Cell Lab

Cell Lab

Learning Objectives

  • Explain the difference in resolving power of light and electron microscopes, and identify which organelles can be visualized with each
  • Use red blood cells as microscopic rulers for estimating sizes of other cells
  • Describe the location, function, and staining characteristics of the major cytoplasmic organelles.
  • Differentiate regions of high and low cellularity based on the number of nuclei.
  • Identify mitotic cells based on the structure of the chromosomes.

Keywords

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Pre-Lab Reading

Introduction

The basic principles of histology and cell structure serve as an important background for the study of specific cells, tissues, and organ systems. This laboratory serves as an introduction to the rest of the course – you will use the principles you learn here in every subsequent lab.

Histological Staining Methods

Cells are difficult to see by light microscopy. To help us visualize the structure and features of cells, dyes are used to impart a particular color to cells. These dyes react with different chemical features of proteins, nucleic acids and carbohydrates and can be used to highlight certain cellular structures.

Hematoxylin and eosin (H&E) staining is the standard method of staining in histology. Hematoxylin is a basic dye (positively charged) that binds to negatively charged DNA and RNA and is blue in color. Eosin is pink in color and is an acidic dye (negatively charged) that binds to positively charged particles like the mitochondria and many components of the cytoplasm. Positively charged structures are therefore said to be "eosinophilic." While H&E staining is widely used, it is limited in its ability to differentiate between cytoplasmic organelles and many other tissue components.

The periodic acid-Schiff method (PAS) is useful for staining structures rich in polysaccharides (glycogen), mucopolysaccharides (ground substance, basement membrane, mucous), glycoproteins (thyroglobulin), and glycolipids. In this method, periodic acid oxidizes 1,2-glycols and 2,2-amino alcohols to aldehydes, which are then stained reddish purple by the Schiff reagent.

Osmium staining blackens lipids and stains the Golgi apparatus under the light microscope. It is also used as a fixative for electron microscopy.

The Cell

The cell is the fundamental unit of living organisms. Cells grow, adapt to their environment and reproduce, processes which characterize life. Cells also assemble into groups to form complex structures. Cells and the extracellular material they make comprise the tissues of our bodies. Several different types of tissues then organize to form organs. The cells in an organ communicate and work together to perform the functions of that organ.

The cell is limited by the cell membrane, also known as the plasma membrane. The cell’s content is divided into two main compartments: the nucleus and the cytoplasm that surrounds the nucleus. Cytoplasm is further divided into organelles, cytosol and inclusions. Organelles are assemblies of specific macromolecules organized to carry out complex functions. Many organelles are surrounded by a membrane that separates their internal environment from the cytoplasm. Membrane-bound organelles concentrate enzymes and reactants, increasing biochemical efficiency and isolating harmful proteins and molecules from the rest of the cell. Cytosol is a gel-like substance that contains dissolved macromolecules, organic compounds and ions. In addition, cytosol contains the cytoskeleton (microtubules, actin filaments and intermediate filaments) that organize the organelles and provide mechanical support. Lastly, inclusions are insoluble substances in the cytosol such as glycogen and lipid droplets.

Dimensions of Cells

One of the most important skills that you will take away from this course is the ability to understand the dimensions of cells and their subcellular components. When it comes to size, there are three key facts that you must remember:

  • Most eukaryotic cells have a diameter of 7-20 microns (µm), while prokaryotic cells are smaller (0.2 - 5 µm).
  • Red blood cells have an average diameter of 7.2 µm, and are a useful reference for size approximation.
  • The approximate diameter of a secretory granule is 1 µm.

With the unaided eye, one can only see exceptionally large cells, such as the human ovum, which has a diameter of 100 µm. Therefore, we must use a microscope to visualize cells in a tissue. Microscope images in this course come from the light microscope (magnification up to 400x) and the electron microscope (magnification up to 500000x). The limit of resolution of the light microscope is 0.2 µm, while the practical limit of resolution of the electron microscope is about 1 nanometer (nm). Thus, light microscopes allow one to visualize cells and their larger components such as nuclei, nucleoli, secretory granules, lysosomes, and large mitochondria. The electron microscope is necessary to see smaller organelles like ribosomes, macromolecular assemblies, and macromolecules.

With light microscopy, one cannot visualize directly structures such as cell membranes, ribosomes, filaments, and small granules and vesicles. Using an appropriate staining technique, however, makes aggregates of these smaller structures or the regions they occupy visible by light microscopy. For example, while it is not possible to see the membranes and ribosomes that compose the surface of the rough endoplasmic reticulum, these structures are represented in light microscope slides by clumps of basophilic material in certain regions of the cell. It is important to be able to correlate the appearance of cells at the light microscope level with the structures visible in electron micrographs.

Organelles

The cell membrane is about 10 nm thick and cannot be resolved by the light microscope. The limits of the cell can be visualized with the light microscope when there is a heavy concentration of glycoproteins or proteoglycans at the cell surface. The presence of large amount of carbohydrate on the cell membrane makes Periodic acid-Schiff (PAS) an effective method to stain the cell membrane.

The nucleus is limited by a nuclear envelope that consists of a two membrane bilayers and nuclear pores that allow passage of material into and out of the cell. Chromatin, complexes of DNA and protein, is the major component of the nucleus and consists of two histological structures. Heterochromatin is condensed chromatin scattered throughout the nucleus or accumulated along the inner surface of the nuclear envelope. Heterochromatin is considered transcriptionally inactive. In contrast, euchromatin in abundant in cells engaged in transcription. Euchromatin is dispersed and not easily stained.

The nucleus often contains one or more nucleoli that are spherical or oval bodies composed chiefly of ribonucleoproteins. Nucleoli are usually stained with basic dyes because of their high RNA content and are prominent in cells that are actively participating in protein synthesis.

The endoplasmic reticulum (ER) is a system of interconnected membranous sacs, channels, or cisternae in the cytoplasm. It has two subtypes: rough endoplasmic reticulum (RER) and smooth endoplasmic reticulum (SER). The RER is a ribbon-like structure surrounding the nucleus near the base of the cell. Its surface appears rough due to the ribosomes attached to its membrane and it is the first organelle into which membrane-bound or extracellular proteins are inserted. SER lacks ribosomes and participates in lipid synthesis and detoxification.

The Golgi apparatus is a system of membranous cisternae and vesicles arranged in stacks near the nucleus. The Golgi processes and modifies sugar side chains on proteins that are being secreted or destined for the plasma membrane or other membrane-bound organelles like the lysosome. Therefore, the Golgi apparatus is particularly prominent in cells synthesizing large amounts of glycoproteins and proteoglycans, such as goblet cells that produce mucous in the gut epithelium. The Golgi can be stained with osmium or silver stains and appears as a network of black-staining tubules or clusters of granules.

Secretory vesicles or granules usually contain specific substances synthesized by cells that are exported to the extracellular medium. They include zymogen granules, mucous droplets, and mast cell granules.

Mitochondria are organelles that vary greatly in number, size, and shape between different cells. They are unusual in that they contain their own mitochondrial DNA and ribosomes; mitochondrial proteins come from genes in both the nuclear and mitochondrial DNA. These organelles also undergo self-replication. Structurally, two features characterize mitochondria: double bilayer membranes, and cristae, folds that project from the inner membrane into matrix.

Lysosomes also vary in size and shape, but can be recognized as membrane-bound organelles containing granular material. There are more than 40 lysosomal enzymes that are active at acidic pH.

Mitosis

Animal and plant cells undergo a precise type of division called mitosis. Before cell division, the entire genome is copied. This appears at the light microscope level as a duplication of chromosomes. During mitosis, the two sets of chromosomes are precisely separated and each daughter cell receives one complete set. The final result is the production of two daughter cells identical in their genomic content. In the timeline of mitosis, division of the nucleus (karyokinesis) precedes division of the cytoplasm (cytokinesis).

Mitosis involves 4 distinct phases: prophase, metaphase, anaphase, and telophase. Each mitotic division is separated by interphase.

  • Interphase is the stage between two successive divisions, and is divided into two gap phases (G1 and G2) separated by a synthesis (S) phase in which DNA is replicated. Cells in the stationary phase of growth, like those of the adult liver, are arrested in G1. The chromosomes during interphase are usually unraveled and exist as chromonemata or chromatin fibers that are ill-defined, delicate threads dispersed throughout the nucleus.
  • In early prophase, the chromatin fibers thicken to form defined, heavily staining threads visible with the light microscope. This is the result of a series of successive coilings. The final result is the appearance of a diploid set of deeply staining chromonemata, each consisting of two sister chromatids that are not yet resolvable by light microscopy. At this stage in animal cells, there is a division and migration of the centrioles in the cytoplasm to initiate the formation of a spindle and asters. Recall that the spindle fibers are composed of microtubules.
  • During mid prophase, the nucleoli disappear and a "double-ness" can be observed in the chromonemata because the sister chromatids begin to separate.
  • In late prophase, the nuclear membrane breaks down and the chromonemata shorten and thicken to form chromosomes. Each prophasic chromosome is composed of two chromatids which are exact duplicates of each other in genetic content and which are closely associated throughout their length without actually being fused.
  • At metaphase, the spindle has been completely formed and the chromosomes have become arranged at the equatorial plate of the spindle.
  • At anaphase, the chromatids separate from each other and a diploid number or complete set of chromosomes moves toward each pole. This occurs through the activity of the microtubular spindles.
  • In early telophase, the chromosomes have arrived at the poles of the spindle and a nuclear envelope begins to assemble around them.
  • Cytokinesis occurs in late telophase and involves the division of cytoplasm between daughter cells. In animal cells, this is achieved by constricting a region of the plasma membrane near the equatorial plate. Opposite sides of the squeezed membrane fuse, resulting in two distinct daughter cells. In plant cells, a cell plate is formed in the middle of the spindle and later extends across the whole cell.

After cytokinesis, chromosomes unravel and reassume the thread-like appearance of chromatin. The nucleoli reappear and the nuclear envelope is fully reconstituted.

Pre-Lab Quiz

  1. Place the following in order of increasing size: bacterium, nucleus, secretory granule, red blood cell, human ovum, eukaryotic cell, thickness of plasma membrane.
  2. Answer:
  3. Trace the production of a trypsin, a digestive enzyme produced by the pancreas, through the cellular organelles beginning with the trypsin gene in the nucleus and ending with its release into the lumen of the pancreatic duct.
  4. Answer:
  5. What might be the difference in cellular activity between a cell containing large amounts of RER versus large amounts of SER?
  6. Answer:
  7. Explain what color you expect the RER and the SER to stain under H&E. If there is a difference, why?
  8. Answer:

Slides

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  1. Cell Membrane
  2. Nucleus
  3. Euchromatin and Heterochromatin
  4. Rough Endoplasmic Reticulum
  5. Rough Endoplasmic Reticulum EM
  6. Smooth Endoplasmic Reticulum
  7. Smooth Endoplasmic Reticulum EM
  8. Golgi
  9. Golgi EM
  10. Secretory Granules
  11. Secretory Granules EM
  12. Prophase
  13. Prometaphase and Metaphase
  14. Anaphase
  15. Telophase

Virtual Microscope Slides

  1. High and Low Cellularity
  2. Using the density of nuclei as a guide, find regions of high and low cellularity. This can be done at low magnification by thinking about the color that nuclei appear in H&E stains. Zoom in to confirm your answer. Note the magnifications at which you can distinguish cells and identify nuclei and rough ER.
  3. Rough Endoplasmic Reticulum
  4. Find cells in the slide that have large amounts of rough endoplasmic reticulum. What kind of material do you think these cells produce?
  5. Cell Membrane
  6. Find a region where you can discern the cell membrane between neighboring cells.
  7. Secretory Cells
  8. Zoom in to locate cells with secretory granules in their cytoplasm. Note that a portion of the cytoplasm of most cells contains closely packed 1 µm diameter acidophilic secretory granules.
  9. Mitosis
  10. Identify different stages of the cell cycle based on the structure and location of the chromosomes.

Pathology

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  1. Proliferation of Smooth ER
  2. Red Ragged Fibers

Quiz

  1. Identify the structures indicated by the arrows.
  2. Answer: 1: Nuclear membrane. 2: Nucleolus. The nucleus and and nucleolus stain dark blue in H&E samples due to the presence of DNA and rRNA, respectively. If you thought 1: plasma membrane and 2: nucleus, remember that the plasma membrane does not stain dark blue.
  3. Estimate the sizes of these cells and their nuclei.
  4. Answer: Cells: ~ 15 µm wide and ~ 21 µm tall. Nuclei: ~ 7 µm. Use red blood cells on right side of image as ruler.
  5. What is the function of these structures?
  6. Answer: Protein synthesis for secretion. This in an electron microscope image of rough ER. Note the dense circular structures (ribosomes) on the membrane.
  7. Identify the structures in the cytoplasm of these cells.
  8. Answer: Secretory granules.
  9. Which cells are more likely to be involved in protein secretion?
  10. Answer: Cells in panel A. Note the cytoplasm of cells in panel A is stained dark-blue, indicating a large amount of rough ER.
  11. Identify cells undergoing cell division.
  12. How might you design an experiment to trace the steps in the transport and processing of a lysosomal hydrolase? What would you expect your results to be? How does this differ from the results you would expect for a similar experiment designed to trace the steps in the processing of a protein component of the ribosome?
  13. Answer: Whatever experiment you design (ask Dr. J!), you would expect the lysosomal hydrolase to be translated on the RER and then enter the Golgi and lysosome. A ribosomal protein would be translated in the cytoplasmic ribosomes, transported to the nucleus for assembly into a ribosome, then transported out of the nucleus back to the cytoplasm.
  14. An alcoholic patient with epilepsy, who has been taking anti-seizure medication and who has not consumed alcohol since his diagnosis, relapses and begins to drink again. He is brought to the hospital because his seizures are becoming more frequent. Explain the etiology of the more frequent seizures on the cellular level.
  15. Answer: The SER breaks down the anti-seizure drugs so that they are no longer functional. An alcoholic patient would have an increased amount of SER because it is needed to detoxify the alcohol. The increased SER would cause the faster breakdown of the anti-seizure medication, which means that it wouldn't last as long as usual and he would be vulnerable to seizures during periods in which the drug has been metabolized but he has not yet taken another dose.