Practical 1 - TSV

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Prac 1 – Microscopes and Unicellular Life– Student Manual Practical 1 – Microscopes and Unicellular Life Pre-laboratory work: Please read this manual and Chapters 4–4.7, 27.1–27.5 and 28. Watch the video on Working Effectively in Groups. Download the Field Assignment sheet, Teamwork Checklist and Project Planning sheet from LearnJCU. Ensure you wear enclosed shoes . Practical 1’s focus on the subject learning outcomes: • Demonstrate practical skills • Practice observation, recording, evaluating and reporting of scientific information Objectives of today’s practical session: • Learn about Workplace Health and Safety Laboratory rules at JCU • Lean how to work effectively in groups • Learn how to use a microscope correctly, and construct a scale bar • Examine Protists from three different habitats (freshwater, saltwater and inside an organism) • Examine different types of locomotion in Protists • Examine Prokaryotic and Eukaryotic cells and understand similarities and differences • Explore key characteristics that differentiate Protist phyla, including: ➢ cell surface arrangement ➢ interior arrangement of some Protists ➢ means of locomotion Workplace Health and Safety Laboratory rules at JCU [10 minutes] The University staff, management and students have mutual obligations in relation to maintaining a safe workplace and learning environment. Students are required to follow directions specific to sites and safety procedures as directed by staff. Failure to follow safety instructions may result in that person being denied access to the facility. SECTION 1: WORKING EFFECTIVELY IN GROUPS Exercise 1: Meet your lab and group project team [10 minutes] The people you are seated with today will be your lab and field assignment team throughout semester. Aside from coming to lectures and practicals, the very best thing you can do for your success in first year is make friends with the people you are studying with. You will be working with 3-4 people: ➢ Swap names and contact details ➢ Spend a few minutes discussing your strengths and what you each think you can bring to the team. 1

Prac 1 – Microscopes and Unicellular Life– Student Manual ➢ Spend a few minutes discussing your weaknesses and how this might affect the team. ➢ Set up your first meeting some time this week. In this meeting, you will need to read through the field assignment, and then plan how you will complete the assignment as a team. You will be required to bring the completed Teamwork Checklist and Project Planning sheet to the practical next week on the 6 th March 2023. SECTION 2: MICROSCOPES AND HOW TO USE THEM Exercise 1. Microscope mastery [10 minutes - guided] Introduction In this practical, you will learn how to use one of the most valuable tools in the biologist’s toolbox: a microscope. Follow along with your demonstrator, take notes and tick off sections as we progress. Ensure you know the various parts of the compound microscope and their function. Although not covered today, we will use a stereo microscope in future sessions. Learning Objectives: • Identify and understand functions of selected microscope parts • Learn proper care and handling techniques of the microscope • Become familiar with field of view and magnification • Learn how to construct a scale bar Table 1. Parts of a microscope and their function (See Figure 1 on the next page) Microscope parts Function Base Broad and heavy and supports the microscope Body Houses the prisms, eyepiece tube (ocular), and lenses Arm Supports the body tube and lenses. See “Correct handling of the microscope” on the next page. Revolving Nose Piece Located at the lower end of the body tube. It contains the objective lenses and can be rotated to select different magnifications. Eyepiece/Ocular What you look through to see the specimen. It magnifies the object 10 times . Objective lenses Located on the revolving nosepiece. The objective lenses vary in magnification from 4x to 10x to 40x . The selected lens is rotated into position by turning the nosepiece. Stage The horizontal platform where the microscope slide is placed. It moves up and down when you move the focus knobs. Condenser Lens Beneath the stage. It concentrates light before it passes through the specimen to be viewed. Iris diaphragm Regulates the amount of light passing through the specimen Light Provides illumination of the specimen Mechanical stage Small circular knobs adjacent to or below the stage. Allow the observer to move the slide across the stage either forwards, backwards or laterally. Coarse adjustment knob Located on either side of the body. Moves the stage up or down to 2

Prac 1 – Microscopes and Unicellular Life– Student Manual bring the specimen into focus. It should only be used when using the low powered objective . Fine adjustment knob Located within the coarse adjustment knob. Moves the stage up or down small distances. Allows fine focus of the specimen. Slide adjustment knob Allows you to move the slide up and down and from side to side Figure 1. Labelled diagram of a high-power (compound) microscope Correct handling of the microscope: Ensure you know the correct way to carry and set up the microscope. Tick each box as you complete each step.  Always carry the microscope with both hands. Grasp the body of the microscope with one hand and place your other hand under the base. Do not drag it along the bench.  Set the microscope on the bench with the stage facing you. Plug the microscope cord into the electrical outlet and turn the microscope on.  Ensure that the condenser is in the highest position (adjacent to the stage). The condenser gathers light from the light source and concentrates it into a cone of light that illuminates the specimen.  While looking down the microscope, adjust the light levels and contrast with the iris diaphragm. Engage the objective to be used in the light path.  Place the slide onto the stage, coverslip up.  Find the specimen using the slide adjustment knob (moves the slide laterally, and up and down).  Always begin viewing the slide using the low (4 x) objective lens. Focus using the coarse adjustment knob first, and then use the fine adjustment knob. 3

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Prac 1 – Microscopes and Unicellular Life– Student Manual  Adjust the distance between the eyepieces to suit your eyes (eyepieces move in and out laterally). When you have finished with the microscope:  Turn off the light.  Ensure that the lowest power objective is clicked into position.  Raise the stage to its highest point.  Remove the last slide from the stage.  Wipe any water or other materials from the stage.  Neatly wrap the electrical cord around the microscope, and place the plastic cover over the microscope (if there is one available).  Return the microscope to the cupboard where you found it. This is good lab etiquette and is very important to practice in every lab class. Exercise 2. Explore the field of view [10 minutes] Field of view The field of view (Figure 2) is the diameter of the circle of light that you see when looking into a microscope. As magnification increases (goes from 4 x to 100 x), the field of view gets smaller . Figure 2. Image of field of view from https://www.microscopeworld.com/t microscope_field_of_view.aspx Magnification The compound microscopes we use have two lenses: a lens in the eyepiece, and an objective lens. Combining two lenses creates higher magnifications. The total magnification of the microscope is equal to the magnification of the eyepiece (x10) multiplied by the magnification of the objective. Q1. What are the magnifications on the objectives of your microscope? Objective lens Magnification a. b. c. Q2. Calculate the total magnification of your microscope when using the 10 x eyepiece with each of the three objectives. 4

Prac 1 – Microscopes and Unicellular Life– Student Manual Objective lens magnification Eyepiece magnification Total magnification a. b. c. Following your demonstrator’s instructions, collect a sample of Amoeba sp . and place it on a slide for use in this exercise. Keep your Amoeba for the next exercise. The circles below represent the field of view (FOV) in a microscope. Examine an Amoeba sp. under 4 x, 10 x and 40 x magnification and, in the circles below, draw the approximate size of the Amoeba sp. under these various magnifications. Q3. How much of the FOV does the Amoeba sp. occupy at each magnification? 4 x 10 x 40 x Q4. Explain what happens to the size of the Amoeba sp. when you change from 4 x to 10 x magnification. Exercise 3. Scientific drawing of Amoeba sp. [10 minutes] In the space provided on the next page, complete a biological diagram of an Amoeba sp. Follow the guidelines below. Guidelines for scientific drawings Title: Must be as informative as possible (e.g. Whole mount of… / transverse section of…) and must have a figure caption. The title comes below the figure. Style: Pencil. Do not shade or colour in, and use solid lines. Classification: Place the full classification in the top left corner. Genus and species names must be underlined if hand-written or italicized if typed. The genus name is capitalised, while the species name is not capitalised. 5

Prac 1 – Microscopes and Unicellular Life– Student Manual Size: Should be approx. half an A4 page (easier to label and see detail). Labels: Use a ruler, do not cross lines, and identify all relevant features (see list below). Biological notes (annotations): Observations (e.g. colour, how did it move) of relevant biological information. Placed next to the label. Scale bar: see next exercise and add to your drawing List of terms: Plasma membrane: The outer cell surface. Protects the organism from the outside environment. Ectoplasm: The clear outer region of the protoplasm. The colourless gelatinous material, including the cytoplasm, comprising the living part of a cell. Endoplasm: The inner, granular protoplasm. The more fluid, granular inner layer of the cytoplasm. Nucleus: The large, round, dark structure in the endoplasm. Functions in cellular control. Contractile vacuole: Transparent vesicle that excretes excess water and wastes. Food vacuoles: Cavities in which food is digested. Keep your Amoeba sp. for the next exercise. Ensure you turn the light off in between each exercise. Why might you need to do this? ________________________________________________________ Exercise 4. Construct a scale bar for your drawing [10 minutes] A scale bar is a comparison of the actual organism and your drawn image. It gives your viewer a reliable estimate of the real-life size of your specimen, regardless of how large you have drawn your 6

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Prac 1 – Microscopes and Unicellular Life– Student Manual image or what magnification you used. It is very simple once you understand, but can be tricky to grasp! Table 2. Estimated field of view (mm) for different object lenses of a compound light microscope Ocular Objective Approx. field of view (mm)* 10 x 4 x 4.5 10 x 1.85 40 x 0.45 * You can ask a demonstrator for a slide ruler so that you can see these for yourself. 𝐅𝐢𝐞𝐥𝐝 𝐨𝐟 𝐯𝐢𝐞𝐰 (𝐦𝐦) 𝐒𝐢𝐳𝐞 𝐨𝐟 𝐬𝐩𝐞𝐜𝐢𝐦𝐞𝐧 (𝐦𝐦) = # 𝐭𝐢𝐦𝐞𝐬 𝐬𝐩𝐞𝐜𝐢𝐦𝐞𝐧 𝐟𝐢𝐭𝐬 𝐚𝐜𝐫𝐨𝐬𝐬 𝐭𝐡𝐚𝐭 𝐯𝐢𝐞𝐰 (Equation 1) 𝑺𝒊𝒛𝒆 𝒐𝒇 𝒔𝒑𝒆𝒄𝒊𝒎𝒆𝒏 (𝒎𝒎)𝒙 𝒅𝒆𝒔𝒊𝒓𝒆𝒅 𝒍𝒆𝒏𝒈𝒕𝒉 𝒐𝒇 𝒔𝒄𝒂𝒍𝒆 𝒃𝒂𝒓 (𝒎𝒎) 𝑺𝒄𝒂𝒍𝒆 𝒃𝒂𝒓 = 𝑳𝒆𝒏𝒈𝒕𝒉 𝒐𝒇 𝒚𝒐𝒖𝒓 𝒅𝒓𝒂𝒘𝒊𝒏𝒈 (𝒎𝒎) (Equation 2) Example 1. If you drew your Amoeba sp. under the 10 x objective, the field of view for that magnification is 1.85mm (see Table 2). 2. On an appropriate magnification, determine approximately how many times the specimen will fit across the field of view, if you multiplied it or laid it end to end. 3. Insert the numbers into the first equation: Size of specimen = 1.85/4 = 0.4625 4. Measure your drawing along the longest edge – i.e. ‘nose to tail’. Let us say it is 150 mm. 5. Pick a size for your scale bar. 10 mm, 20 mm or 25 mm are the usual sizes. 6. If you would like your scale bar to be 20 mm long, insert the numbers into the second equation: Scale bar = (0.4625 x 20)/150 = 0.061 7. Draw a 20 mm line underneath your diagram, ended with bars, and write your final calculated number above it: Following the above steps, calculate the scale bar for your drawing. SECTION 3: STUDYING PROTISTS 7

Prac 1 – Microscopes and Unicellular Life– Student Manual Kingdom Protista Protists are a diverse group of organisms, comprising those Eukaryotes that cannot be classified in any of the other kingdoms (fungi, animals, or plants). The Protists are a paraphyletic clade, rather than a natural (monophyletic) group, and do not have much in common besides a relatively simple organization (unicellular, or multicellular without highly specialized tissues). Essentially, the Kingdom Protista comprises organisms that cannot be classified into any other Kingdom. Although found wherever life exists, they always require moisture, which restricts them to a narrow range of environmental conditions in freshwater or marine habitats, the soil, decaying organic matter or inside the bodies of plants and animals. In this section of the practical, we will be examining Protists from three habitats: • Freshwater habitats - Amoeba sp. , Euglena sp. and Paramecium sp. • Marine habitats - Foraminifera (“forams”) and Radiolaria (radiolarians) • Living inside other organisms - Trichonympha sp. and Babesia sp. Some Protists have traditionally been treated as animals, as they are able to move and are heterotrophic. In this practical, we will be examining three types of locomotion in a number of Protists: • Pseudopodia • Flagella • Cilia Q1. Protists were the first eukaryotes. Briefly explain the endosymbiosis theory The endosymbiosis theory explains how eukaryotic cells, including the first protists, evolved from simpler prokaryotic cells. The theory suggests that mitochondria and chloroplasts were once free-living prokaryotes that were engulfed by other cells, forming a symbiotic relationship. Over time, the host cell and the endosymbiont became dependent on each other, and the endosymbiont evolved to become a specialized organelle within the host cell. Various lines of evidence support the endosymbiosis theory, including the fact that mitochondria and chloroplasts have their own DNA and replicate independently of the host cell. Protists have a range of feeding strategies: • They can be heterotrophic (eat other organisms) or autotrophic (produce their own food) or both (e.g. Euglena sp. ) 8

Prac 1 – Microscopes and Unicellular Life– Student Manual • Among the heterotrophs, some are phagotrophs . They ingest particles of food by pulling them into intracellular vesicles called food vacuoles or phagosomes. Lysosomes then fuse with the food vacuoles, introducing enzymes that digest the food particles in the vesicles. Digested molecules are absorbed across the vacuolar membrane and distributed throughout the organism. Exercise 1. Freshwater protists: Amoeba sp. , Euglenia sp. and Paramecium sp. [20 minutes] 1. Amoeba sp. Amoeba are single celled Protists. Some protozoan’s move by mobile extensions called pseudopodia ("false feet") that result from the flowing of cytoplasm within them, allowing them to creep along the substratum. Protozoans that form pseudopodia have two types of cytoplasm, an outer, more viscous portion called the ectoplasm , and an inner, more fluid portion called the endoplasm . When a pseudopodium begins to form, a clear space at the leading edge of the pseudopodium, called the hyaline cap , appears. After this occurs, endoplasm begins to flow into this space, causing the pseudopodium to be pushed forward through the medium. Obtain an Amoeba sp. and place it on a microscope slide. Examine your Amoeba sp. under low power 4 x magnification. Change to the 10 x objective and examine your specimen again. Observe its constantly changing shape with movements of pseudopodia and streaming of the cell contents. Identify the different structures, and label the drawing of Amoeba sp. below (and your earlier drawing if you need), using the terms provided on Page 5. 9

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Prac 1 – Microscopes and Unicellular Life– Student Manual Q1. Does this diagram meet the criteria for a good scientific drawing? If so, why? If not, why not? Title: Must be as informative as possible (e.g. Whole mount of… / transverse section of…) and must have a figure caption. The title comes below the figure. Style: Pencil. Do not shade or colour in, and use solid lines. Classification: Place the full classification in the top left corner. Genus and species names must be underlined if hand-written or italicized if typed. The genus name is capitalised, while the species name is not capitalised. Size: Should be approx. half an A4 page (easier to label and see detail). Labels: Use a ruler, do not cross lines, and identify all relevant features (see list below). Biological notes (annotations): Observations (e.g. colour, how did it move) of relevant biological information. Placed next to the label. Scale bar: see next exercise and add to your drawing Q2. What is the function of the contractile vacuole? Why are contractile vacuoles important to the Amoeba sp. ? the contractile vacuole plays a vital role in regulating water balance and preventing the cell from bursting due to osmotic pressure. This is particularly important for single-celled organisms like Amoeba sp., which live in freshwater environments and rely on the contractile vacuole to maintain a stable internal environment. 10

Prac 1 – Microscopes and Unicellular Life– Student Manual Q3. Amoeba sp. consists of how many cells? How does it respire? Amoeba sp. is a single-celled organism, which means it is made up of just one cell. Amoeba sp. respires aerobically, meaning it requires oxygen to carry out cellular respiration and generate energy. The oxygen is absorbed through the cell membrane and into the cell. Q4. Describe the locomotion of Amoeba sp . How can it move if it does not have any muscles? In your description, use words such as pseudopod, hyaline cap, and endoplasmic streaming. Amoeba sp. moves using pseudopods, temporary extensions of the cell membrane and cytoplasm that allow the cell to move and change shape. The process involves the formation of a hyaline cap, which anchors the pseudopod to a surface, and endoplasmic streaming, which pulls the rest of the cell along the pseudopod. Q5. Describe how Amoeba sp. feeds. You will need to do a little research! Amoeba sp. feeds by engulfing its food through phagocytosis, forming a food vacuole that is brought into the cell's interior and fused with a lysosome. Digestive enzymes in the lysosome break down the food into smaller molecules, which are absorbed into the cell and used for energy or to build new cell structures. Undigested waste material is eliminated from the cell through exocytosis. During exocytosis, the undigested waste material is packaged into a vesicle and transported to the cell membrane. The vesicle then fuses with the cell membrane, releasing the waste material outside the cell. 11

Prac 1 – Microscopes and Unicellular Life– Student Manual Q6. Given that this is a single celled organism, list the various life functions carried out by this simple organism. 1. Nutrition: Amoeba sp. feeds on other organisms through phagocytosis, breaking down food molecules into smaller units for energy and growth. 2. Respiration: Amoeba sp. respires aerobically by taking in oxygen through the cell membrane and releasing carbon dioxide as a waste product. 3. Transport: Nutrients and waste materials are transported within the cell through the process of endoplasmic streaming. 4. Excretion: Undigested waste materials are eliminated from the cell through exocytosis. 5. Response to stimuli: Amoeba sp. can respond to stimuli in its environment, such as changes in light or temperature, by changing its movement or behavior. 6. Reproduction: Amoeba sp. reproduces asexually by binary fission, dividing into two identical daughter cells. 7. Homeostasis: Amoeba sp. maintains a stable internal environment by regulating its internal conditions, such as pH and temperature. 2. Euglenia sp. (Phylum Euglenozoa) Euglena is a common genus of freshwater unicellular organisms in the Kingdom Protista. They have chloroplasts and can make their own food by photosynthesis. They are not completely autotrophic, however, as they can also absorb food from their environment. Some species move using flagella to pull them through the medium. Euglena have both plant (chloroplasts) and animal features (flagella). Place a Euglena sp. on a clean slide, but do not apply a cover slip. Examine and observe the spiralling movement of the organism. Now add a drop of methyl cellulose, apply a cover slip and re-examine. Q1. What does the methyl cellulose do? Why does it have this effect? Methyl cellulose is a commonly used ingredient in the food, pharmaceutical, and cosmetic industries as a thickening agent, emulsifier, and stabilizer. It works by absorbing water and swelling, which increases its volume and viscosity, creating a more substantial texture without altering taste or adding calories. It also acts as a stabilizer, keeping ingredients from separating or settling. Methyl cellulose is a viscous substance that can be added to a sample to slow down the movement of organisms and provide a more stable observation under a microscope. Methyl cellulose is a type of 12

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Prac 1 – Microscopes and Unicellular Life– Student Manual polymer that is soluble in water and has a high molecular weight. When added to a sample, it increases the viscosity of the surrounding medium, which creates a thickened, more gel-like consistency. This thickened consistency slows down the movement of organisms in the sample, including Euglena, which makes them easier to observe under a microscope. In summary, the addition of methyl cellulose increases the viscosity of the surrounding medium, which slows down the movement of the Euglena, making it easier to observe its characteristics and behaviors under the microscope. Using the live specimen, identify and label the following on the diagram below: Pellicle: The flexible outer covering. Flexibility allows them to change shape. Flagellum: A long, whip-like strand. Functions in locomotion. Reservoir: A depression at the base of the flagellum used for storage. Stigma: A light-sensitive structure (the dark organelle near base of flagellum) used to orientate. Chloroplasts: Green bodies containing chlorophyll. Used to produce food. Nucleus: Contains the cell's DNA and controls the cell's activities. Contractile vacuole: A clear organelle located near the stigma. Functions to remove excess water from inside the organism, and empties it into the reservoir. 13

Prac 1 – Microscopes and Unicellular Life– Student Manual Q2. How does Euglena sp. obtain food? You will need to do a little research! Euglena sp. can obtain food through photosynthesis. It has chloroplasts that can capture light energy and convert it into chemical energy in the form of glucose. This process occurs in the presence of sunlight and involves the absorption of carbon dioxide and the release of oxygen. Euglena sp. can also obtain food through phagocytosis, which involves engulfing small particles or organisms. Euglena sp. has a specialized structure called a stigma or eyespot, which allows it to detect the presence of light and move towards it. This same structure can also detect the presence of food particles, such as bacteria or other protists, which the Euglena sp. can then engulf using its phagocytic mechanism. Q3. What is the function of the stigma, and how is this related to the presence of chloroplasts? The stigma in Euglena sp. helps the organism to detect and move towards or away from light, a process known as phototaxis. The presence of chloroplasts in Euglena sp. is related to the function of the stigma as it helps to position the organism for optimal light absorption, maximizing photosynthesis efficiency, which is the primary means of obtaining energy and producing food. Additionally, the stigma can detect food particles, allowing Euglena sp. to engulf them using its 14

Prac 1 – Microscopes and Unicellular Life– Student Manual phagocytic mechanism, making it a crucial component of the organism's sensory system for locating light and food sources and ensuring its survival. Q4. What are the animal characteristics of Euglena sp. ? Euglena sp. exhibits several animal-like characteristics, including mobility, heterotrophy, lack of a cell wall, the presence of contractile vacuoles, and phagocytic feeding. However, it is primarily considered a photosynthetic organism, like plants. Heterotrophy: While Euglena sp. primarily obtains its energy through photosynthesis, it can also behave heterotrophically, meaning it can ingest and digest other organisms to obtain nutrients, like an animal. 3. Paramecium caudatum (Phylum Alveolata) Paramecium is a common genus of largely freshwater (only one marine species) unicellular organisms in the Kingdom Protista. They are covered with minute hair--like projections called cilia . Cilia are used in locomotion and during feeding. The fastest protozoans move by cilia, which are short, hair-shaped organelles embedded in a protein coat called the pellicle . The pellicle provides a semi-rigid structure that helps to transmit the force of their beating along the entire surface of the protozoan. Paramecium have two nuclei, a macronucleus and a micronucleus. The macronucleus operates the cell's activities, while the micronucleus is used for reproduction. Place a few strands of cotton wool on a clean slide, making sure that the strands do not cross. Add a drop of Paramecium caudatum culture to the slide. Apply a cover slip and examine under low power. Try to follow one individual as it moves about. Q1. What happens when the organism encounters an obstacle, i.e. a strand of cotton wool? Does it seem to learn from the experience? When Paramecium caudatum encounters an obstacle, such as a strand of cotton wool, it typically responds by changing its direction of movement. Paramecium has cilia, which are hair-like structures that cover its surface and help it to move through its environment. When it encounters an obstacle, the cilia on one side of the organism's body stop beating, while the cilia on the other side continue to beat, causing the organism to turn and swim in a new direction. Paramecium caudatum does not have a central nervous system, and so it does not have the capacity to learn from its experiences in the same way that more complex organisms do. It can, however, adapt its behavior to better navigate its environment over time. For example, if Paramecium encounters an obstacle multiple times, it may learn to avoid that area altogether or adjust its swimming behavior to avoid the obstacle more effectively. This adaptation is not due to conscious learning or decision-making but rather a process of trial and error, where the organism's behavior is modified based on its past experiences. 15

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Prac 1 – Microscopes and Unicellular Life– Student Manual Add a drop of methyl cellulose to a fresh drop of Paramecium caudatum culture on a clean slide and apply a cover slip. Observe the beating cilia. Q2. Are their movements of Paramecium caudatum coordinated? Q3. Describe how Paramecium caudatum feeds. You will need to do a little research! Overall, Paramecium caudatum uses its cilia to create a feeding current that brings food particles into contact with the oral groove. The organism then takes up the food particle, encloses it in a food vacuole, and digests it using lysosomal enzymes. Any waste products are eliminated through the anal pore. Examine a live Paramecium caudatum under a microscope. Identify and label the diagram on the next page: Pellicle: A thin, firm, elastic and colourless cuticular membrane. Ectoplasm: The outer layer of the protoplasm Endoplasm: The inner, granular layer of the protoplasm. Oral groove: Funnels food towards mouth; Food vacuoles: Small circular organelles used for ingesting food. Contractile vacuole: Small circular organelles used for regulating water balance. Micronucleus: For reproduction. Macronucleus: Control centre of the cell. Used for day-to-day functioning (main metabolic activities). 16

Prac 1 – Microscopes and Unicellular Life– Student Manual Exercise 2. Marine protists: Foraminifera and Radiolaria [10 minutes] 1. Foraminifera (Phylum Retaria) “Forams” are amoeboid marine protozoa that secrete a calcareous, many-chambered test in which to live, and then extrude protoplasm through pores to form a layer over the outside. The animal begins with one chamber and, as it grows, it secretes a succession of new and larger chambers, continuing this process throughout life. Forams are very abundant in the ocean. There are planktonic and benthic forms. When they die, the dead bodies settle on sea floors, forming thick deposits of limestone. In fact, the limestone that makes up the White Cliffs of Dover, and was quarried to build the great pyramids of Egypt, was formed from the bodies of countless Foraminifera! Examine the demonstration of mixed Foraminifera shells. Note the range of forms and the apertures for pseudopodia. 2. Radiolaria (Phylum Actinophyta) The radiolarians are marine forms with beautiful tests made of silica that are often perforated with tiny holes and long pointed spines. The pseudopodia, which are thin and stiff, radiate out through apertures in the skeleton. Most radiolarians are pelagic and, when they die, their dead skeletons sink, contributing to marine oozes. 17

Prac 1 – Microscopes and Unicellular Life– Student Manual Examine the demonstration of mixed Radiolaria shells. Q1. How do the shells and skeletons of Foraminifera and Radiolaria differ from one another? The shells of Foraminifera and Radiolaria differ in their composition and structure, with Foraminifera having tests that are typically composed of calcium carbonate or organic material and divided into multiple chambers, while Radiolaria have tests that are composed of silica and typically consist of a single capsule with elaborate ornamentation. 18

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Prac 1 – Microscopes and Unicellular Life– Student Manual Exercise 3. Protists Living in Animals [15 minutes] There are a range of associations between organisms of different species. These associations are described by a variety of terms that are often difficult to define precisely. Some of these terms and their common definitions are: • Commensalism - both members benefit, but each can live independently. Alternately, commensalistic relationships are often described as one member benefitting, but the other being neither harmed nor helped. • Mutualism - both members benefit and are unable to live independently. • Parasitism - one member benefits at the expense of the other (the other is harmed). 1. Trichonympha sp. (Phylum Axostylata, Class Parabasalea) Termites eat wood, which is largely cellulose, but they cannot digest it. Instead, they rely on a whole community of microorganisms from all three domains of life to do the job for them. In the termite gut, you will find Bacteria, Archaea and Eukarya in the form of protists. Specifically, Trichonympha are Protists and symbionts that digest the cellulose found in the wood and plant fibres eaten by their host termites. Trichonympha live in the intestines of many termite species. They ingest their food by phagocytosis, and they produce cellulose, an enzyme that digests cellulose. Trichonympha have flagella that make them motile. Reproduction is asexual (by division) or sexual. Collect a termite and a glass slide (a well slide will work best). Using forceps, hold the termite’s head and use a dissecting needle to extract the contents of the gut onto the slide. Examine the contents of the termite gut and see if you can find protists in it. Q1 . Can you see food vacuoles containing wood in Trichonympha sp. ? Do they make up a large percentage of the volume inside Trichonympha sp. ? The exact composition of the food vacuoles inside Trichonympha sp. can vary depending on the specific microorganisms that are present in the insect gut at any given time, but it is unlikely that wood would make up a large percentage of the volume inside these vacuoles. Q2. In what way do the termite depend on the protist? In what way does the protist depend on the termite? In summary, the relationship between termites and their gut protists is one of mutual interdependence, where each organism relies on the other for survival. The termites rely on the protists to help them break down and digest the cellulose and other complex carbohydrates found in the wood they eat, as they lack the necessary enzymes to do so themselves. In return, the protists are provided with a warm, protected environment in the termite gut and a steady supply of nutrients from the wood ingested by the termites. 19

Prac 1 – Microscopes and Unicellular Life– Student Manual Q3. Which of the above terms (commensalism, mutualism or parasitism) is applicable to this association, and why? In summary, the relationship between termites and their gut protists is one of mutual interdependence, where each organism relies on the other for survival. Q4. Can you see any Bacteria or Archaea? Why/Why not? Yes, bacteria and archaea can be seen inside the termite gut through the use of a microscope. In fact, the termite gut contains a diverse microbial community that plays a crucial role in the termite's digestive process. These microorganisms are responsible for breaking down the wood and other materials that termites consume into simpler molecules that can be absorbed by the termite for energy and nutrition. Some of these microorganisms include cellulolytic bacteria, which are capable of breaking down the cellulose found in wood, and methanogenic archaea, which produce methane gas as a byproduct of their metabolism. The presence of these microorganisms is essential to the termite's survival, as they provide the necessary enzymes and metabolic pathways to digest the termite's food. 2. Babesia canis (Phylum Apicomplexa) Babesia canis is a protist that parasitizes erythrocytes, causing anaemia in its host. It is the causal organism of babesiosis in dogs (primary host), especially puppies. Infection occurs when a Babesia infected tick bites a dog and releases Babesia sporozoites into the dog’s bloodstream. Examine the demonstrations of blood infected with Babesia canis . Note the appearance and size of the parasite relative to the size of the red blood cell. Make a note of your observation. Babesia canis is a protozoan parasite that infects the red blood cells of dogs and causes a disease known as canine babesiosis. The parasite is typically pear-shaped or round, and ranges in size from 1 to 2.5 micrometers in length and 0.5 to 1.5 micrometers in width. In comparison, red blood cells in dogs are generally between 5 to 7 micrometers in diameter. This means that the parasite is significantly smaller than the red blood cells it infects, and may be difficult to see without the use of a microscope. 20

Prac 1 – Microscopes and Unicellular Life– Student Manual 21

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Prac 1 – Microscopes and Unicellular Life– Student Manual Exercise 4. Bacteria: Gram-positive and -negative cells [5 minutes] 22

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Prac 1 – Microscopes and Unicellular Life– Student Manual Bacteria: Gram-positive and Gram-negative Figure 3. Gram-positive and Gram-negative cells, showing the difference in peptidoglycan layer size. Below demonstrates the Gram staining slide preparation process. Examine the Gram-positive and Gram-negative bacteria slides provided, and answer the following question. Q1. In step three of the slide preparation process (Figure 3 on the previous page), which type of cell retains the crystal violet dye? How is this related to the structure of the cell wall? During the alcohol wash (decolorization) stage in the Gram staining process, Gram-positive cells retain the crystal violet dye, while Gram-negative cells lose the dye. This is related to the structure of the cell wall in these two types of bacteria. Gram-positive bacteria have a thick peptidoglycan layer in their cell wall that retains the crystal violet stain when exposed to the decolorizing agent. In contrast, Gram-negative bacteria have a thinner peptidoglycan layer and an outer membrane that contains lipopolysaccharides. The alcohol in the decolorizing agent dissolves the outer membrane of the Gram-negative bacteria and removes the crystal violet dye from the thin layer of peptidoglycan, making them appear colorless under the microscope. 23

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Prac 1 – Microscopes and Unicellular Life– Student Manual Therefore, the retention or loss of the crystal violet dye during the alcohol wash stage is an important characteristic used to differentiate between Gram-positive and Gram-negative bacteria. 24

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Practical 1 - TSV

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