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Importance of the Microscope Important for hematology, microbiology, TB, and malaria testing Compound microscope used in bacteriology, biology, and medicine to examine minute objects such as bacteria, other unicellular organisms, and plant and animal cells and tissue Advances in fluorochrome stains and monoclonal antibody techniques caused growth in use of fluorescence microscopy in both biomedical analysis and cell biology

 Brightfield microscopy is very simple to use with fewer adjustments needed to be made to view specimens.  Some specimens can be viewed without staining and the optics used in the bright-field technique don’t alter the color of the specimen.  It is adaptable with new technology and optional pieces of equipment can be implemented with bright-field illumination to give versatility in the tasks it can perform..

 Certain disadvantages are inherent in any optical imaging technique.  By using an aperture diaphragm for contrast, past a certain point, greater contrast adds distortion. However, employing an iris diaphragm will help compensate for this problem.  Bright-field microscopy can’t be used to observe living specimens of bacteria, although when using fixed specimens, bacteria have an optimum viewing magnification of 1000x.

 Bright-field microscopy has very low contrast and most cells absolutely have to be stained to be seen; staining may introduce extraneous details into the specimen that should not be present.  Also, the user will need to be knowledgeable in proper staining techniques.  Lastly, this method requires a strong light source for high magnification applications and intense lighting can produce heat that will damage specimens or kill living microorganisms.

 Resolution  Ability to distinguish (resolve) two close- together points as separate.  Contrast  Differences in intensity between two objects, or between an object and background  Important in determining resolution  Staining increases contrast

A. Bright-field microscopes B. Dark-field microscopes C. Phase microscopes D. Fluorescent microscopes

 Brightfield microscopy is the most elementary form of microscope illumination techniques and is generally used with compound microscopes.  The name "brightfield" is derived from the fact that the specimen is dark and contrasted by the surrounding bright viewing field. Simple light microscopes are sometimes referred to as brightfield microscopes.

 Bright field microscopy is best suited to viewing stained or naturally pigmented specimens such as  stained prepared slides of tissue sections or living photosynthetic organisms.  It is useless for living specimens of bacteria, and inferior for non-photosynthetic protists or metazoans, or unstained cell suspensions or tissue sections.

 Prepared slides, stained - bacteria (1000x), thick tissue sections (100x, 400x), thin sections with condensed chromosomes or specially stained organelles (1000x), large protists or metazoans (100x).  Smears, stained - blood (400x, 1000x), negative stained bacteria (400x, 1000x).  Living preparations (wet mounts, unstained) - pond water (40x, 100x, 400x), living protists or metazoans (40x, 100x, 400x occasionally), algae and other microscopic plant material (40x, 100x, 400x). Smaller specimens will be difficult to observe without distortion, especially if they have no pigmentation

Using a bright field microscope

 Mount the specimen on the stage  Optimize the lighting  Adjust the condenser  Think about what you are looking for  Focus, locate, and center the specimen  Adjust eyepiece separation, focus  Select an objective lens for viewing  Adjust illumination for the selected objective lens

 In bright-field microscopy a specimen is placed on the stage of the microscope and incandescent light from the microscope’s light source is aimed at a lens beneath the specimen. This lens is called a condenser.  The condenser usually contains an aperture diaphragm to control and focus light on the specimen; light passes through the specimen and then is collected by an objective lens situated in a turret above the stage.

 The objective magnifies the light and transmits it to an oracular lens or eyepiece and into the user’s eyes. Some of the light is absorbed by stains, pigmentation, or dense areas of the sample and this contrast allows you to see the specimen.  For good results with this microscopic technique, the microscope should have a light source that can provide intense illumination necessary at high magnifications and lower light levels for lower magnifications

BASIC COMPONENTS OF THE MICROSCOPE AND THEIR FUNCTIONS

Power switch Light intensity control Condenser Stage Objectives Eyepiece lens Stage motion control knobs Course and fine adjustments knobs Field diaphragm ring Aperture diaphragm

Care and Use of the Bright Field Microscope Malaria parasite Mycobacterium tuberculosis

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  Cover when not in use with vinyl or plastic dust cover  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  Cover when not in use with vinyl or plastic dust cover

CLEANING THE MICROSCOPE Routine Cleaning Supplies:  Commercial lens tissue for optics  Caution: Do not use paper towels or other rough paper products  Cotton swabs with wooden shaft (optics)  70% isopropyl alcohol  Dilute methanol is satisfactory  Mild detergent and soft cloth for stage and base of microscope

CLEANING OCULARS AND OBJECTIVES  Unplug the microscope  Wash hands  Remove dust from optical glass surfaces  Carefully remove eyepieces, objectives, condenser, and filters–one at a time  Excessive rubbing can cause damage to iridescent coating on lens  Clean and replace as completed  Do Not take eyepiece or objectives apart

 Unplug microscope and allow bulb to cool  Carefully place microscope on its side  Open bulb house; use tissue to remove bulb  Use tissue (to avoid fingerprints) to pick up new bulb  Insert new bulb and close bulb house Replacing Microscope Bulb

 Plug in microscope and turn on illuminator. Rotate nosepiece to lock 10X objective in place  Place smear on stage and center it under the 10X objective  Open the field diaphragm all the way and close condenser diaphragm all the way  Move up (rack up) stage to its highest position  Adjust the oculars for interpupillary distance so that only one circle of light is seen  Rack up condenser as high as possible Setting the Koehler Illumination

Close field diaphragm half way and focus smear at 10X Close field diaphragm until diameter of illuminated image is smaller than the field of view Lower condenser with positioning knob until you have a sharp, focused image of the edges of the field diaphragm Adjust condenser using centering screws so that the circle of light is centered in field Open field diaphragm until illuminated image is just larger than the field of view. If more light is needed, use the transformer. Koehler illumination is now set. It is important not to move the condenser up or down or change the field diaphragm. Setting the Koehler Illumination (continued)

OPERATION OF THE MICROSCOPE – EXAMINING SMEARS  Put smear on stage and center it under the 10X objective  Adjust intensity of the light to a comfortable level with the transformer  Open condenser diaphragm about 70% to achieve a good balance of resolution and contrast  Adjust oculars for interpupillary distance so that when looking with both eyes only one circle of light is seen

Examining Smears (continued) Adjust sharpness of image by moving adjustment ring on adjustable ocular Once 10X focus is achieved, rotate nosepiece so that the 40X objective is in place Readjust the intensity of light to a comfortable level using the transformer Use the fine adjustment knob to focus up and down through the different planes of the field

Microscope Problems – Troubleshooting 1 Problem: Black Field Possible Causes: Microscope not plugged in Power not available at outlet Illuminator not turned on Bulb burned out Objective not clicked into place Condenser too low with diaphragms closed

Microscope Problems – Troubleshooting 2 Problem: Field only partially illuminated Possible Causes: Objective not clicked into position Condenser not centered correctly Condenser too low Field diaphragms closed too much

Problem: Difficulty focusing with 10X objective Possible Causes: Wrong objective in place Objective not screwed into place Not in correct plane of focus Microscope Problems – Troubleshooting 3

Microscope Problems – Troubleshooting 4 Problem: Difficulty focusing with 40X objective Possible Causes: Not in correct plane of focus Not initially focused at 10X

Microscope Problems – Troubleshooting 5 Problem: Blurry image at 10X or 40X Possible Causes: Dirty objective Dirty slide Dirty coverslip Problem: Ground glass appearance Possible Causes: Condenser too high

  Advanced Light Microscopy vol. 1 Principles and Basic Properties by Maksymilian Pluta, Elsevier (1988)  Advanced Light Microscopy vol. 2 Specialised Methods by Maksymilian Pluta, Elsevier (1989)  Introduction to Light Microscopy by S. Bradbury, B. Bracegirdle, BIOS Scientific Publishers (1998)  Microbiology: Principles and Explorations by Jacquelyn G. Black, John Wiley & Sons, Inc. (2005)  Microscopy and Imaging Literature Microscopy and Imaging Literature  html html