Reading: RR&K, Chapter 2
SLIDE 65a liver, human (H&E)
(RR&K p521, Fig. 1,2; p523, Fig. 1)
This slide contains a section of the human liver embedded in the paraffin and routinely stained with H&E. In this exercise you will compare a thick (6 um) paraffin section (SLIDE 65a) with a thin (1 um) plastic section (SLIDE 65e) described on the next page. Use the atlas in your textbook to orient yourself on the slide. Focus your attention on the hepatocytes which are primary cell types in the liver. Study these cells at low power (10X) and then with the high dry objective (40X) to determine which organelles are visible in this preparation (see table of organelles printed at the end of this lab). Usually, the hepatocytes have a polygon shape. The easiest organelle to see in this slide is the centralized round nucleus. The nucleus contains a distinct basophilic nucleolus surrounded by pale staining euchromatin. Basophilic heterochromatin is usually found at the periphery of the nucleus. Some cells contain round unstained lipid droplets. Why is the lipid unstained?
Check list for SLIDE 65a:
SLIDE 65e liver, rat (toluidine blue)
(RR&K p523, Fig. 2)
This slide contains a thin (1 um) plastic section of the rat liver, fixed in glutaraldehyde and osmium and stained with toluidine blue. Using the lowest magnification observe the general arrangement of the hepatocytes in the liver. Next, with high dry (40X) objective, examine the selected hepatocyte and then change to the oil immersion lens (100X). Observe the nucleus with darker-stained nucleolus and heterochromatin. The pale-stained euchromatin is clearly evident. Some cells contain round blue-stained lipid droplets. Why is the lipid stained? The numerous mitochondria are found in all hepatocytes. These light-blue organelles are smaller than the lipid droplets and do not stain as intensely as lipids. The mitochondria are best visualized using the oil immersion lens (100X). Areas containing glycogen stain magenta (purple) due to metachromasia. Compare images from the last two slides seen in your microscope with the electron micrograph of a conventionally prepared liver section found in your EM package FIGURE 01 (see next page for EM descriptions). Which preparation provides the sharpest cellular detail? and Why? Which organelles can be clearly seen in the paraffin section, the plastic section, and the electron micrograph? Can you recognize these in the electron micrographs?
Check list for SLIDE 65e:
FIGURE 01 Liver, Hepatocyte (EM) (RR&K p508, Fig. 17.10)
This figure represents two electron micrographs obtained at different magnifications. It can be seen in the upper panel at a lower magnification (5,000X) that the hepatocyte contains a centralized nucleus with many mitochondria and abundant cisternae of rough endoplasmic reticulum. In the lower panel at a higher magnification (20,000X), the nuclear envelope with nuclear pores can be observed. Find the mitochondria and observe the outer and inner membranes. In addition, mitochondrial cristae can be identified. The differences between the size and organization of membrane-bound ribosomes and glycogen is also evident. Smooth endoplasmic reticulum can be observed between glycogen granules.
Check list for FIGURE 01:
SLIDE 02a ascites, human (Pap)
A collection of fluid in the peritoneal cavity is called ascites. The peritoneal cavity can accumulate a large volume of ascitic fluid, which in turn produces a massive abdominal distention. Because of the discomfort for the patient and also for diagnostic purposes, fluid is drained by abdominal tapping. This slide provides you with the opportunity to observe the many single cells that were suspended in this abdominal fluid. Fluid for your slide was obtained from a 67-year-old white male with liver disease. Ascitic fluid usually contains squamous epithelial (mesothelial) cells, lymphocytes, red blood cells, neutrophils and may also contain free floating cancer cells. The suspension of cells on your slide was obtained by centrifugation of the ascitic fluid (cytospin), and the slide was stained with Pap stain (see stain section). Scan the slide with the 10X objective lens. On this slide you are looking at whole cells rather than sections, as observed in SLIDE 65e at the beginning of this laboratory exercise. Note the large number of red stained erythrocytes. The numerous small cells with little cytoplasm and darkly stained spherical nuclei are the lymphocytes. Occasionally, larger cells are observed that have a "spongy" cytoplasm and large nucleus. These cells have been referred to as reactive mesothelial cells. With the 40X objective lens in place, observe nucleoli within the nuclei of the mesothelial cells. Sometimes the mesothelial cells may occur in small clusters. The cytological diagnosis obtained from this slide was inflammation with reactive mesothelial cells and absence of abnormal (cancer) cells.
Check list for SLIDE 02a:
| Organelles and Cytoplasmic Inclusions: A key to LM and EM Identification | |||||
|---|---|---|---|---|---|
| Organelle or Inclusion | Size (um) | Observe in LM? | LM Features | EM Features | |
| nucleus | 3-10 | + | 1. largest structure inside cell 2. distinct boundary evident 3. nucleoli and chromatin often evident inside |
1. double bilayer and nuclear pore complexes 2. regions of dark and diffuse chromatin staining (hetero- and euchromatin) |
|
| nucleolus | 1-2 | + | dense, roughly circular region inside nucleus | dense, roughly circular region inside nucleus | |
| plasma membrane | 0.008-0.10 | - | none | trilaminar appearance of an inner and outer electron-dense regions with an intermediate electron-lucent region | |
| rough endoplasmic reticulum (rER) | often spans area ~5-10 on a side | -/+ | general region often observed as "ergastoplasm" | flattened sheets, sacs, and tubes of membranes with attached ribosomes | |
| smooth endoplasmic reticulum (sER) | throughout cytoplasm | - | none | flattened sheets, sacs, and tubes of membranes without attached ribosomes | |
| Golgi complex | spans area ~5-10 on a side | -/+ | general region sometimes observable as negative-staining region | stack of flattened membrane sheets, often adjacent to one side of the nucleus | |
| secretory vesicles | 0.050-1.0 | -/+ | only when very large (e.g. zymogen granules in slide 64a) | many relatively small, membrane-bounded structures of uniform diameter. Often polarized on one side of cell | |
| mitochondria | diameter ~2; length variable (<10) | -/+ | sometimes observed in favorable situations (e.g. slide 65e) as very small, dark dots | 1. organelle with two membrane systems: outer membrane and inner membrane (cristae) 2. in steroid secreting cells: tubular cristae |
|
| endosomes | 0.02-0.5 | - | none | tubules and vesicles containing electron lucent material or other smaller vesicles | |
| lysosomes | 0.2-0.5 | - | none | membrane-bounded region of dense staining | |
| peroxisomes | 0.2-0.5 | - | none | membrane-bounded region of dense staining | |
| cytoskeletal elements | 0.008-0.025 | -/+ | only observed when organized into large structures (e.g. muscle fibrils) | long, linear staining pattern with a width characteristic of each filament type | |
| ribosomes | 0.025 | - | none | very small, dark dots; often associated with the rough ER | |
| glycogen | 0.010-0.040 | -/+ | can see a purple haze (metachromasia) in an area containing glycogen with toluidine blue staining | very dense grape-like structures | |
| lipid droplets | 0.2 - 5, up to 80 | +/- | 1. readily visible when very large (e.g. adipocytes) 2. large hole in section (lipid itself is usually removed by embedding solvents) |
1. not surrounded by membrane 2. generally appears as a void in the section | |
| Organelles and Inclusions Functions and Pathologies | ||||
|---|---|---|---|---|
| Organelle or Inclusion | Function | Examples of Associated Pathologies | ||
| nucleus | storage and utilization of genome | including: 1. inherited diseases 2. environmentally-induced mutations |
||
| nucleolus | synthesis of ribosomal components | ? | ||
| plasma membrane | ion and nutrient transport, recognition of environmental signals, adhesion to ECM and other cells | cystic fibrosis | ||
| rough endoplasmic reticulum (rER) | chemical modifications of proteins destined for secretion or membrane insertion | pseudoachondroplasia calcium phosphate dihydrate crystal deposition disease |
||
| smooth endoplasmic reticulum (sER) | lipid metabolism | hepatic endoplasmic reticular storage disease | ||
| Golgi complex | chemical modification of proteins and sorting of molecules for secretion or transport to other organelles | I-cell disease polycystic kidney disease |
||
| secretory vesicles | transport of secreted proteins to plasma membrane | Lewy bodies of Parkinson's disease proinsulin diabetes |
||
| mitochondria | aerobic energy supply (oxidative phosphorylation; ATP) | mitochondrial myopathies such as: Myoclonic Epilepsy and Ragged Red Fibers Syndrome (MERRF1)Mitochondrial Myopathy, Encephalopathy, Lactic Acidosis, and Stroke-Like Episodes Syndrome (MELAS2), Kearns-Sayre Syndromes and Laber's hereditary optic atrophy |
||
| endosomes | transport of proteins | mannose-6-phosphate receptor deficiency | ||
| lysosomes | digestion of macromolecules | glycogen storage disease Type II Tay-Sachs disease metachromatic leukodystrophy |
||
| peroxisomes | oxidative digestion, e.g. fatty acids | Zellweger's Syndrome | ||
| cytoskeletal elements | various, including cell motility, intra- and extracellular transport, cellular skeleton | immotile cilia syndrome, Alzheimer's disease, epidermolysis bullosa | ||
| ribosomes | protein synthesis | note that many antibiotics selectively act on bacterial ribosomes | ||
| glycogen | storage of metabolites as carbohydrates | there are at least nine known glycogen storage diseases, including major groups of hepatic-hypoglycemic and muscle-energy pathophysiologies | ||
| lipid droplets | storage of metabolites in a high energy form | lipid storage diseases such as Gaucher's and Niemann-Pick disease | ||
Organisms consist of various structures and organs that are specialized for certain functions. Similarly, the cell contains many structures and organelles for various purposes. This unit is an introduction to cellular components.