ExerciseⅠ Bright-field Light Microscope
Materials per Student；compound microscope；lens paper and lens cleaner；immersion oil；prepared stained slides of several types of bacteria；（rods，cocci，spirilla），flagella，capsule；glass slides；coverslips；dropper with bulb；tweezers
Learning Objectives
Each student should be able to:
1.Identify all the parts of a compound microscope.
2.Know how to correctly use the microscope—especially the oil immersion lens.
3.Learn how to recognize different shapes of bacteria.
Principles
The bright-field light microscope is an instrument that magnifies images using two lens systems.Initial magnification occurs in the objective lens.Most microscopes have at least three objective lenses on a rotating base，and each lens may be rotated into alignment with the eyepiece or ocular lens in which the final magnification occurs.The objective lenses are identified as the low-power，highdry，and oil immersion objectives.Each objective is also designated by other terms.These terms give either the linear magnification or the focal length.The latter is about equal to or greater than the working distance between the specimen when in focus and the tip of the objective lens.For example，the low-power objective is also called the 10×，or 16 mm，objective；the high-dry is called the 40×，or 4 mm，objective；and the oil immersion is called the 90×，100×，or 1.8 mm，objective.As the magnification increases，the size of the lens at the tip of the objective becomes progressively smaller and admits less light.This is one of the reasons that changes in position of the substage condenser and iris diaphragm are required when using different objectives if the specimens viewed are to be seen distinctly.The condenser focuses the light on a small area above the stage，and the iris diaphragm controls the amount of light that enters the condenser.When the oil immersion lens is used，immersion oil fills the space between the objective and the specimen.
Because immersion oil has the same refractive index as glass，the loss of light is minimized（Figure 1）.The eyepiece，or ocular，at the top of the tube magnifies the image formed by the objective lens.As a result，the total magnification seen by the observer is obtained by multiplying the magnification of the objective lens by the magnification of the ocular，or eyepiece.For example，when using the 10×ocular and the 43×objective，total magnification is 10×43=430 times.
Figure 1.1 The Oil Immersion Objective
An oil immersion objective lens operating in air and with immersion oil.Light rays that must pass through air are bent（refracted），and many do not enter the objective lens.The immersion oil prevents the loss of light rays.
Because this is a microbiology course and most of the microorganisms studied are bacteria，this is an excellent place to introduce three basic bacterial shapes：cocci，rods，and spirilla.By gaining expertise in using the bright-field light microscope，everyone should be able to observe these three bacterial shapes by the end of the lab period.
Procedure for Basic Microscopy：Proper Use of the Microscope
1.Always carry the microscope with two hands.Place it on the desk with the open part away from you.
2.Clean all of the microscope's lenses only with lens paper and lens cleaner if necessary.Do not use paper towels or wipes；they can scratch the lenses.Do not remove the oculars or any other parts from the body of the microscope.(https://www.daowen.com)
3.Cut a lowercase e from a newspaper or other printed page.Prepare a wetmount as illustrated in Figure 2.Place the glass slide on the stage of the microscope and secure it firmly using stage clips.
If your microscope has a mechanical stage device，place the slide securely in it.Move the slide until the letter e is over the opening in the stage.
Figure 2 Preparation of a Wet-mount Slide
（a）Add a drop of water to a slide.（b）Place the specimen（letter e）in the water.（c）Place the edge of a coverslip on the slide so that it touches the edge of the water.（d）Slowly lower the coverslip to prevent forming and trapping air bubbles.
4.With the low-power objective in position，lower the tube until the tip of the objective is within 5 mm of the slide.Be sure that you lower the tube while looking at the microscope from the side.
5.Look into the microscope and slowly raise the tube by turning the coarse adjustment knob counterclockwise until the object comes into view.Once the object is in view，use the fine adjustment knob to focus the desired image.
6.Open and close the diaphragm，and lower and raise the condenser，noting what effect these actions have on the appearance of the object being viewed.Usually the microscope is used with the substage condenser in its topmost position.The diaphragm should be open and then closed down until just a slight increase in contrast is observed（Table 1）.
Table 1 Troubleshooting the Bright-Field Light Microscope
7.Use the oil immersion lens to examine the stained bacteria that are provided（Figure 3a-d）.The directions for using this lens are as follows：First locate the stained area with the low-power objective and then turn the oil immersion lens into the oil and focus with the fine adjustment.An alternate procedure is to get the focus very sharp under high power，then move the revolving nosepiece until you are halfway between the high-power and oil immersion objectives.Place a small drop of immersion oil in the center of the illuminated area on the slide.Continue revolving the nosepiece until the oil immersion objective clicks into place.The lens will now be immersed in oil.Sharpen the focus with the fine adjustment knob.Draw a few of the bacteria in the spaces provided.
8.After you are finished with the microscope，place the low-power objective in line with the ocular，lower the tube to its lowest position，clean the oil from the oil immersion lens with lens paper and lens cleaner，cover，and return the microscope to its proper storage place.
Figure 3 Examples of Bacterial Shapes as Seen with the Bright-field Light Microscope
（a）Staphylococcus aureus cocci；singular，coccus（×1，000）.（b）Bacillus subtilis rods or bacilli；singular，bacillus（×1，000）.（c）A single，large spirillum；plural，spiralla（Spirillum volutans；×1，000）.（d）Numerous，small spirilla（Rhodospirillum rubrum；×1，000）