1

MICROSCOPY AND PROTISTA

MICROSCOPY Since a many animals differences and characteristics can be microscopic, the competent and careful use of the microscope is essential. In this lab, you will need to use two different types of microscopes, a dissecting or stereomicroscope and a compound microscope. Read all of the introductory information carefully before beginning the exercises. There is a series of short exercised that you should accomplish during today’s lab: 1) achieve Koehler illumination, 2) learn how to use a dissecting microscope, 3) familiarize yourself with the art of producing wet mount, 4) the art of viewing specimens and 5) taking care of your microscopes Exercise I – Koehler Illumination Koehler illumination is proper alignment of the incident or illuminating light for microscopy. Every time you use the microscope you should align the condenser lens to assure Koehler illumination is optimal or you will end up with poor resolution, contrast artifacts, and unevenly lit pictures. Köhler illumination is simple to achieve with any microscope equipped with a field diaphragm, which in your microscopes will be located in the base just beyond the lamp.

With the new microscopes Köhler Illumination is quickly and easily achieved in five easy steps 1. Locate the specimen and bring it into focus (usually done with the condenser iris closed to yield maximum contrast.)

2. With the 10X or 40X objective, close the field diaphragm in the base sufficiently to be able to see its edges. This is done by rotating the control ring on top of the lens where the light leaves the base. Notice that the edges are NOT in sharp focus. It should look like this:

3. Using the condenser focus knob on the left side beneath the stage, raise or lower the condenser to bring the edges of the field diaphragm into sharp focus. When the edges are in sharp, clear focus it should look like this:

4. Open the field diaphragm. 5. Now use the condenser iris control to achieve best balance between contrast and resolution. Incidentally, another use for the field diaphragm is when viewing one small object in a particularly densely packed field. So many small objects can be confusing. Closing the field diaphragm down to include only area surrounding the object of interest can be a great help. A very important point!!! The condenser iris is NOT intended to control brightness!! That should be done by the rheostat (“dimmer control”) or with filters. When changing objectives use the knurled ring on the turret, don’t grab the objectives. The manufacturer has extended this ring to make it easier to grasp than are the objectives. Exercise II – The use of the dissecting microscope 1. Adjust the magnification to its lowest power with the magnification knob on the top or side of the microscope body. 2. Adjust the inter-pupillary distance of the ocular lenses. Look through the ocular lenses. If you see one image, no adjustment is necessary. If you see two images, or a lot of black, adjust the distance between the ocular tubes until you see one image. You also may need to move your eyes closer to or farther away from the ocular lenses so that the specimen's image fills the lenses. You may need to move the ocular lenses far apart or close together. 3. Focus on the specimen. This is a two-step process. In the first step, you will roughly focus on the specimen with the objective lens. In the second step, you will compensate for any differences in strength between your eyes to obtain the sharpest image possible. 4. Rough focus . Lower the microscope body to its lowest point with the focusing knob on the sides of the microscope arm. Use the focus knob to raise the microscope body until the specimen image is the sharpest. Compensate for any differences in strength between your eyes. (The following directions are written for microscopes with diopter adjustment rings on the right ocular tube. Obviously, if you have a scope with the diopter adjustment on the left ocular tube, you will start with your right eye closed.) a. Close your left eye. Adjust the diopter adjustment ring until the image is in focus for your right eye. You may want to adjust the ring back and forth (i.e., in and out of focus) a few times until you are sure you have the best focus for yourself. b. The first time through the diopter adjustment you may want to repeat steps a through d-sometimes our eyes automatically compensate for out-of-focus images seen in the microscope, and eyestrain results. Who wants a headache in marine bio lab?? 5. If you change the magnification, you may need to adjust the focus again. 6. When you are finished with the microscope, unplug it and lightly wind the cord around the arm, clean the stage (this is pertinent when working with marine organisms and cultures because seawater is incredibly corrosive) and lower the body all the way down before storing it away. Exercise III – Preparing Wet Mounts The purpose of this exercise is to prepare usable wet mounts that may be viewed under the microscope using both microscopy cultures and macroscopic preparations. Use the life protest cultures to hone your wet-mount skills. 1. Using the dropper, place a few drops of pond water onto the center of a clean, dry slide.

2. Hold the side edges of the coverslip and place the bottom edge on the slide near the drop of pond water.

3

3. Slowly lower the coverslip into place. The water should spread out beneath the coverslip without leaving any air bubbles. If air bubbles are present, you can press gently on the coverslip to move the air bubbles to the sides.

Exercise IV - Viewing specimens 1. View the specimen with the low-power objective: a. Turn the nosepiece until the low-power objective locks into place. b. Place the slide on the stage and center it over the condenser. c. Looking from the side, turn the coarse adjustment knob until the low-power objective is in its lowest possible position. d. Looking through the ocular lens, slowly turn the coarse adjustment knob in the other direction. This raises the low-power objective away from the slide. Continue until a clear image appears. e. Slowly turn the fine adjustment knob until the object comes into sharp focus. f. Adjust the light for maximum contrast using the iris diaphragm. g. Move the slide around on the stage using your fingers or the control knobs until you find a microorganism. h. Open the iris diaphragm until optimum contrast is achieved. 2. If the microscope has an oil immersion objective, then view the microorganism using this lens. a. Turn the nosepiece so that no objective lens is directly over the slide. Then place a small drop of immersion oil on top of the coverslip. b. Turn the nosepiece so that the oil immersion objective is locked into place over the specimen, making sure that the lens is immersed into the oil. c. Slowly turn the fine adjustment knob until the image comes into focus. d. Adjust the iris diaphragm to achieve maximum contrast. e. When you are finished, remove the oil from the oil immersion lens using lens paper. f. Remove the oil from the slide by wiping it gently with a paper towel.

PROTISTA While they are not true animals, the Protozoa represent the first eukaryotic organisms (bearing a nucleus) to evolve. The organisms referred to as protozoans (“first animals”) compose a diverse group of eukaryotic (mostly) unicellular organisms. In protozoans all life functions are carried out within the confines of a single cell. Although there are obviously no organs or tissues in protozoans, they are far from “simple” organisms as they are sometimes described. In fact, the cells of some species show the greatest complexity and internal organization of any organisms on Earth! About 10,000 species have close are found in all environments, fresh, marine and brackish water, sewage, moist soil and associated with animals and plants. They have (symbiotic) relationships with animals or plants. These relationships may be mutualistic (both partners benefit), commensalistic (one benefits, while the other is neither helped nor harmed) or parasitic (the parasite benefits; the host is harmed). In fact, some of the most important diseases of humans and domestic animals are caused by parasitic protozoans!

4

We examine members of the Protozoa because though most of them are unicellular, they can carry out all of the basic life processes in the housing of a single cell. This means everything from cellular respiration to reproduction occurs within a single cell. An observation you want to make during lab is the relative size of the cells of protozoans. Some will be quite large, whereas others will be small. Think about what the minimum size is for a cell and it can still carry out all life processes. Next, what is it about the protozoa that have small cell size? Have they given up some life processes for smaller size or have they acquired something that allows for smaller size. In later labs, compare the cell size of protozoans to the cells of multicellular eukaryotes. Another topic of observation in this lab exercise is the complexity and diversity of the Protozoa. Note protistan systematics (Table 1). This is a complex group despite their apparently unicellular simplicity. Table 1. Current systematics of the Protista with representative genera (only representatives from phyla highlighted blue are available in lab) and main characteristics. Phylum General Information Representative Genera Euglenida 1,000 species

Euglenids formerly Sarcomastigophora generally noncolonial, pellicle, may be autotrophs or heterotrophs

Euglena, Phacus

Kinetoplastida 600 species Trypanosomes and kin formerly Sarcomastigophora free living, pellicle, 2 flagallae, no plastids

Trypanosoma, Leptomonas

Ciliophora 12,000 species The Ciliates Pellicle of alveolar vesicles, ciliation, sexual reproduction

Didinium, Paramecium, Stentor, Tetrahymena

Apicomplexa 5,000 species Gregarines, coccidians, heamosporidians and piroplasms Pellicle of alveolar vesicles, gliding locomotion, absence of cilia, flagellae, gametic sexual reproduction

Cryptosporidia, Plasmodium

Dinoflagellata 4,000 species Dinoflagellates formerly Sarcomastigophora cellulose theca, two flagellae, entirely photosynthetic

Amphidinium, Ceratium, Zooxanthella

Stramenopila (Heterokontophyta) 9,000 species Diatoms, brown algae, golden algae, etc. Plasma membrane, 2 flagellae in some lifecycle stages

Fucus

Rhizopoda 200 species Amoebans and kin formerly Sarcomastigophora

Amoeba, Chaos

5

plasma membrane, pseudopodia Actinopoda 4,240 species

polycystines formerly Sarcomastigophora locomotion mostly passive, plasma membrane

Actinosphaerium

Granuloreticulosa 40,000 species Foraminiferans formerly Sarcomastigophora test outside of plasma membrane

Rhizoplasma

Diplomonadida 100 species Diplomonads formerly Sarcomastigophora various number of flagellae, 2 nuclei

Enteromonas

Parabasilida 300 species Hypermastigotes and trichomonads formerly Sarcomastigophora flagellae (4-thousands)

Trichonympha, Trichomonas

Cryptomonada Cryptomonads formerly Sarcomastigophora biflagellated, cell surface of proteinaceous plates

Chilomonas

Microspora 800 species Microsporans Intracellular parasites in all animal phyla

Nosema

Ascetospora Ascetosporans Exclusively parasitic

Haplosporidium

Choanoflagellata Choanoflagellates formerly Sarcomastigophora stalked colonies

Monosiga

Chlorophyta The green algae Photosynthetic

Chlamydomonas, Eudorina

Opalinida Opalinids formerly Sarcomastigophora homokaryotic (identical nuclei)

Opalina

Note the magnification you are using to observe protozoans and draw an image that reflects what you are seeing using the field of view to provide perspective of scale (hint: the 40X or 100X objective will provide the best view on most, although 10X might be sufficient for the larger taxa). Always start with the scanning (4X objective) when first trying to find something on a glass slide. I. Phylum Euglenida – Movement through 2 flagellae (one too small to visualize), pellicle, may be photosynthetic 1. Euglena, a unicellular free-living organism found in freshwater or marine environments. A) Prepared slides – Find the single flagellum? What is the problem with flagellae in microscopic preparations?

6

Identify the nucleus? Where is the nucleus found compared to the flagellum? Can you find any other organelles, such as the contractile vacuoles, the stigma (eyespot, light sensitive structure) etc.?

B) Live specimens = make a wet mount by carefully placing 1 drop of solution (you should stir the Euglena in solution prior to use) onto a clean slide and carefully covering it with a clean coverslip (Hint: carefully lower the coverslip or else you will produce too many air bubble. Observe the motion of the organism without any interference but if specimens of Euglena are too fast to properly observe, add a drop of Protoslo® in order to view flagellar motion, organelles, coloration etc. Notice the coloration of live organisms. Are these organisms heterotrophic or autotrophic? What pigment produces the color of the organism? How is the color an indicator of their lifestyle? Find the eyespot or stigma. How does its color differ from that of the rest of the cell? What pigments are responsible for the production of the color? Find the flagellum. Describe the flagellar motion! Where is the flagellum located (anterior or posterior location)? What does that tell you about overall movement? II. Phylum Kinetoplastida - movement through 2 flagellae, no photosynthesis 1. Trypanosoma, a blood parasite responsible for African Trypanosomiasis (sleeping disease), which is spread by Tsetse, flies. Have a long flagellum attached by an undulating membrane to achieve motion. Slide is a blood smear so you’ll see many red blood cells or erythrocytes and occasional white blood cells or leucocytes; you will need high magnification to view prepared slides! Something to think about: Sleeping sickness is always fatal without treatment! There is currently no preventative vaccine or drug treatment and travelers going to Africa should be extra careful to avoid Tsetse fly bites.

7

A) Prepared slides - Find the flagellum? Can you observe the undulating membrane that connects them without observing live specimens? Can you localize the nucleus? What is the overall size? Hint: Compare the parasites to the blood cells (erythrocytes = 6-8µm; leucocytes 8-18µm) III. Phylum Ciliophora (Ciliata) This large and diverse group includes some of the most complex protozoans known such as Paramecium, Stentor, Spirostomum and Vorticella. Locomotion is always by cilia, and all forms are multinucleate, having at least one macronucleus (responsible for metabolic and developmental functions of the cell) and one or more micronuclei that are involved in sexual reproduction). Most are holozoic but a few forms are parasitic and cause damage to their hosts, including humans. Several parasitic species can cause serious problems for aquarium fish and fish in farm enclosures. In addition to a number of complex organelles, many ciliates have a sculptured, rigid outer covering called a pellicle. Embedded in the pellicle are the cilia plus a number of thread-like structures called trichocysts. Upon mechanical or chemical stimulation, these trichocysts can be discharged to produce long, sticky protein threads that remain attached to the organism. Although the function of these structures is probably defensive, it has been hard to demonstrate this. 1. Paramecium - The paramecium genus of protozoa of the phylum Ciliophora, is often called slipper animalcules because of their slipper-like shape. Paramecia are unicellular organisms usually less than 0.25 mm (0.01 in) in length and covered with minute hair-like projections called cilia. Cilia are used in locomotion and during feeding. When moving through the water, paramecia follow a spiral path while rotating on the long axis. When a paramecium encounters an obstacle, it exhibits the so-called avoidance reaction: It backs away at an angle and starts off in a new direction. Paramecia feed mostly on bacteria, which are driven into the gullet by the cilia. Two contractile vacuoles regulate osmotic pressure and also serve as excretory structures. A paramecium has a large nucleus called a macronucleus, without which it cannot survive, and one or two small nuclei called micronuclei, without which it cannot reproduce sexually. The pellicle, a stiff but elastic membrane that gives the paramecium a definite shape but allows some small changes. Covering the pellicle are many tiny hairs, called cilia. On the side beginning near the front end and continuing half way down is the oral groove. The rear opening is called the anal pore. The contractile vacuole and the radiating canals are also found on the outside of a paramecium. Inside the paramecium cell contains the cytoplasm, food vacuoles, the trichocyst, the macronucleus, and the micronucleus. Reproduction is usually asexual by transverse binary fission, occasionally sexual by conjugation, and rarely by endomixis, a process involving total nuclear reorganization of individual organisms. Macronuclear DNA in Paramecium has a very high gene density. The macronucleus can contain up to 800 copies of each gene. Paramecia most abundant in freshwater ponds throughout the world; one species lives in marine waters. They are easily cultivated in the laboratory by allowing vegetable matter to stand in water for a few days. The common species Paramecium caudatum is widely used in research. A) Prepared slides - Compare the overall size to the other prepared specimens you have observed (Hint: You do not need to “measure” the specimens but should take note of the magnification used and how much of the field of view is by any given specimen) Find organelles such as the macronucleus and check for the presence of food vacuoles. Are these organisms autotrophic or heterotrophic? How can you tell? B) Live specimens – Observe the locomotion of these organisms. How do they compare to the locomotion and overall directionality of movement of the remainder of the protozoans you observed live? Can you observe any feeding structures? 2. Tetrahymena - A ciliated single-celled protozoan, Tetrahymena is a freshwater organism that inhabits

8

streams, lakes, and ponds and can be found almost everywhere, in a range of climates. The cells are large (40-50 um) and their complexity rivals human cells, making them a good alternative to human tissues. The cells are inexpensive to grow, requiring little more than a shaker, and they grow rapidly to high density in a variety of media and conditions. Tetrahymena possesses many core processes conserved across a wide diversity of eukaryotes (including humans) that are not found in other single-celled model systems (such as yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe). Conventional tools for genetic analysis, and molecular genetic tools for experimental analysis of gene function have been developed for use in Tetrahymena.

A) Live cultures – Prepare and examine a wet mount. You should be able to find the macronucleus and the vacuoles without a problem. Vacuoles can be digestive and contractile to determine which ones are which, try to “feed” india ink (if available in lab) to the organisms by adding particles to a slide. Tetrahymena will phagocytize the particles of india ink which will allow you to determine between different vacuole types and potentially visualize the oral apparatus. Viewing Tetrahymena und oil magnification will allow you to see the ciliary beat. 3. Didinium – Didinium are from 80-200 um long, fast moving carnivorous protozoans that feed almost exclusively on live Paramecium. When its oral cone strikes a Paramecium it latches on with a threadlike trichocyst. Once captured and paralyzed, Didinium devours the Paramecium whole. The "C"-shaped structure inside the body is a band shaped nucleus and can sometimes be seen with a regular light microscope. Didinium will encyst when the food source is depleted and excyst when the food returns.

A) Live cultures – Prepare and examine a wet mount and observe the ciliation which is very different compared to Paramecium and Tetrahymena. Find the C-shaped nucleus, the oral cone and the vacuoles. Notice that Didinium generally has fewer vacuoles than the other ciliates. Add Didinium to a wet mount of Paramecium and view and document the feeding process. IV. Phylum Dinoflagellata

9

Dinoflagellates are marine phytoplankton that bloom to produce red tides, some contains toxins responsible for fish kills and some are bioluminescent.

How do dinoflagellates differ other protozoans in terms of their overall shape, flagellae, cell structure and physiology? V. Phylum Rhizopoda These are the transparent shape shifters. They move and gather food using foot-like cytoplasmic extensions called pseudopodia (pseudopodium = singular). These are not true “feet” but simply extensions of the cytoplasm. 1. Amoeba proteus - Amoeba proteus is a large protozoan with an ever-changing shape and is approximately 500-1000 um long and may almost be seen with the naked eye. Amoeba proteus is the classic specimen used in the classroom to demonstrate the pseudopods in action. They can sense light and tends to move away from it. Just before they reproduce (asexually by fission), it rounds up into a ball with tiny pseudopodia extensions.

A) Prepared slides - Compare the overall size to the other prepared specimens you have observed (Hint: You do not need to “measure” the specimens but should take note of the magnification used and how much of the field of view is by any given specimen) Find the pseudopodia, the nucleus and the food vacuoles. What is the function of the food vacuoles? B) Live Specimens –You will again be required to make a wet mount (see above). If the life specimens are too fast, you may again want to use a drop of Protoslo® to slow them down. VI. Phylum Actinopoda All with axopodia; pseudopodia with microtubular cores; elaborate endoskeletal systems generally present; tubular mitochondrial cristae; complex central capsule characteristic of many; primarily marine. 1. Actinosphaerium - Actinosphaerium is a protist (protozoan) and looks like a sea urchin and is from 200-1000 micrometers wide, which is quite large for a protists. The image below shows a close up of the

10

endoplasm and ectoplasm, the water expelling vesicles (WEV) and how this protists uses the radial arms to move by flowing the protoplasm into the arms. These are also called Heliozoans, or "Sun Animals". The body is spherical with stiff unbranched arms radiating in all directions.

A) Live specimens – Prepare and examine a wet mount. Find the axopodia, which unlike pseudopodia in Amoeba proteus are not always associated with locomotion. Locomotions in the actinopoda is generally passive. If the axopodia are present, what may their function be? In addition, Actinosphaerium is generally divided into endo and ectoplasm. Can you distinguish between the two? Find the water expelling vesicles and determine their function (other than the obvious). 2. Radiolarians - Radiolaria can range anywhere from 30 microns to 2 mm in diameter and contain a skeleton composed of silica. Just like all other actinopoda their skeletons are penetrated by arm-like cytoplasmic extensions that resemble spikes, the axopodia, which are used both to increase surface area for buoyancy and to capture prey. Most radiolarians are planktonic, and get around by coasting along ocean currents. Most are somewhat spherical, but there exist a wide variety of shapes, including cone-like and tetrahedral forms (see the image above). Besides their diversity of form, radiolarians also exhibit a wide variety of behaviors. They can reproduce sexually or asexually; they may be filter feeders or predators; and may even participate in symbiotic relations with unicellular algae.

A) Prepared slide of radiolarian tests – Examine the prepared slide, which will contain only the radiolarian test composed of silica, not longer any cellular materials. Notice the intricate lattice shape of the tests as well as their different shapes. Some tests may also contain silica spines (picture above left), not to be confused with axopodia. VII. Phylum Parabasilida The parabasalids are a group of flagellated protists within the supergroup Excavata. Most of these organisms form a symbiotic relationship in animals; these include a variety of forms found in the intestines of termites and cockroaches, many of which have symbiotic bacteria that help them digest cellulose in woody plants. Other species within this supergroup are known parasites, and include human pathogens.

11

1. Trichonympha Termites, of course, are famous as the organisms that eat wood - often the wood in your house. However, termites could not feed on wood without the help of symbiotic protists living in their guts. These protists take in wood particles: in the picture of Trichonympha below, in the lower portion of the cell, you can see a mass of granular material that is in fact wood particles being digested. (The actual cell is about 300 microns long.) Trichonympha is only one of several such protists found in the guts of termites. A) Prepared slide – Examine the slide of Trichonympha. Find the large nucleus and the location where flagellae may be attached. Can you see any food particles inside the unicellular organisms?