1. Fundamental Tissues and Mitosis
Medical School Histology
Larry Johnson Texas A&M University
Part I
Types of microscopy, tissue preparation, and staining

2. Objectives Part I Operate the “virtual microscope”
Describe steps for tissue processing and discuss different types of microscopy
Interpret cytological significance of differential staining by hematoxylin and eosin (H&E)
Recognize and identify cells in microscopic tissue preparations
Understand that morphology reflects the function of cells
Identify stages of mitosis and cellular structures associated with mitosis
From: Douglas P. Dohrman and TAMHSC Faculty 2012 Structure and Function of Human Organ Systems, Histology Laboratory Manual

3. The Microscope Light (bright field) used with stained specimens

4. Kohler illumination Focus specimen Close field diaphragm
Focus condenser
Center condenser
Open field diaphragm

5. Types of Microscopy Light (bright field) Phase Contrast
Most common, used to observe stained sections.
Phase Contrast
Used to observe living cells, cells appear as black and white image
Nomarski (differential interference)
Similar to phase contrast, but greater resolution of 3D appearance and optical sectioning
Dark Field
Illuminates cells and tissues against dark background to allow for contrast
Transmission Electron Microscopy (TEM)
Magnifies image to allow visualization of intracellular structures
Scanning Electron Microscopy (SEM)
Scans cell surfaces at great magnifications providing highly resolved surface details

6. Bright Field Phase Contrast Nomarski Dark Field
Stained dead cells
Phase Contrast
Live unstained cells
Nomarski
Differential interference contrast
Dark Field
Dark, contrasting background

7. Types of Electron Micrographs Conventional TEM, SEM carbon replica TEM
EM 2b
EM 4a
EM 18b
Types of
Electron
Micrographs
EM 6
EM 7
EM 8

8. Types of Electron Micrographs Conventional TEM, SEM carbon replica TEM
EM 2b
EM 4a
EM 18b
Types of
Electron
Micrographs
EM 6
EM 7
EM 8

9. Types of Electron Micrographs Conventional TEM, SEM carbon replica TEM
EM 2b
EM 4a
EM 18b
Types of
Electron
Micrographs
EM 6
EM 7
EM 8

10. Transmission Electron Microscopy (TEM)

11. Scanning Electron Microscopy (SEM)

12. Transmission Electron Microscopy (TEM)
Comparing Magnifications
EM 8f 9,000x
EM 12a 13,200x
Compare the magnification by
comparing sizes of
membranes
ribosomes
mitochondria
EM 4c 60,000x
EM 7 80,000x

13. EM 6a 200,000x
EM 2b 60,000x
Compare sizes of
membranes
ribosomes
mitochondria
EM 7 80,000x

14. Tissue Preparation
Fixation – Chemically prevents autolysis of cells and preserves morphology
Dehydration – organic solvents
Embedding –paraffin or plastic used to stiffen tissue
Sectioning – cutting thin sections (1-10 microns thick)
Staining – different stains react with different biochemical components of cells
Mounting – preserve section
Histological viewing – using microscope

15. Sample Preparation Fixation Embedding Sectioning
A. Paraffin
B. Plastic
Sectioning
A. 0.5 um for Light Microscopy
B nm for Electron Microscopy

17. Differential Staining Properties
Slide 46
Hematoxylin and Eosin (H&E) are the most widely used stains
Hematoxylin
Blue-black dye with basic pH, preferentially binds acid molecules
Nuclear DNA and cytoplasmic ribosomes darkly stain with hematoxylin based on density of material
Eosin
Red dye with acidic pH, preferentially binds basic molecules
Proteins (amines) stain with eosin based on density of material
Dark blue nuclei of lymphocytes
Slide 55
Red cytoplasm of smooth muscle cells

18. Hematoxylin and Eosin (H&E) Stain
Slide 32: Kidney (H&E)

19. Periodic Acid-Schiff (PAS)
Used to observe saccharides (hot pink of brush border and basement membrane)
Slide 33: Kidney (PAS)

20. Staining Light Microscopy Electron Microscopy (TEM)
Hematoxylin and Eosin (H&E)
B. Periodic Acid/Shiff (PAS)
Toluidine Blue
Electron Microscopy
(TEM)
A. Osmium
Lead Citrate

21. Staining Color provides clues Shape Size Intensity of staining
Light Microscopy
Hematoxylin and Eosin (H&E)
Periodic Acid/Shiff (PAS)
C. Toluidine Blue
Color provides clues
Shape
Size
Intensity of staining

22. Staining Light Microscopy Electron Microscopy (TEM)
Hematoxylin and Eosin (H&E))
B. Periodic Acid/ (PAS)
Electron Microscopy
(TEM)
A. Osmium
Lead Citrate
Lead citrate

23. Artifacts
Artifacts are man-made distortions and aberrations of tissue structure
Artifacts are fairly common, beware!
Slide 32 : Kidney
Artifacts

24. Identifying Cells
Cells < Tissues < Organ < Organ System < Human
Somatic vs. Germ Cells
Morphology accomplishes specific function
Distinct features of cell
Nucleus: shape is indicative of cell morphology
Cytoplasm: Contains various organelles such as RER, Golgi, and mitochondria
diFiore’s Atlas of Histology with Functional Correlations
Victor P. Eroschenko

26. Fundamental Tissues and Mitosis
Medical School Histology
Part II
Images of light and electron microscopy

27. Objectives Part II Operate the “virtual microscope”
Describe steps for tissue processing and discuss different types of microscopy
Interpret cytological significance of differential staining by hematoxylin and eosin (H&E)
Part II
Recognize and identify cells in microscopic tissue preparations
Understand that morphology reflects the function of cells
Identify stages of mitosis and cellular structures associated with mitosis
From: Douglas P. Dohrman and TAMHSC Faculty 2012 Structure and Function of Human Organ Systems, Histology Laboratory Manual

28. Laboratory Experience
Primary purpose of this lab is to gain a general impression of similarities and differences in cells and tissues and what can be inferred from that
Do not be too concerned about detailed structure, but rather try to gain an overall appreciation for histology
USE
YOUR
ATLAS!

29. Slide 29: Skin
Epithelial skin cells
Collagen fibers

30. Slide 29 : Skin

31. Slide 51: Tongue (cross section)
Skeletal muscle
Serous glands
Adipocytes

32. Slide 51 : Tongue (cross section)
Serous glands
Adipocytes
Skeletal muscle

33. Slide 32 : Kidney
Proximal convoluted tubules with microvilli lining lumen

34. Slide 75: Thyroid
Spherical epithelial follicles containing thyroglobulin
Simple cuboidal epithelium
Extracellular collagen fibrils
Blood vessel with red blood cells
Simple squamous epithelium lining blood vessel

35. Slide 70: Pancreas Zymogen granules Pancreatic acinus
Glandular cell with euchromatic nucleus and basophilic base

36. Slide 70: Pancreas Zymogen granules Pancreatic acinus
Glandular cell with euchromatic nucleus and basophilic base

37. Endoplasmic Reticulum
Granular ER
Granular ER
Nuclear envelope
Granular ER
Nuclear pore
Nuclear pore
Nucleolus
Nucleus
Nuclear envelope

38. Granular vs. Agranular Endoplasmic Reticulum

39. What is the structure of the plasma membrane?

40. What is the structure of the plasma membrane?

41. Plasma Membrane

42. Identify pinocytotic vesicles
Plasma Membrane
Identify pinocytotic vesicles

43. Identify mitochondria

44. What is the structure and function of mitochondria?

45. Mitochondria & Microbodies
Could mitochondria and microbodies be confused morphologically?
What is the structure and function of microbodies?

46. Observe the ultrastructure of the Golgi complex

47. Digestion and Storage Vesicles
Lipid in SER
Secretory granules
Lysosomes

48. Digestion and Storage Vesicles
Lipofuscin granules
Glycogen
Glycogen
Microbodies

49. Axonemes encased in membrane
Cytoskeleton
Microtubules
Axonemes encased in membrane
Centrioles

50. Cytoskeleton
The microvilli in this micrograph contain what type of cytoskeletal element?
Hint: What is supporting the microvilli?
Lipid in SER

51. Cytoskeleton
The microvilli in this micrograph contain what type of cytoskeletal element?
Hint: What is supporting the microvilli?
Slide 33
Lipid in SER

52. Max Planck Institute for Neurological Research
Clinical Correlation
Cell death can occur due to apoptosis or necrosis
Apoptosis
Programmed cell death
Necrosis
Accidental cell death resulting from pathological condition
Max Planck Institute for Neurological Research

54. Fundamental Tissues and Mitosis
Medical School Histology
Part III
Images of mitosis

55. Objectives Part III Operate the “virtual microscope”
Describe steps for tissue processing and discuss different types of microscopy
Interpret cytological significance of differential staining by hematoxylin and eosin (H&E)
Recognize and identify cells in microscopic tissue preparations
Understand that morphology reflects the function of cells
Part III
Identify stages of mitosis and cellular structures associated with mitosis
From: Douglas P. Dohrman and TAMHSC Faculty 2012 Structure and Function of Human Organ Systems, Histology Laboratory Manual

56. Mitosis Mitosis is cellular multiplication
This is the means by which organisms grow
Cancer is essentially uncontrolled mitosis
We will look at onion root tip and fish egg cells to identify the stages of mitosis

57. Slide 1 : Onion root tip Interphase
Interphase nucleus

58. Slide 1: Onion root tip Prophase

59. Slide 1 : Onion root tip Metaphase
Spindle fibers

60. Slide 1 : Onion root tip Anaphase
Centrosomal area

61. Slide 1 : Onion root tip Telophase

62. Slide 2: Fish egg mitosis Interphase
Interphase nucleus

63. Slide 2 : Fish egg mitosis Prophase

64. Slide 2 : Fish egg mitosis Metaphase
Spindle fibers

65. Slide 2 : Fish egg mitosis Anaphase
Centrosomal area

66. Slide 2 : Fish egg mitosis Telophase

67. Describe the composition of the nucleolus and the nucleus.
EM 25 & 26
Describe the composition of the nucleolus and the nucleus.
What happens to the nucleolus and nuclear membrane (envelope) during mitosis?

68. EM 31
What is the function of centrioles?

69. Clinical Correlation Malignant vs. benign tumors
Increased mitotic figures and abnormal mitoses helps distinguish
Anti-mitotic drugs are used in cancer chemotherapy
Ex: Taxol
These drugs also effect normal cells with high proliferation rates causing undesirable consequences

70. Study Questions
Does a highly active cell contain a euchromatic or heterochromatic nucleus?
Would the cytoplasm of a cell that contains large quantities of protein stain eosinophilic or basophilic?
Is a rounded (spherical) nucleus indicative of a squamous, cuboidal or columnar cell?
Does hematoxylin preferentially stain chromatin or collagen? (2-3 sentences)
Are polysaccharides and mucopolysaccharides best stained with eosin, Sudan black, hematoxylin, or periodic acid – Schiff?
At which stage of mitosis do sister chromatids separate from each other?
During which phase of interphase does the first “checkpoint” occur? What is the significance of the p53 protein?
From: Douglas P. Dohrman and TAMHSC Faculty 2012 Structure and Function of Human Organ Systems, Histology Laboratory Manual

71. Use your atlas! Use your atlas! pancreas blood stomach
Atlas of Normal Histology by DiFiore
testis

72. Many illustrations in these VIBS Histology YouTube videos were modified from the following books and sources: Many thanks to original sources!
Bruce Alberts, et al Molecular Biology of the Cell. Garland Publishing, Inc., New York, NY.
Bruce Alberts, et al Molecular Biology of the Cell. Garland Publishing, Inc., New York, NY.
William J. Banks, Applied Veterinary Histology. Williams and Wilkins, Los Angeles, CA.
Hans Elias, et al Histology and Human Microanatomy. John Wiley and Sons, New York, NY.
Don W. Fawcett Bloom and Fawcett. A textbook of histology. W. B. Saunders Company, Philadelphia, PA.
Don W. Fawcett Bloom and Fawcett. A textbook of histology. Chapman and Hall, New York, NY.
Arthur W. Ham and David H. Cormack Histology. J. S. Lippincott Company, Philadelphia, PA.
Luis C. Junqueira, et al Basic Histology. Lange Medical Publications, Los Altos, CA.
L. Carlos Junqueira, et al Basic Histology. Appleton and Lange, Norwalk, CT.
L.L. Langley, et al Dynamic Anatomy and Physiology. McGraw-Hill Book Company, New York, NY.
W.W. Tuttle and Byron A. Schottelius Textbook of Physiology. The C. V. Mosby Company, St. Louis, MO.
Leon Weiss Histology Cell and Tissue Biology. Elsevier Biomedical, New York, NY.
Leon Weiss and Roy O. Greep Histology. McGraw-Hill Book Company, New York, NY.
Nature ( Vol. 414:88,2001.
A.L. Mescher Junqueira’s Basis Histology text and atlas, 13th ed. McGraw
Douglas P. Dohrman and TAMHSC Faculty 2012 Structure and Function of Human Organ Systems, Histology Laboratory Manual - Slide selections were largely based on this manual for first year medical students at TAMHSC

73. The End!