Module_5_Histology&Lab_Reports_2024

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Feb 20, 2024

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Module 5: Histology & Lab Reports Adapted from Professor Hartmut Doebel and Kajal Patel’s “BISC 1125 Lab Manual 2019,” Tiffini Smith and Jackie Leidig’s “BISC 1112 Lab Manual 2018,” & Materials provided by Caitlin Dicara Objectives & Purpose: Part 1 of Module 5 is all about leveraging student knowledge of the scientific method in order to design experiments and communicate results to a scientific audience. Specifically, students will take a deep dive into experimental design, reading scientific literature, and the art of writing in science. Ultimately, students should understand what information can be found in sections of a typical research article in order to read more efficiently and better construct their own lab reports. All of this will prepare students for Module 6, where students will be generating their own hypotheses and performing an experiment (partially of their own design) to address how certain factors related to exercise affect various physiological responses in human beings. Part 2 of Module 5 will introduce students to histology, or the study of tissues and specialized cell types using microscopy. By the end of part 2, students will understand the basic structures and functions of different types of human tissues. Students will gain this understanding by comparing and contrasting a subset of varying types of human tissues, using prepared slides viewed with light microscopy. Special emphasis will be placed on muscular and connective tissues in order to give students a solid foundation in the cellular structures that will govern physiological processes related to exercise that will be measured for the first lab report in Module 6 next week. Students may wish to review Module 1 guidelines on how to properly format microscope drawings and estimate sizes of objects under the scope. *Students must bring a PENCIL and their LAPTOPS to class for this Module! Students are also required to read the peer-reviewed article posted to Blackboard by their lab instructor PRIOR to the start of lab.* Part 1 - Scientific Writing & Lab Reports: Generation of the hypothesis is one of the beginning stages of a scientific process (after identifying a problem or question). It refers to a supposition, based on reasoning and evidence. The researcher examines it through observations and experiments, which then provides facts and forecast possible outcomes. The hypothesis can be inductive or deductive, simple or complex, null or alternative. The null hypothesis is the hypothesis which is to be actually tested, whereas the alternative hypothesis is usually stated in opposition to the null hypothesis. More specifically, a null hypothesis is a statistical hypothesis in which there is no significant difference between the set of variables. It is the original or default statement, with no effect, often represented by H 0 (H-zero). It is always the hypothesis that is tested. A null hypothesis can be rejected, but it cannot be accepted just on the basis of a single test. In contrast, an alternative hypothesis is a statistical tool used in hypothesis testing, which states that there is a significant difference between the set of variables. It is often referred to as the hypothesis other than the null hypothesis, often denoted by H 1 (H-one). It is what the researcher seeks to support in an indirect way, by using the test to gather supporting evidence. The support of the alternative hypothesis depends on the rejection of the null hypothesis i.e. until and unless the null hypothesis is rejected, an alternative hypothesis cannot be well supported in your experiment. Researchers can generate many hypotheses, design rigorous experiments, and collect pristine data, but the last step of the scientific process is often considered the most important. Scientific work of any sort is useless unless the results can be clearly communicated to others. An entire writing style has been dedicated to this purpose, called technical writing. Technical science writing differs from other types of writing, such as creative writing, in that it is meant to convey technical information to a specific audience for a specific purpose. However; like creative writing, your report should tell a scientific story. When writing anything, you should always start by identifying your audience and purpose. For reports in this lab, your audience will be other scientists (your lab instructor and UGTA specifically) and the purpose is to convey the results of the experiments you have performed in the laboratory. Clarity in the structure of your lab report will prevent reader confusion. Remember, your entire purpose is to convey your work to the reader. Sentences should be non-convoluted and meaningful to the 1

overall message of the lab report, not just added to fulfill a length requirement. Word choice should also be carefully thought out. Modifiers such as very, quite, rather, and somewhat are not necessary to convey the message of your work. Other words in lab reports are frequently misused. The word affect and effect have distinct and different uses. Affect is a verb meaning to act on and effect is a noun meaning a resulting condition, use them accordingly. Also, stating that a result is significant means that you performed a statistical analysis and based on this statistical analysis there is a difference between the two or more groups (p<0.05 for this lab class). Additionally, the terminology of null and alternative hypotheses can only be used if a statistical analysis is performed. Prove is misused frequently and should not appear in science writing. Proof indicates that there is a finite end to the conclusion (as in mathematical proofs), however; science is constantly changing and evolving into a better knowledge base. Scientific evidence, obtained through well designed experiments, can support, but not definitively prove a hypothesis. It is important to be able to critically evaluate the resources you are using in your paper. A peer- reviewed article is a piece of scientific literature containing original experimental work that has undergone the peer review process and is published in a journal. A review is also peer reviewed and published, but is a summary of several original pieces of scientific literature on related topics. Books are acceptable resources and contain more established theories and hypotheses, but tend to be many years out of date with the recent discoveries of peer reviewed articles depending on the field. Websites should be carefully evaluated and used with extreme caution as a source of information. It is often difficult to determine who has published the website or if their information is accurate and this should be taken into consideration. Opinion pieces and blog posts should not be used at all. Each section of the report should have a subheading (except for the title page), and the report should be typed and carefully proofread. Here are a few links recommended by the GWU Writing Center on how to write a formal lab report that you should review: ● https://www.craftofscientificwriting.org/laboratory-reports.html ● https://projects.ncsu.edu/labwrite//res/res-homepage.htm Lab reports in BISC 1112 should include the following sections: Title Page: In this page you should include your name as it appears on Blackboard, your lab partners’ names, course designation & number, submission date, lab section number/lab instructor name, the title of your report and the abstract. The title should be specific and informative, relating to the experiment that was performed. “The effect of caffeine on blood pressure” is a better title than “Human Physiology Report”. Abstract: The abstract is a summary of the entire experiment performed, similar to a plot summary or teaser on a novel. It is usually 300 words or less and includes brief background information, methods, results and conclusions. The aim is to succinctly state the experiment purpose, key findings of experiment, significance of findings, and major conclusions. No numbers, no statistics, and no citations in abstracts! The abstract should be typed ON the title page. Introduction: The introduction tells the purpose of the study and gives a brief summary of the known facts pertaining to your experiment. You should research and provide any background information that the reader may need to understand the purpose of your experiment. Do not forget to cite your references in-line & in a reference list . Explain the purpose of your experiment and include a brief description of how the results will be interpreted. Be sure to clearly state the null and alternative hypotheses being tested. The topics should be structured in a funnel organization. The introduction should funnel from the broad information that is known about the topic, to more specific details, then finally to what is unknown and the question being answered by performing the experiments, culminating in your hypotheses (null and alternative). This organization will help your reader follow the thought process for the experiment and also set the stage for your scientific story. This section should catch the attention of the reader, establish the purpose of the report and justify why it is important. 2

Methods (do not make a materials list) : The methods section clearly describes how you performed your experiments with the materials used, and how to reproduce your experiment. It should be written in the past tense. This section is crucial because it gives the necessary information for someone else to repeat the experiment. You should try to strike a balance between an over-detailed description of even the most trivial items and a statement that provides insufficient information. An indicator of a well written methods section is if another worker of similar training and ability, following your description, should get the same results. Note that this section should be written as a description of what you did (including any statistical analyses and software used for data analysis), not as a set of instructions. In other words, a methods section of a paper is structured differently than the protocol section of a lab manual. Methods sections MUST be written in third person passive voice. Results: The results section presents the data you collected in your experiment or observational study, any required graphs or tables of the results, and the results of all statistical analyses that were performed on the data. It does not include any discussion or interpretation of the results !! This means you should NOT make a statement about “rejecting the null hypothesis” or “failing to reject the null hypothesis” in this section! In this section you should describe the important qualitative and quantitative observations of your work. Tell the reader exactly what you want them to see when they look at your graph or table. The text should summarize, in past tense, the important findings in the data; it does not simply repeat raw data from the graphs or tables. If you are using graphs and tables, refer to them by Figure number or Table number within the text. Captions for figures go below the graph with a brief description of that figure; captions for tables will go above the table in the paper. Arrange them in the order in which you refer to them in the text of the results section. Use words to draw the reader’s attention to the key patterns in your data. First decide exactly what you want your reader to see when looking at each graph or table, and then stick the reader’s nose right in it. This section is not a list of your raw data. All raw data should be clearly labeled and represented in a table and/or summarized in a graph when appropriate. You may use any type of graph to show your results —whatever works best for your data. Don’t forget to label all axes in your graph including appropriate units. Each table and graph should have a figure legend that describes what’s in the graph or table. Graphs are not self-explanatory and the figure legend summarizes for the reader what is in the table or graph. The figure legend is freestanding text that goes below the figure and includes the title of the graph and additional information to make the data more comprehensible. The first sentence of the legend typically is a succinct statement that summarizes what the entire figure is about; for example, "Figure 1: Distribution of Daphnia in the presence of light and dark habitats." The first sentence is then followed with particulars of the figure contents, explanations of abbreviations etc. By convention, table legends go above a table and figure legends go below a figure. Discussion and Conclusions: This is where the data are fully discussed and interpreted, and the null hypothesis is rejected or not with supporting data. You may also say that your alternative hypothesis was supported by your results (even that your some results supported and others did not) but you NEVER say that the alternative and/or null hypothesis was “accepted” or “proven.” This section also includes a critique of the experimental design. Does it adequately address the hypotheses being tested? Were there faulty assumptions in the design that confound your interpretation of the data? Would you do the experiment differently if you did it again? Did your observations of the subjects/focus of the experiment beforehand give you any insights into the results? Discuss what you would do to improve the design or how you would change if you were to repeat the experiment. Also discuss any confounding factors in your design. Typically, discussion sections are formatted in a “reverse funnel” format from specifics of what you found in your experiment to broad implications. This is in contrast to an introduction section which is formatted from broad to specific. The discussion section is also where researchers are expected to connect their work to the broader 3

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field of study, which is accomplished through the use of citations to other scientific articles or reports. A relevant and reasonably complete discussion of how this novel research project relates to others’ work in the field is necessary to provide scientific context. Along with the introduction, this is the section where writers are expected to have the most relevant and robust citations to peer-reviewed research. References: There are not a correct number of references, but at least two references from peer-reviewed journals are expected in this class for a chance at full credit. Any time you refer to a previously reported idea, result, method, etc., you must always insert a citation. For example, you may wish to reference the textbook or lab handout for your lab report. Every paraphrased statement that is not common knowledge must be referenced. Not to do so is, at best, a violation of scientific and literary ethics and, at worst, plagiarism. A warning about direct quotations (i.e. copying a sentence or a paragraph in quotation marks): direct quotes are not acceptable in the scientific literature—perhaps with the exception of quoting a known statement of a very famous scientist to emphasize a certain point. Sometimes, it seems that there are almost as many reference styles as there are cells in the human body! An exaggeration for sure, but ultimately when a scientist intends to publish their work in a peer reviewed journal, the scientist must use the reference style of that journal. In this class, we will use APA style citations for our lab reports. APA style always demands the same sets of information in the same order for entries to the reference list or “works cited” section of a paper. Here is a useful template for a scientific journal article : Author Surname, Author Initial. (Year Published). Title. Publication Title , Volume number (Issue number), Pages Used. doi:DOI Number. The first line of each citation is left adjusted and every subsequent line of that entry is indented 5-7 spaces. Below are a few examples of how APA style citations would look in the reference list at the end of your lab report. Make sure the list is in ALPHABETICAL order. Blechschmidt, J., M. Wittmann, and C. Blüml. (2020). Climate Change and Green Sea Turtle Sex Ratio- Preventing Possible Extinction. Genes 11 (5): 588. https://doi.org/10.3390/genes11050588 Crozier, L., J. Siegel, L. Wiesebron, E. Trujillo, B. Burke, B. Sandford, and D. Widener. (2020). Snake River sockeye and Chinook salmon in a changing climate: Implications for upstream migration survival during recent extreme and future climates. PloS One, 15 (9): e0238886–e0238886. https://doi.org/10.1371/journal.pone.0238886 Freeman, S., Eddy, S. L., McDonough, M., Smith, M. K., Okoroafor, N., Jordt, H., & Wenderoth, M. P. (2014). Active learning increases student performance in science, engineering, and mathematics. Proceedings of the national academy of sciences , 111 (23), 8410-8415. https://doi.org/10.1073/pnas.1319030111 For all scientific reports and papers, the reference list at the end of the document is only half the story. Writers must also use in-line citations to specifically note which ideas presented are not original to the writer or common knowledge. An in-line citation in APA takes the following format at the end of the cited statement (Author or Authors, Date of publication). Essentially, citing is the crediting of sources used in academic work. When another source contributes to your work you must acknowledge the original author with an accurate reference, unless it is common knowledge (e.g., the mitochondria is the powerhouse of the cell). Failing to cite all of your sources or citing them incorrectly constitutes plagiarism, which is considered a serious academic offense and will result in a referral to the Academic Integrity Council here at GWU. It is important to remember that information doesn’t just belong to anyone who happens to stumble upon it and crediting the minds that generated the information you used in your paper is the ethical responsibility of any writer. Not all the sources you cite will be peer-reviewed journal articles. Sometimes you will use books or textbooks, presentations or lectures, interviews, dissertations, etc…. Certainly some of these will be more 4

common than others in science writing but all of them have ever so slight variations in what information must be included in the reference list Here ( https://www.citethisforme.com/citation-generator/apa ) is a wonderful resource you can use to explore various sources cited in APA style. Please note that while the use of auto-generation programs for reference is typically fine, remember that these programs can only work with the information you give them. In other words, you as the writer are ultimately responsible for any errors in your citation lists even if you used a program. DOUBLE CHECK THE OUTPUTS IF YOU USE AUTOMATIC REFERENCE SOFTWARE. Formatting: The report should be typed and double spaced in 12 point font Times New Roman. It should contain correctly formatted citations in a reference list; as well as, in the body of the report, in APA style. Proper terminology, grammar, spelling, and sentence structure is used throughout the report. Do not use contractions, as they are considered unprofessional. Each section of the report should have a subheading (except for the title page). Finally, a note about form and style. Philosophically, science should be independent of scientists, their times, and places of work. That is the actual discoverer of a principle is less important than the principle itself. Thus, traditionally reports have been written in the third person, rather than the first. It is also traditional to write scientific papers in the passive, rather than the active, voice. For example, one should avoid statements like, "I counted the number of Daphnia in the dark half of the tube." Instead write: “The number of Daphnia in the dark half of the tube were counted.” Scientific Writing & Lab Report Protocol: *Bring your laptop to class to complete this section of the protocol!* 1. Open your lab Blackboard page and download the peer-reviewed paper of the lab TAs choosing from the appropriate tab. 2. Take 10-15 minutes and independently read through the Abstract and Introduction sections. In your lab notebook, identify the “big question” or problem the research is trying to address (Think not “What is this paper about”, but “What problem is this entire field trying to solve?”) and any specific questions/problems the particular paper addresses. 3. In your lab notebook, paraphrase the purpose of the paper, the key findings, the significance of the findings, and the conclusions drawn. 4. Take a few minutes and discuss with your bench mates the types of information included in the introduction. Take special note of what information the author cited. How does the information cited relate to the central question of the paper? After having read the abstract and introduction, do you have a solid understanding of what the author was trying to convey to the audience? What, according to the authors, needs to be done to address the “big question” or problem? Try explaining the purpose of the paper in your own words. 5. Summarize the discussion you had with your bench mates in your own words, in your lab notebook. 6. Take a careful look at the results section and any graphs/tables summarizing the data. Note any time the words “significant” or “non-significant” are used. These have precise statistical meanings regarding any statistical analyses that were applied to the data. NOTE: the results section of the paper is composed of visual depictions of data (fully labeled tables & graphs) AND written descriptions of the data and analyses performed. Your lab reports must also follow this convention. Think about/discuss with your lab mates what the presentation of the data and the analyses are trying to convey to the reader. Write your thoughts in your lab manual. 7. Read the discussion/conclusions section: What do the authors think the results mean? Do you agree with them? Can you come up with any alternative way of interpreting them? Do the authors identify any 5

weaknesses in their own study? Do you see any that the authors missed? (Don’t assume they’re infallible!) What do they propose to do as a next step? Do you agree with that? Write your thoughts in your lab manual. 8. Note how the discussion/conclusion section of the paper attempts to contextualize the findings of the study with the broader field by robustly incorporating previous findings from peer-reviewed citations. Students will be expected to do this in their own lab reports. Part 2 - Introduction to Histology: Next week in the lab, students will be partially designing their own experiment assessing the effects of physical activity on various physiological metrics like heart rate, blood pressure, and/or reflexes. As an introduction to the concepts of human anatomy, exercise science, and the health field in general, it is helpful to have a solid basis in cellular structures comprising the relevant tissues and organ systems. The study of specialized cell types, along with tissue structure and function on a microscopic level is known as histology . Studying “normal” tissues allows scientists and medical professionals to recognize different diseases, their cause, and formulate treatment options. Tissues are large clusters of specialized cells that together complete a shared function. Organs arise from the interaction of several tissue types and keep the body operating. Since tissues are the building blocks of virtually everything in the body, understanding histology gives you the necessary tools to predict and understand organ behavior and function. Animals are unique among the kingdoms of life in the immense diversity of their specialized cell types, resulting in an unparalleled complexity of tissue and organ systems. That is to say, while other multicellular kingdoms of life, like plants, also have true tissues and organs, no group compares to animals in this regard. As an animal embryo grows, specialized cell types develop from the three primary germ layers through a process known as differentiation, until eventually all the adult tissues are formed. Recall that invertebrate echinoderms (e.g. the sea star) are the closest evolutionary relatives of the chordates (e.g. human beings). Fittingly, both are deuterostomes, and thus have all three of the primary germ layers: ectoderm, mesoderm and endoderm. In the deuterostome clade, the blastopore forms the anus and the body cavity (coelom) forms through the process of enterocoely. Differentiation is the process of cells specializing in structure and function through differences in gene expression. Remember all the cells made by mitosis have essentially the same DNA. However, these differentiated cells look and function differently, due to the fact that different genes will be turned on (or off) in the differentiated cell types. For example, liver cells will express different genes compared to pancreatic cells, giving each of them their different form and function. Most of these differentiated cells lose the ability to return to their original embryonic state as stem cells. Embryonic stem cells can produce any type of cell in the body, and they are found only in the early embryo. In the United States there are serious restrictions on the use of embryonic stem cells for research, and the creation of new embryonic stem cell lines. This is not the case with adult stem cells; seemingly differentiated cells from tissues that retain the ability to divide indefinitely, but usually only produce their same cell type. For example, blood stem cells produce only more blood cells, not muscle or bone cells. Adult stem cells are not easy to detect by their appearance as they look exactly like the surrounding cells. Some adult stem cells have been found to carry different molecular markers on their cell surface, and can be identified by these markers. Prior to starting the protocol below, make the following table in the “Data tables & observations” section of your lab notebook: 6

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Type of Microscope ID Number of Microscope used on [Date] Compound Light Dissecting (Stereo) Histology Protocol: In the lab today, students will be primarily looking at prepared microscope slides of human tissues, which are similar to tissues found throughout the animal kingdom. As one might expect from our current understanding of evolutionary relationships among the animal kingdom, tissues of organisms which share a more recent common ancestor with humans would have more similar tissues than those animals that share a more distant common ancestor with us. Thus humans share more tissue and organ similarity with chimps or cats, than they do with snails or worms. There are four basic types of human tissues: epithelial, connective, muscle and nervous tissues, as well as many subtypes within these four classes. Today, students will be focusing entirely on the various types of muscle and connective tissues, to gain a better understanding of the organ systems they will be assessing the performance of in next week’s experiments. *For each of the prepared slides below, students are responsible for making a properly labeled and formatted microscope drawing of the requested cell/tissue type. See Module 1 for microscope drawing guidelines. Additionally, students will estimate & record the size of the focal cells in their drawings, again using guidelines from Module 1. Read slide labels carefully to take notes on which tissues and organs have specific types of cells. Finally, below each numbered slide below is some brief information to orient students or provide further context.* 1. Muscle Tissue - All muscle cells contract (or shorten) when stimulated. You can read about the mechanism underpinning this phenomenon called the “Sliding-Filament Model of Muscle Contraction” in your Urry et al. 2021 Campbell Biology textbook on pages 1126-1127 of Ch. 50. Slides of muscle cells appear very different in cross- sections (c.s.) compared to longitudinal sections (l.s.). Think of a braided rope viewed from the side (l.s.), or viewed from the end (c.s.). For the purposes of this lab, you will only be responsible for identifying muscle tissue sectioned longitudinally, i.e. cut along the lengths of the cells. The three types of muscle tissues in the body you need to be familiar with are: skeletal, smooth and cardiac. Refer to figure 1 below for a snapshot of muscular tissue diversity. 7

Figure 1 . The three types of muscle tissue represented as simplified cartoons and as longitudinal cross sections of dyed tissue slides, like those viewed in the lab today. Note the differences in arrangement between each muscle type, and the notable features of the cells like the darkly stained nuclei. A. Smooth Muscle - Smooth muscle surrounds many tube-like organs in your body, such as blood vessels, the digestive tube, the uterus, vas deferens in the male, etc. In the esophagus, stomach and intestines it is the contraction of the smooth muscle that causes the food to move down the digestive tube in the process known as peristalsis. Smooth muscle contraction around your arteries is responsible for raising your blood pressure. Contractions of these smooth muscles are largely involuntary (they happen without you thinking about it). Smooth muscle can be found on many of your slides, but you will be responsible for locating it only in certain areas. Notice how each individual smooth muscle cell has a single nucleus and how elongated nuclei tend to be relative to other cells you have looked at this semester. The cells themselves are somewhat like a tapered sausage (or spindle) in shape and are arranged fairly regularly. The lighter pink stained portions of the tissue are mainly the cytoplasm of the cells, and no striations are seen internally due to a lack of organization in the thick and thin filaments. Look for smooth muscle tissue on these slides: #18 Smooth Muscle - Smooth muscle surrounds all arteries and arterioles - Auto-rhythmic, meaning it contracts spontaneously (involuntary) #3 Gall Bladder - Find patches of smooth muscle beneath the epithelium and connective tissue layers #2 Epithelium 3 Types - Locate the large bands of smooth muscle in the esophagus and small intestine pieces. B. Cardiac Muscle - This is the muscle of the heart and it appears striated under the microscope because the cytoskeletal elements of the cells (thick and thin filaments) are arranged in a regular and repeating pattern. Contractions of the cardiac muscle tissues are involuntary; good thing too, because having to think about keeping your heart pumping would be very difficult! Cardiac muscle tissue has distinctive junctions between 8

cells that are actually folded pieces of cell membrane; they appear as dark vertical bands between cells and are called intercalated discs. Notice how the ends of the cardiac muscle cells branch and lead directly to another muscle cell. This branching pattern is distinct among muscle types and is found ONLY in the muscle tissues that comprise the walls of the heart. Despite this unique branching shape, cardiac muscles have only a single nucleus, just like the smooth muscle cells. #19 Cardiac Muscle - Auto-rhythmic - Striated - Intercalated discs - Branching C. Skeletal Muscle - Skeletal muscle is sometimes referred to as striated muscle, but skeletal muscle is the preferred term since both skeletal and cardiac muscles are striated (for the same reason; thick and thin filaments are arranged into regular repeating patterns). Skeletal muscles allow us to move our arms and legs as well as our diaphragm. Since we have conscious control over the contraction of this muscle type it is often called “voluntary” muscle. As you could guess from the name, most skeletal muscles are attached to the skeletal system in some way, usually via connective tissues called tendons. You will easily see why it is called striated: the proteins actin (thin filament) and myosin (thick filament) that contract muscle cells form very distinct bands or striations in this muscle type, making it one of the easiest tissues to identify. Another interesting characteristic of skeletal muscle cells is that they are considered a syncytium , a single cell that contains many nuclei. Because a skeletal muscle cell can be very long and thin, each cell is often called a muscle fiber. As with smooth muscle, you will only be responsible for identifying skeletal muscle sectioned longitudinally, so that the striations are evident. The striations cannot be seen when the cell is cut transversely or in cross-section. #20 Striated Muscle - Striations - Many nuclei #21 Skeletal Muscle - Find the threads of striated skeletal muscle - This slide also contains smooth muscle surrounding the arteries, as well as pockets of adipose tissue #22 Skeletal Muscle Cross Section - Long striated strands that look like rope (longitudinal cut) - Bundles of muscle look like isolated pockets (cross sectional cut) 2. Connective tissue - Connective tissue has many functions within the body. It acts as filler tissue and support for other tissues, forms sheaths around soft organs, connects one tissue with another, serves as a place for blood vessels and nerves to pass from one area of the body to another, offers protection against infection, and aids in tissue repair. There are many types of connective tissue including loose connective tissue, dense connective tissue, adipose tissue, cartilage, bone and blood. Connective tissue has three components: the cells, the fibers, and the 9

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extracellular matrix. Both the fibers and the extracellular matrix are outside of the cells, but have been secreted by their accompanying cells. A. Basic Connective Tissue - Basic or unmodified connective tissue can be seen immediately beneath epithelial tissues, as well as in many other areas of the body. In most cases it consists of a large amount of extracellular material— fibers and extracellular matrix with scattered cells of different types. One major component of basic connective tissue is the fibrous protein collagen, the most abundant protein in the human body! This strong, yet flexible protein gives connective tissue strength and resilience. Just about everything you see will be collagen fibers, and an occasional cell that makes the fibers. Sometimes collagen fibers are also called white fibrous connective tissue. Refer to figure 2 below to orient yourself as you look through the prepared slides. Figure 2 . Dyed histological sections of dense regular, dense irregular, and loose connective tissues. Note that darker dyed (blue-purple) areas tend to be nuclei, whereas lighter pink-purple areas tend to be cytoplasmic contents. White areas are often extracellular space with secreted fibers of the extracellular matrix. Loose Connective Tissue slides: #10 Areolar Tissue - Thin Fibers - Loosely packed #2 Epithelium 3 Types - Small intestine piece - Thin collagen fibers directly beneath the layer of simple columnar epithelial tissue 10

Dense Connective Tissue slides: #8 Human Skin - Dermis layer, directly beneath the stratified squamous epithelial tissue - Intensely stained pink collagen fibers - Tough thick sheets of material with fibers running in all directions - Also called dense irregular connective tissue #11 Muscle Tendon Junction - Attach muscle to bone - Thick ropes of collagen - All of the fibers run in the same direction - Also called dense regular connective tissue B. Adipose Tissue - Adipose tissue consists of accumulations of fat in cells called adipocytes. Compared to other cells in the body, fat cells are quite large. Originally, each cell contained a large droplet of fat (mostly triglycerides) but all the fat dissolved during the tissue processing, so the central part of the cell appears unstained. Fat cells look like white circles on your slides, and often appear in clumps. Liposuction literally sucks out fat cells with an instrument that is really just a little vacuum cleaner. Fat is a very efficient way to store energy, taking up less space and weight than equivalent energy stores of carbohydrates or proteins. Since animals have to carry their energy reserves around with them, animals mostly use fat to store excess energy. # 12 Adipose Tissue - White fat is the most common type of fat - Used for: Energy storage, Insulation, & Protection (Soles of feet, palms, and butt) C. Bone - Bone is a special form of connective tissue where the extracellular matrix becomes hardened by the formation of calcium salts. Bones serve to protect some areas of the body (e.g. skull protects the brain), as well as allow for movement of the limbs (e.g. muscles attached to the femur help move the leg). Bones also provide the body with a readily available source of calcium, and protect the blood-cell forming tissues of the body, the bone marrow. Your bone slide was made from a piece of dried bone that was ground down to a thin wafer-like slice. Thus, you will not be able to see any cells, only the hard parts of the bone. In the living animal, bone is laid down in concentric layers called lamellae, which are readily visible on your slide. In the very dense outer part of all bones, called compact bone, new bone is laid down in concentric rings around a central cavity. These ring- shaped structures are called Haversian systems . Use figure 3 below to orient yourself while viewing the bone slice slides. Bones are continuously being remodeled throughout one’s life. Osteoclasts are cells that dissolve older bone tissue and osteoblasts are the cells that lay down new bone cells and extracellular matrix. Once the osteoblasts are trapped in the matrix, they are called osteocytes. Osteoporosis results from a shift in the balance between osteoclasts and osteoblasts, such that more bone is removed by the osteoclasts than is replaced by the osteoblasts. 11

Figure 3 . Cartoon bone cross section to orient students when looking at the wafer-thin slice of bone under the microscope. Note the orientation of the Haversian systems, surrounded by their concentric rings of lamellae. #13 Dry Bone Find a Haversian system on your slide and notice the dark spots in the lamellae. - In the living animal, these are actually spaces (i.e. lacunae) in the hard bone in which the bone cells (osteocytes) live. - The hole in the center of each Haversian system is called the Haversian canal; it serves as a tube through which blood vessels and nerves pass. #14 Decalcified Bone - Stained pink - Loose disjointed structure D. Blood - Plasma, the liquid part of blood, does not stain, but all the cells of the blood are evident. Of the six main blood cell types, we will only identify the three most common cell types. By far the majority of cells on your slide are made up of the red blood cells or erythrocytes . Platelets (thrombocytes) are the smallest blood cells. When thrombocytes touch the rough surface of a torn blood vessel, they stick to it and each other. Platelets release factors that form a thread- like substance (fibrin) which forms a clot. The clot hardens into a scab, which covers the breakage until the vessel heals. White blood cells or leukocytes contain nuclei and function mainly in the defense system of the body. The two most common white blood cells are the neutrophil and the lymphocyte . Much larger than an erythrocyte, the neutrophil contains a very constricted, odd-shaped nucleus that appears to have lobes to it. The lymphocyte, slightly larger than a red blood cell, is mostly filled with the round nucleus. Some types of lymphocytes produce antibodies that fight infections (B cells), while other types of lymphocytes are responsible for tissue rejection when someone receives a transplant from another person (T cells). 12

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Figure 4 . Cartoon image of a human blood smear under the microscope. Note the different types of labeled blood cells, including the common red erythrocytes, the various “white” blood cell types important for the immune response, and the colorless cell fragments called platelets which help our blood form clots that prevent bleeding. #15 Human Blood Smear Red Blood Cells - Stains light pink - No nuclei to make more room for the protein hemoglobin whose function is to carry oxygen and carbon dioxide - Look like donuts Platelets - Cell Fragments (do NOT contain a nucleus) - Darkly stained White Blood Cells - Neutrophils and Eosinophils: lobulated nucleus - Lymphocyte: large round nucleus with almost no cytoplasm E. Cartilage - Cartilage is a tough but flexible support material that resists compression. It is made up of cells and an extracellular matrix. Chondrocytes are the cells in cartilage that secrete the matrix. They reside in lacunae, just as in bone tissue, and give cartilage a distinctive appearance. Chondroitin sulfate laced with collagen fibers give cartilage its distinctive structure. Cartilage connects the ribs to the sternum, and forms the discs between vertebrae in the spine. The long bones in young children have significant sections of cartilage in the growth plates between the diaphysis and the epiphyses. As long as the rate of chondrocyte division outpaces the rate of bone formation the child will grow in height. However, eventually all the cartilage in the growth plates will be replaced with bone and growth will cede. Your ears or the tip of your nose are other places where cartilage can be found. While there are several different types of cartilage depending upon the predominant type of fiber in the extracellular matrix, you are only responsible for identifying the generic cartilage tissue. Refer to figure 5 for a general idea of the cartilage types and the differences between them. 13

Figure 5 . Cartoon representations of three common cartilage types: Hyaline cartilage, Elastic cartilage, and Fibrous cartilage. The primary difference between these three types of cartilage is the identity, quality, and amount of fibers in the extracellular matrices of the chondrocyte cells. #16 Hyaline Cartilage - Most common type of cartilage in the body found in the trachea, vertebral discs, and developing bone - Withstands compression - Strong and Flexible - Easy to identify as it stains a blue color - Notice the pink stained nucleus of the chondrocyte surrounded by the blue matrix # 17 Hyaline and Elastic Cartilage - Elastic cartilage is largely responsible for giving the external portions of the ear (pinnae) its shape - Note: the darkly stained elastic cartilage Clean Up: 1. Return all slides to the slide boxes at student benches in an organized fashion. 2. Turn the light on your microscope off by flipping the switch 14

3. Unplug your microscope and wrap the cord appropriately 4. Remove any garbage or debris from the benchtop (lens paper, scraps, garbage), and dispose in the correct receptacles 5. Check the floors around your benchtop for garbage or debris, and dispose of ion the correct receptacles 6. Spray down the benches with ethanol and wipe down the bench tops with paper towels 7. Push in your chairs fully before you are dismissed Homework (to be written in your lab notebook along with our normal pre-lab prep) : 1. Pick a group of four total peers and exchange contact information BEFORE leaving the lab! 2. Read through the introductory section of Module 6: Human Physiology when it is posted to Blackboard on Friday afternoon. Pay particular attention to sections describing equipment available for this lab. 3. The broad question addressed in Module 6 is “How does exercise affect physiological metrics, such as heart rate, blood pressure, or reflexes, in humans?” 4. As a group, generate null and alternative hypotheses addressing these questions. When preparing your pre-lab notebook material, write these in a section labeled “Group Hypotheses.” 5. As a group, brainstorm how you might design an experimental protocol to address the broad question in a quantitative way. Think critically about what your independent variable, dependent variable, and controls would have to be to make your hypothesis testable. What types of “exercises” would you like to do? How would you ensure this is a controlled experiment? 6. Write down the protocol you brainstormed with your group in your lab notebook and label it “Brainstormed Protocol.” 7. Prepare the rest of your pre-lab materials in your lab notebook as you normally would. 15

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