Plan of Action

The microbiologist may study employ different laboratory equipment like the microscope in the study of microbes. He may study the microbes in various ways. Generally, there are five standard procedures being followed or the identification and evaluation of microbes in the microbiologic laboratory.

First in the procedure is through direct microscopic examination. Microbial cells or their colonies are too tiny to be visible to the unaided eye. The microbiologist must use an instrument of precision. This instrument is the microscope. With the use of the microscope the microbes can be viewed clearly. The microscope magnifies the minute microorganisms, hence they can be studied well. A test specimen is prepared for microscopic study. Some specimen is applied to or spread thinly on a glass slide in a manner that light rays from the light source on the scope can pass through the material and allow or visualization. Sometimes the microscopic preparation is viewed unstained. More often, the slide is stained by one of the several methods o staining before it is viewed under the microscope.

Second is through culture. The microbiologist may make a culture from the test specimen. This is done by allowing microorganisms to multiply sufficiently through media to form visible growth. He then studies the physical pattern of that growth with the use of the microscope. Just like in direct microscopic observation method, the culture may or may not be stained by microscopic preparations.

Third method is done by biochemical test. The colonial growth of the microbes which is obtained from the original specimen is studied under the microscope to determine their biologic properties. The growth is then subjected to a series of biochemical tests from which identifying characteristics of the microbes emerge.

Fourth is through animal inoculation. The microbes recovered from the test specimen by cultural methods are injected into a suitable laboratory animal, just like the rat. The animal is then observed for further reactions.

The fifth method is through immunologic reactions. The microbiologist may opt to use this method which uses antigen-antibody tests for the identification of microorganisms recovered.

Pathogenic microbes must be handled with utmost caution and in accordance with well-known principles of conduct in microbiologic laboratories. These pathogenic microbes are disease-producing agents and can be very dangerous. The accidental laboratory infections can be fatal. In a study conducted which surveyed 1300 infections among laboratory workers, 39 ended fatally. In many instances the infections occurred in research workers and highly trained technologists.

“`Man is a tool-using animal … without tools he is nothing, with tools he is all….” Carlyle.

Tools for the Study

The microbiologist has many instruments of precision and laboratory apparatus at his command. Some are in constant use while others are needed only in special investigations.

The Compound Light (Bright-field) Microscope

The instrument most often used by the microbiologist is his study of various microorganisms is the compound microscope. It is very important that the microscope should be handled with utmost care. Its workmanship should be of highest quality.

There are two kinds of microscopes namely the simple microscope and the compound microscope. A simple microscope is a title more than a magnifying lens. A compound microscope on the other hand incorporates two or more lens systems so that the magnification of one system is increased by the other. Practically, the compound microscope consists of two parts, the supporting stand and the optical system.

The supporting stand includes (1) a base and pillar, (2) an arm to support the optical system and house the fine adjustment, (3) a platform or stage on which the object to be examined rests, and (4) a condenser and mirror fitted beneath the stage. The condenser and mirror focus the light from either an external source such as a special microscope lamp or from an illuminating system fitted into the base of the scope. Where present, the built-in base illuminator which is a self-contained substage illuminator, houses the light source, a collecting lens system, mirror, and a condenser lens. An iris field diaphragm, a variable transformer attached to the system, and properly placed filters permit adjustments of the light.

The optical system consists of a body tube that supports the ocular lenses or the eyepiece at the top end and the objective lenses attached to a revolving nosepiece at the other end. The optical system is connected to the arm of the supporting stand by an intermediate slide, which moves up and down on the arm in response to movement of the fine adjustment. The intermediate slide contains the rack and pinion for the coarse adjustment, which acts directly on the tube of the optical system. The platform of the microscope is usually equipped with a mechanical stage to hold the glass microslide firmly. The object is mounted on the microslide so that it can be moved from place to place by set screws. The advantages of this device are that the specimen can be examined systematically and, unless moved, the specimen remains in a fixed position.

The magnification of an objective is usually designated by its equivalent focal distance in inches or millimeters. By equivalent focal distance is meant the focal distance of a lens having the same magnification as the objective. The higher the number of the objective, the less is its magnification. American microscopes are usually fitted with 16 mm., 4 mm., and 1.8 mm. objectives. The last is an immersion objective because for the best results there must be a liquid either oil or water between the objective and the object being examined. This objective is best known as an oil-immersion objective because only rarely would its lens system be immersed in water. Most modern immersion oil is highly refined mineral oil having the same refractive index, (.52 as glass. The 16 mm. objective magnifies 10 times; most 4 mm.. objectives magnify 43 times; and most 1.8 mm. objectives magnify 97 times.

The oculars of a microscope are given 6x, 10x, and similar designations to indicate that they increase the magnification of the objective 6, 10, or more times, ` respectively. To obtain the magnification of any combination of ocular and objective lenses, multiply the magnification of the objective by that of the ocular. Remember that magnification refers to both the length and the width of an object; that is, a magnification of 100 means that the object is made to appear 100 times as long and 100 times as wide.

The use of the microscope requires simple instructions as follows:

1. Keep both eyes open. A very little practice will enable you to do this.

2. Most modern microscopes have self-contained substage illuminators. If yours does not and you must work with the mirror, avoid direct sunlight. North light is advantageous. Good results are obtained with daylight.

3. When you place a slide on the stage, see that it lies flat against the platform. Adjust the light so that the object is evenly illuminated.

4. Learn to focus with the low-power objectives (such as the 16 mm. objective). Use the coarse adjustment to move the low-power objective until it nearly (but not quite) touches the cover glass or upper surface of the mounted specimen. Then focus up until the object comes plainly into view. Complete focusing with the fine adjustment. Keeping the specimen in focus, use very short back and forth strokes of the fine adjustment to produce an illusion of depth in the specimen. When the immersion objective is used, first place a drop of immersion oil on the object so that it can be clearly brought into view. Parfocal objectives are designed so that when switching from one objective to another the microscopist can keep the specimen essentially in focus; most modern microscopes are equipped with them.

5. Keep the microscope clean and handle all parts with care. Do not touch the glass parts of the microscope. Do not allow chemicals to contact the microscope, since they may injure it. Clean the mechanical parts with an application of olive oil on gauze; wipe the oil off with chamois or lens paper. Keep optical glass parts especially ocular lenses, condenser lens system, and non immersion objective lenses of the microscope clean by frequent use of lens paper. Remove immersion oil from oil-immersion objective lens carefully right after you have finished your microscopic study. At times it may be necessary to remove dried immersion oil with lens paper moistened with xylol. Do this as rapidly as possible to prevent injury to the optical settings of the oil-immersion objective lens system.

6. Clean the microscope thoroughly when you are finished. Leave the, lowest power objective in the working position; thus the least expensive objective would be injured should the optical system be jammed down accidentally. Keep the microscope covered when you are not using it.