INTRODUCTION
These activities are intended to provide fundamental materials for students in marine microbiology. The approach use in these laboratory exercises is physiological and ecological to enable you clearly understand the composition of microorganisms as well as the reaction they produce. You will be exposed to the role of marine microorganisms in the degradation and recycling of nutrients in the marine ecosystem and biotechnological application of some important forms. I hope these activities can serve as solid foundation for your advanced works later on.
ASEPTIC TECHNIQUES TO BE OBSERVED IN THE LABORATORY
1. Wear a protective gown made of material easily cleaned and sterilized if possible and avoid wearing it outside to prevent possible spread of contaminants to the outside.
2. Disinfect your working area before and after use.
3. Disinfect your hands before and after each laboratory manipulations.
4. Avoid putting anything in your mouth during laboratory exercises.
5. Always flamed-sterilize forceps, rods, loops and needles before laying them aside.
6. Avoid any kind of contaminated materials from spilling and contacting surfaces, hands or clothing. Notify the laboratory instructor immediately if accident happens even a minor one.
7. Keep personal items in place designated and away from working area.
8. Arrange culture and infectious materials in a secure place of the table. Do not allow unused equipment to accumulate in your working area.
9. Dispose infectious materials/cultures and contaminated materials carefully as prescribed by the instructor. Note location of special containers for disinfectants used regularly.
10. Keep glassware and equipment clean and in their proper places. Be as clean and as orderly as possible at all times.
LABORATORY PROCEDURES
1. Keep laboratory manual on hand. Read the procedure thoroughly before starting with the exercise. Always think and plan ahead. Outline the laboratory procedures in your data notebook.
2. Keep only equipment/materials in use on the table. Be careful in handling them. They are expensive.
3. Perform the exercise. Pay attention to the preliminary briefing of your instructor. Check and ask your instructor for clarification. Take note of your results and records all measurements and observations.
4. Label your cultures and study specimens as indicated by your instructor. Include the following data:
Name or initials
5. Use references, Check related materials in textbooks and outside references. Make conclusion about the data you have gathered. How does it relate to the works done in the field? Have other problems been raised by the data your exercise generated?
6. Dispose of contaminated and infectious materials. Locate proper jars and pans for discards. Heat them to boiling before discarding.
7. Replace equipment, glassware and materials where you find them.
8. Turn off the water light, gas outlet when not in use.
1. Keep laboratory manual on hand. Read the procedure thoroughly before starting with the exercise. Always think and plan ahead. Outline the laboratory procedures in your data notebook.
2. Keep only equipment/materials in use on the table. Be careful in handling them. They are expensive.
3. Perform the exercise. Pay attention to the preliminary briefing of your instructor. Check and ask your instructor for clarification. Take note of your results and records all measurements and observations.
4. Label your cultures and study specimens as indicated by your instructor. Include the following data:
Name or initials
Name of organisms
Culture media used
Date of culture
6. Dispose of contaminated and infectious materials. Locate proper jars and pans for discards. Heat them to boiling before discarding.
7. Replace equipment, glassware and materials where you find them.
8. Turn off the water light, gas outlet when not in use.
Laboratory Exercise No 1
MICROSCOPY
INTRODUCTION
The microscope is one of the indispensable tools of biologists in observing and probing the structural basis of life. Microscopy is the techniques used designed to help you become familiar with and proficient in the use of microscope. This consists of a system of lenses and appropriate mechanical fixtures for focusing and observing live or stained specimens. The objective lenses are close to the object and produce magnified image of it. The ocular lenses are near the eye and magnify the image. Magnification is the enlargement of the image and is the end result of the combined effects of the objective and ocular lens system. The objective lens provides an enlarge image of the object from 4 to 100x and the ocular lens magnify the image n more than 10 to 15 times. The total magnification of the lens system is the product of the objective’s magnification and the ocular’s magnification.
The resolution of a bright field microscope is defined as the ability of lens system to produce an image that allows the viewer to distinguish between two structures near each other in a specimen. The resolving power of the microscope is determined by the wavelength of light used to illuminate the object, index of refraction between the specimen and objective lens and half the angle of the cone of light entering the lens.
The testing, calibration and critical use of a microscope demands a precise alignment of all optical components
The testing, calibration and critical use of a microscope demands a precise alignment of all optical components
For microscopic observations to have meaning, one must have a standard means of measurement and a way to determine the size of the specimen under investigation. The metric system has been adopted by the scientific community in order to establish a uniform standard of measurements and is now used by most scientists for all quantitative determinations (Appendix A, Table 1)
OBJECTIVES
1. To explain how various types of microscopes work and their importance in the field of microbiology;
2. To let the students understand the principles involved in order that the instrument maybe employed to greater advantage;
3. To impress to the students the importance of proper care and maintenance of such precision instrument;
METHODOLOGY
A. Parts of the Microscope
Place the microscope in proper position. Locate all parts and learn the functions of each.
Figure 1. The Compound Microscope
B. Manipulation of Microscope
Focusing and estimation of the microscope field of view
1. The field of view of the microscope is the area of the slide when you look through a microscope and is very important in estimating the size of the specimen. Carefully place a thin metric ruler on the stage (where a slide would usually go) and focus under the 4x or 10x objectives. Adjust the eyepieces of the microscope to your own personal measurements. Look through the eyepiece and adjust the distance between the eyepieces until one circle of light appears.
2. Close the iris diaphragm so only a minimum of light enters the objective lens.
3. Lower the condenser until a distinct circle of light is visible. Open the iris diaphragm until the light just fills the field.
4. Measure the field of view in millimeters and estimate the total field of view. Convert your millimeter value to micrometers.
5. When an image has been brought into focus with low power and have completed your observations, swing the high dry objective into position and focus. The specimen will remain almost in focus and only slight adjustment is needed. You may need more light for better resolution.
6. Move the high dry out of position and place a drop of immersion oil on the area of the slide you are observing. Carefully click the oil immersion lens into position. Focus with the fine adjustment. When observations are completed swing back to low power objective and clean the objective with lens paper.
Estimation of the size of specimen
1. Focus on the prepared slide of Spirulina sp provided and estimate the size of specimen in relation to the field of view. If the specimen takes up half of the field of view, that is ½ x the field of view. The field of view of the high power cannot be measured directly because the high power objective is so close to the stage. But we can use the width of view under low power (using the steps above) and the relationship between the low and high power magnification to mathematically calculate the width of the field of view under high power..
Calibration of the Microscope
Microscope objects can be measured by means of ocular micrometer. Both must be calibrated first with stage micrometer.
1. Remove the eyepiece from the drawtube and replace it with the micrometer eyepiece.
2. Place the micrometer stage in the stage and focus in its scale. Arrange the stage micrometer so that one line in it coincides with the line in the ocular.
3. Count the number of divisions in the ocular micrometer coinciding with line in the stage scale.
4. Count the number of divisions in the ocular micrometer subtended by the number of divisions in the stage micrometer. It is important that the line coincides properly.
5. Use the following formula to determine the value of one division in the ocular micrometer.
stage micrometer division value of one stage
subtended by ocular micrometer x micrometer divisions (µm)
CF= ocular micrometer divisions subtended by stage micrometer
6. Measurement of the specimen
Measure the specimen by counting the number of divisions it will cover in the ocular micrometer. Compute the size of the specimen using the calibration factor,
7. Care of the microscope, Clean the microscope after using. Be sure to remove the oil from the oil immersion
objective. Use lens paper only on optical glass parts (objective)
objective. Use lens paper only on optical glass parts (objective)
RESULTS AND DISCUSSION
Guide Questions for Discussion
1. How can each objective be identified if identification marks are removed?
2. As you go from the low power to the high power what happens to the:
a. diameter of the field of vision
b. Amount of light reaching your eye
c. Working distance between lens and slide.
3 What does it mean when your microscope is parfocal?
4. What is the numerical aperture of a lens? What are its functions?
5. Define resolving power of a microscope. How can you use this to better advantage?
6. Why oil often put between stained specimens and the objective lens?
7. Of what use is a dark-field microscope? Why are the field dark and the object light?
8. Of what use is the phase-contrast microscope? Explain why small objects are dark compared to bright field.
9. Of what use is the fluorescence microscope? What kind of light is used to illuminate the specimen? Explain why the field is dark and the object colored?
10. Computation of Drawing Magnification
a. Draw your specimen in a 10 x 10 cm square. Using the actual size of your specimen compute for your drawing magnification by dividing the size of your drawing by the actual size of your specimen
REFERENCES:
LABORATORY EXERCISE 1 MICROSCOPY
Name:
Date Performed:
Rating:
RESULTS
OCULAR | OBJECTIVES | DIAMETER (mm) | RADIUS | MEAN AREA | ||
mm | mm | mm2 | mm2 | |||
.2. Magnification
DIAMETER OF THE FIELD (LPO) | ESTIMATE WIDTH OF SPECIMEN | ||
mm | µm | mm | µm |
a. What is the highest magnification you could get with your microscope?
b. How many cells of Spirulina sp. can be fitted in the low power field of view, how many of those cell would you see under the microscope?
3. Calibration
OBJECTIVE | NUMBER OF DIVISIONS | CALIBRATION FACTOR (CF) | ||
OCULAR | STAGE | STAGE | OCULAR | |
a. Why should the ocular micrometer be calibrated for each object? Can a calibrated ocular micrometer measure the diameter of a field? Why or why not?
APPEDIX A
Table 1. The Metric System
UNIT | MEASURE | SYMBOL | ENGLISH EQUIVALENT |
Linear Measure | |||
1 kilometer | 1,000m (103)m | km | 0.62137mile |
1meter | 100m | m | 39.37inches |
1 decimeter | 1/10 (10-1) | dm | 3.937 inches |
1 centimeter | 1/100m (10-2) | cm | 0.3937inch |
1 millimeter | 1/1000m (10-3) | mMm | |
1 micrometer or micron | 1/1,000,000m (10-6) | mm (m) | |
1 nannometer | 1/1,000,000,000 (10-9) | nm | |
1 angstrom | 1/10,000,000,000 (10-10) | A | |
Measures of capacity (for fluids and gases) | |||
1 liter | |||
1milliliter | 1/1000L | L | 1.0567 |
Measures of Volume | |||
1 cubic meter | m3 | ||
1 cubic decimeter | 1/1,000,000 cubic meter | dm3 | |
1 cubic centimeter | 1,1000,000,000 cubic meter | cm3 or ml | |
1 cubic millimeter | 1/100,000,000 cubic meter | ||
Measures of Mass | |||
1 kilogram | 1,000g | kg | 2.2046 pounds |
1 gram | 1/1,000 kg | g | 15.432grains |
1 milligram | 1/1,000 g | mg | . 01grains |
1 microgram | 1/1,000,000gram | µg (mcg) | |
