenital-in light source to pro-vide direct illumination. Others are provided with a mirror; one side flat and the other concave.

An external lite source, such as a lamp, is placed in front of the mirror to direct the light upward into the lens system. The flat side of the mirror is used for artificial light, and the concave side for sunlight.

Abbe Condenser

This component is found directly nether the phase and contains 2 sets of lenses that collect and concentrate calorie-free passing upward from the light source into the lens sys-tems. The condenser is equipped with an iris diaphragm, a shutter controlled past a lever that is used to regulate the corporeality of lite inbound the lens organisation.

Body Tube

Higher up the phase and attached to the arm of the microscope is the body tube. This structure houses the lens system that magnifies the specimen. The upper cease of the tube contains the ocular or eyepiece lens. The lower portion consists of a movable nosepiece containing the objective lenses. Rotation of the nosepiece posi-tions objectives above the stage opening. The body tube may be raised or lowered with the aid of coarse-adjustment and fine-adjustment knobs that are located to a higher place or beneath the phase, depending on the type and make of the instrument.

Theoretical Principles of Microscopy

To use the microscope efficiently and with minimal frustration, y'all should sympathize the basic principles of microscopy: magnification, resolution, numerical discontinuity, illumination, and focusing.

Magnification

Enlargement or magnification of a specimen is the function of a two-lens system; the ocular lens is found in the eyepiece, and the objective lens is situated in a revolving nose-piece. These lenses are separated by the body tube. The objective lens is nearer the specimen and magnifies it, producing the real image that is projected up into the focal airplane and then magnified past the ocular lens to produce the last prototype.

The nigh commonly used microscopes are equipped with a revolving nosepiece containing iv objective lenses possessing unlike degrees of magnification. When these are combined with the magnification of the ocular lens, the total or overall linear magnification of the specimen is obtained.

Resolving Ability or Resolution

Although magnification is of import, you must be enlightened that unlimited enlargement is not possible past merely increasing the magnifying power of the lenses or by using additional lenses, because lenses are limited by a property called resolving power. By definition, resolving ability is the ability of a lens to show two adjacent objects as discrete entities. When a lens cannot discriminate, that is, when the two objects appear as one, it has lost resolu-tion. Increased magnification volition not rectify the loss, and volition, in fact, blur the object. The resolv-ing ability of a lens is dependent on the wave-length of calorie-free used and the numerical aperture, which is a feature of each lens and imprinted on each objective. The numerical aper-ture is divers as a function of the diameter of the objective lens in relation to its focal length. Information technology is doubled by use of the substage condenser; which illuminates the object with rays of light that pass through the specimen obliquely as well equally directly. Thus, resolving power is expressed mathematically, equally follows:

Resolving power = Wavelength of Light .

ii (Numerical Aperture)

Based on this formula, the shorter the moving ridge-length, the greater the resolving power of the lens. Thus, brusk wavelengths of the electromag-netic spectrum are better suited than longer wavelengths in terms of the numerical aperture.

Withal; equally with magnification, resolving power besides has limits. You might rationalize that but decreasing the wavelength volition automati-cally increase the resolving power of a lens. Such is not the case, because the visible portion of the electromagnetic spectrum is very narrow and borders on the very short wavelengths found in the ultraviolet portion of the spectrum.

The relationship between wavelength and numerical discontinuity is valid only for increased resolving power when calorie-free rays are parallel. Therefore, the resolving power is dependent on some other cistron, the refractive index. This is the bending power of lite passing through air from the drinking glass slide to the objective lens. The refractive index of air is lower than that of glass, and equally light rays pass from the drinking glass slide into the air, they are bent or refracted then that they do not pass into the objective lens. This would crusade a loss of lite, which would reduce the numerical aperture and diminish the resolving power of the objective lens. Loss of refracted calorie-free tin be compensated for by interposing mineral oil, which has the same refractive alphabetize as glass, between the slide and the objective lens. In this fashion, decreased light refraction occurs and more than light rays enter straight into the objective lens, producing a vivid image with loftier resolution.

Illumination

Effective illumination is required for efficient magnification and resolving power. Since the intensity of daylight is an uncontrolled variable, artificial light from a tungsten lamp is the most unremarkably used low-cal source in microscopy. The calorie-free is passed through the con-denser located below the stage. The condenser contains two lenses that are necessary to produce a maximum numerical aperture. The height of the condenser can exist adapted with the con-denser knob. Ever go on the condenser close to the stage, particularly when using the oil-immersion objective.

Between the light source and the condenser is the iris diaphragm, which can exist opened and closed past means of a lever; thereby regulating the amount of light entering the condenser. Excessive illumination may actually obscure the specimen because of lack of contrast. The corporeality of light entering the microscope differs with each objec-tive lens used. A rule of pollex is that as the mag-nification of the lens increases, the distance between the objective lens and slide, called working distance, decreases, whereas the numerical aperture of the objective lens increases.

Utilise and Care of the Microscope

You will exist responsible for the proper care and use of microscopes. Since microscopes are expensive, you must notice the following regu-lations and procedures.

The instruments are housed in special cabinets and must exist moved past users to their laboratory benches. The right and just acceptable way to practise this is to grip the microscope arm firmly with the correct hand and the base with the left hand, and lift the instrument from the cabinet shelf. Carry it shut to the body and gently identify it on the laboratory demote. This will foreclose collision with article of furniture or co-workers and will protect the instrument against impairment.

Once the microscope is placed on the laboratory demote, observe the following rules:

ane.Remove all unnecessary materials such as books, papers, purses, and hats from the laboratory bench.

2.Uncoil the microscope's electric wire and plug it into an electrical outlet.

three.Clean all lens svstems; the smallest bit of grit, oil, lint, or eyelash volition decrease the efficiency ot the microscope. The ocular; scan-ning, low-ability, and high-power lenses may be cleaned by wiping several times with adequate lens tissue. Never use paper tow-eling or fabric on a lens surface. If the oil-immersion lens is gummy or tacky, a piece of lens paper moistened with methanol is used to wipe it clean. If the lens is very dirty it may be cleaned with xylol however the xylol cleansing procedure should exist performed only by the instructor, and only if necessary. Consequent use of xylol may loosen the lens.

The post-obit routine procedures must exist followed to ensure correct and efficient use of the microscope while focusing.

1. Identify the microscope slide with the specimen within the stage clips on the fixed stage. Move the slide to eye the specimen over the opening in the stage directly over the low-cal source.

2. Rotate the scanning lens or the low power lens into position. While watching from the side to insure that the lens doesn't touch the specimen, turn the fibroid focus knob to move the stage as close as it can get to the lens without touching the lens. (Always watch from the side whenever y'all movement a specimen towards any objective lens to make sure the lens doesn't crash through the specimen and go damaged!)

3. At present, while looking through the ocular lens, turn the fibroid focus knob carefully, and slowly motion the stage abroad from the lens until the specimen comes into vague focus. And so, use the fine focus knob to bring the specimen into sharp focus.

four. If this is the kickoff specimen of the day, you should Kohler your microscope at this point (while information technology is in focus). Otherwise, if your microscope has already been Kohlered y'all won't demand to do information technology over again

5. Routinely suit the light source past means of the light source transformer setting, and/or the iris diaphragm, for optimum illumination for each new slide and for each change in magnification.

half dozen. Our microscopes are parfocal, which means that when one lens is in focus, other lenses will also have the same focal length and can be rotated into position without further major adjustment. In practice, however; ordinarily a one-half-turn of the fine-adjustment knob in either direction is necessary for precipitous focus.

seven. One time you have brought the specimen into sharp focus with a low-powered lens, preparation may be made for visualizing the spec-imen under oil immersion. Place a drop of oil on the slide straight over the area to be viewed. Rotate the nosepiece until the oil-immersion objective locks into position. Care should be taken not to allow the high-power objective to touch on the drop of oil.The slide is observed from the side as the objective is rotated slowly into position. This volition ensure that the objective will be properly immersed in the oil. The fine-adjustment knob is readjusted to bring the image into abrupt focus.

8. During microscopic examination of microbial organisms, information technology is always necessary to observe several areas of the preparation. This is accomplished by scanning the slide with-out the application of additional immersion oil. This will require continuous, very fine adjustments past the slow, back-and-along rotation of the fine aligning knob merely.

On completion of the laboratory do, render the microscope to its cabinet in its original condition. The following steps are recommended:

1.Clean all lenses with dry, clean lens paper. If y'all need to, you tin utilize a drop or two of methanol to help clean the lens. Use xylol to remove oil from the stage only.

2. Place the depression-ability objective in position and bring the stage and objectives shut together.

3.Heart the mechanical phase.

4.Coil the electric wire around the body tube and the stage.

5.Carry the microscope to its position in its cabinet in the fashion previously described.

Source: https://www2.hawaii.edu/~johnb/micro/m140/syllabus/week/handouts/m140.2.4.html

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