EXERCISE 4 Lab Procedures I Calibrate your reticle 1 Rotate the scanning 4X objective into viewing position and place a transparent plastic ruler on the stage Focus the microscope and align the ruler until the millimeter lines come into clear focus in the field of view 2 Define the distance between 2 of the lines closest together on the reticle as one reticle unit r u Careful Do not be distracted by numbers on the reticle if present A reticle unit is the smallest unit visible on the reticle 3 Rotate the ocular and move the ruler until the reticle is aligned across 2 adjacent millimeter marks Determine the number of reticle units r u s which correspond to 1mm as seen through the microscope Make sure you count the number of r u s from the middle of one mm line to the middle of the next mm line Reticle units per millimeter for your microscope using the 4X objective Using this information calculate the number of m per r u With this microscope using the 4X objective one reticle unit corresponds to m 4 Rotate the nosepiece to bring the 10X objective into aligned position Note that the reticle appears the same size with its marks the same distance apart as when the 4X objective was in position However the millimeter lines on your ruler appear to be spaced much further apart at higher magnification Determine the m s per r u when using the 10X objective If the reticle in your microscope is not be long enough to reach between 2 adjacent millimeter marks at this magnification use the following formula to calculate m s per r u m using 4X obj 4X r u Mag of the new obj m using new obj r u With this microscope using the 10X objective one reticle unit corresponds to m Also determine the m s per r u when using the 40X objective using the formula above With this microscope using the 40X objective one reticle unit corresponds to m II Examining Prokaryotic Cells 1 Bacterial shapes Because bacterial cells are very small not much more than their basic shape will be visible under the light microscopes available in lab Usually bacteria exhibit one of 3 shapes bacilli rods cocci spheres or spirilli spirals a Place the prepared slide labeled bacterial types on a white background and examine it with your naked eyes You should see 3 distinct pinkish or purplish spots under the coverslip Each spot contains bacteria exhibiting one of the 3 shapes listed above b Place the slide on the stage of your microscope and examine each of the 3 colored spots Focus with low power first and then switch to high power The cells you observe should be stained pink or purple They will be much smaller than the eukaryotic cells you observed last week Note that in some cases the cells may be attached to form clusters or chains Be sure you locate and can recognize all 3 bacterial shapes For assistance check photographs that may be available in lab or in your textbook Exercise 4 Austin Community College BIO 1406 Laboratory Manual 12th Ed 2006 4 5 2 c In your lab notebook draw diagrams of the 3 bacterial shapes observed on your slide and label each shape with its name d Use the reticle to measure the size of a few bacterial cells with each shape Record the results in your lab notebook in reticle units then convert the measurements to micrometers Make sure you mark down the objective you used when making your measurements e When you are finished return the slide to its original location Cyanobacteria Cyanobacteria are a fairly common group of bacteria that carry out a type of photosynthesis that produces O2 Note Some bacteria carry out alternative types of photosynthesis that do not produce O2 Scientists believe cyanobacteria were largely responsible for converting the early earth s atmosphere from a reducing atmosphere into an oxidizing atmosphere Cyanobacteria use a pigment called chlorophyll a to absorb light energy for photosynthesis This type of chlorophyll is also found in photosynthetic eukaryotes However in cyanobacteria the chlorophyll molecules are not located in chloroplasts as they are in eukaryotes Instead they are found on the surface of photosynthetic lamellae located throughout the cytoplasm a Prepare a wet mount of Anabaena making sure you get some of the green filaments from the culture onto your slide Examine Anabaena using low power first and then high power On high power you should be able to distinguish the individual cells that make up each filament For assistance check photographs that may be available in lab or in your textbook b In your notebook draw a filament of Anabaena which clearly shows the shape of the individual cells Do all the cells look identical Note any differences on your diagram c Use the reticle to measure the size of a few Anabaena cells Record the results in your lab notebook in reticle units then convert the measurements to micrometers Make sure you mark down the objective you used when making your measurements d When you are finished remove the coverslip and place it in the discard container provided Wash the slide dry it and return it to its original location III Examining Eukaryotic Cells 1 Mixed Protozoans non photosynthetic protists Protozoans is a general term for a variety of mostly unicellular non photosynthetic protists a Prepare a wet mount of the Mixed Protozoans culture Note Protozoans are relatively large and tend to settle to the bottom of the culture jar You will get the best results if you leave the water in the jar undisturbed and take your sample from the bottom Squeeze some air out of a dropper BEFORE you place it into the jar and then slowly suck in some of the water and debris from the BOTTOM of the jar Do NOT disperse the cells by stirring up the water in the jar or by squeezing air out of the dropper into the water b Examine the slide with your microscope Using the keys and diagrams provided identify at least 2 different protozoans on your slide Careful The protozoan culture may be contaminated with small animals called rotifers or other organisms If you are not sure the organism you are viewing is a protozoan check with your instructor c In your lab notebook draw diagrams of 2 different protozoans and label each with its name Next record the length and width of each organism in reticle units and then convert the measurements into micrometers Finally indicate whether each organism is unicellular or colonial d When you are finished discard the coverslip in the container provided Clean and dry the slide and return it to its original location Exercise 4 Austin