1. HISTOLOGY: INTRODUCTION
“What is going on ?”
Regions
Organs
Systems
Tissues
Parts
Cells
Connections
Organelles
Development
Pulling it together
Molecules
Functions

2. Images versus REALITY - Anatomy
In Anatomy, the source of the evidence - the essential point of reference - is
the cadaver for Gross &
the microscope slide for Histo
Bed-rock
As the physician is knowledgeably comfortable with the patient’s gross & microscopic structure and its implications, you will become confident at the cadaver & the microscope, and with the resulting images
TESTS focus on the cadaver, the slides, and interpreting images - identification, interpretation, & synthesis

3. MICROSCOPIC SLIDE Side view of slide
Glass coverslip
Tissue Section
Mounting medium
Label
Glass slide 1”X3”
Mounting medium: permeates section; fastens coverslip to slide; is clear; has refractive index as for glass

4. SLIDE USE - Cautions
Slides & Microscope remain in the teaching Lab, always!
GLASS IS FRAGILE ! Take care with individual slides & especially with the boxes of slides
The slide must go on the stage coverslip up
The high-dry & oil objectives cannot focus through the thickness of the slide to the section
Label ~
The label may have been put on the non-coverslip side, as shown

5. SLIDE PREPARATION I Steps
Excise & Fix (preserve) the tissue in fixative
Remove the water & replace with wax-solvent
Imbed the oriented specimen in molten wax
After it is solid, hold the wax block & cut slices
Mount the thin slices (sections) on slides
When dry, remove the wax, & stain the section
Remove surplus stain & water; mount coverslip
When mounting medium has set, do microscopy

6. Remove the water & replace with wax-solvent
Imbed the oriented specimen in molten wax
50 % ethanol
70 % ethanol
95 % ethanol
Dehydrating series
label
100 % ethanol
Fresh tissue
10% Formalin fixative
benzene/xylene
Miscible with ethanol; dissolves wax
paraffinwax

7. After it is solid, hold the wax block & cut slices
Knife
Section
Block
Glass slide
MICROTOME - a fancy meat-slicer - holds the wax block, & cuts off thin slices, as the block is slowly advanced mechanically
Water-bath
Mount the thin slices (sections) on slides
Lift out floating section on the slide

8. Mount the thin slices (sections) on slides
For fast biopsy, imbedding is omitted - frozen sections
Knife
Section
Block is the tissue
Glass slide
FREEZING MICROTOME holds the frozen tissue, & cuts off thin slices, as the block is slowly advanced mechanically
Water-bath
Mount the thin slices (sections) on slides
Lift out section on the slide

9. Stain with Hematoxylin - blue
When dry, remove the wax, & stain the section
Dissolve paraffin wax
Stain with Hematoxylin - blue
Wash
Stain with eosin - red
Wash
Nuclei - blue
Cytoplasm- red

10. Stain with Hematoxylin - blue
When dry, remove the wax, & stain the section
Dissolve paraffin wax
Stain with Hematoxylin - blue
Wash
Potassium+ eosinate- stain charged amine, etc, groups on proteins bind -eosin “Acidophilic staining”
Stain with eosin - red
Wash
Nuclei - blue
Cytoplasm- red
“Basophilic”

11. SLIDE PREPARATION III Steps
Excise & Fix (preserve) the tissue in fixative
Remove the water & replace with wax-solvent
Imbed the oriented specimen in molten wax
After it is solid, hold the wax block & cut slices
Mount the thin slices (sections) on slides
When dry, remove the wax, & stain the section
Remove surplus stain & water; mount coverslip
When mounting medium has set, do microscopy

12. KEY TO SLIDE LABELING: Slide SET number: same as cabinet and
Slide number J SET microscope number
PARATHYROID Tissue or organ
Source of tissue Human
H & E Stain

13. SLIDE PREPARATION IV Artifacts
Images versus REALITY Artifacts are appearances not true to the original state of the tissue
Excise & Fix (preserve) the tissue in fixative
Bruising/splitting from cutting; Poor preservation, e.g., gut lining, enzymes, lost fat
Imbed the oriented specimen in molten wax
Misleading orientation, Shrinkage & distortion, Mislabeled
After it is solid, hold the wax block & cut slices
Knife scores, chatter
Mount the thin slices (sections) on slides
Wrinkles, section not flat, splits
When dry, remove the wax, & stain the section
Weak/unbalanced staining
Remove surplus stain & water; mount coverslip
Dirt, hair, bubbles
When mounting medium has set, do microscopy
Dirt on lenses, bad illumination

14. . HS 303 Histology Laboratory: The basic agenda
1 Today, Friday, we’ll have a little theory about how microscope slides are made and marked for identification, and a quick review of the light microscope and its controls and use.
2 Then, we’ll go to the lab, 4023, to find our assigned places with a key to the microscope locker and the drawer with the slide sets.
3 The individual slide sets will be checked against an inventory (not all slides are present) and the completed inventory sheets handed in.
4 Using a well-stained slide, we’ll try using the microscope and getting binocular fusion of the images (not everyone will the first time).

15. 5 On Monday, we shall try the microscope on some slides showing the less usual methods of tissue preparation, and we’ll get some first impressions of how tissues and cells look with various staining methods. What one stains for is what one sees, and the picture is always a very partial one. The figures specified in the text-atlas give one an initial idea of what to expect. The standing assignment is to read in advance the chapter matching the lecture topic, at the least, to have read the text going with the plates. Other textbooks can substitute for Ross & Romrell.
6 Also on Monday, we shall start thinking about cells as the fundamental component of organ systems, and taking a preliminary look at them in some slides. However, the electron microscope (EM) is far better than the light microscope for seeing cell structure, so that here the ‘lab’ exercise extends beyond lab time, being mostly one of your looking at cytology pictures in the textbook and atlases and using the histology computer program (Histology Lab Assistant) in the SBLC.
7 On Thursday, we’ll start the first tissue - Epithelium, and the lab will involve searching designated slides for examples of what has been shown as 35-mm projected slides in the lecture. Read the ‘Epithelium’ chapter beforehand.

16. 8 The point of a lab is to tackle messy reality rather than looking at pretty pre-packaged views. [You know the theory of cardiovascular and respiratory systems, but can you confidently do CPR?] Mixed in with, or at the end of, most lists of things to find in a lab session, are comments on how the material fails to connect with the theory. Skim through these comments before going systematically down the list. By the end of the Module, you will be competent in the skills of microscopy and reading slides, will appreciate the significance of cell and tissue structure for normal function and pathology, and should know enough to pass the boards.
9 The assessments and exams will have typical written questions - you will be provided with examples and can get them from previous years. The histology lab exam involves your going around microscopes and EM figures set up in the lab in numbered sequence, with just over a minute per station. Answer the question on the card. The kinds of question we ask are at the end of the Neural Tissues section. Frame your lab thinking from the start in terms of questions by reformatting the neural examples for each tissue.
10 After the first exam, life gets easier. (a) We are on organ systems, whose components and physiology you know. (b) The light microscope is operating at its most favorable, showing how tissues and special cells are organized to make up an organ. (c) We have left behind the initially difficult mixture of three levels of analysis - cytology, tissues, & organs - to concentrate on organs.

17. CLASS LIGHT MICROSCOPE
Eyepiece/Ocular
Objective lenses
Max MAGNIFICATION
Eyepiece (10X) times ‘Oil’ Objective (100X) = 1000X
Stage
Slide
Body
Condenser
Base
Light source

18. CLASS LIGHT MICROSCOPE Controls I
Eyepiece/Ocular
Inter-ocular distance
Objective selection
Iris diaphragm
Slide
Body
Coarse & Fine focus
Condenser
Moving stage
Light intensity
Base
Field diaphragm
Light
On/Off
left rear

19. CLASS LIGHT MICROSCOPE Controls II
Ocular focusing
Eyepiece/Ocular
Base
Stage clip for slide
Condenser focusing
left-side
Slide
Body
Condenser
Condensercentering
Light

20. Base
Condenser
Eyepiece/Ocular
Slide
Light
Body
Inter-ocular distance
Moving stage
Iris diaphragm
Field diaphragm
Coarse & Fine focus
Light intensity
On/Off
Objective selection
OPERATION I
Without looking down the eyepieces, plug in the cord Turn the light-intensity knob back counterclockwise, Switch on the light, turn the intensity up (about a 90o turn) while observing the light via the field opening Open the field diaphragm wide Move the condenser assembly to its top position Switch the shortest objective lens (X4) into the working position Open the iris diaphragm wide Select any well-stained slide

21. Base
Condenser
Eyepiece/Ocular
Slide
Light
Body
Inter-ocular distance
Moving stage
Iris diaphragm
Coarse & Fine focus
Light intensity
On/Off
Objective selection
OPERATION II
Field diaphragm
Pull back the clip & place slide, cover-slip up, on the stage Use the stage controls to bring the stained section over the light Focus, using coarse, then fine adjustments Close the iris diaphragm to take the glare out of the view Push (pull) the eyepieces together to match your eye spacing Shut one eye, focus with the fine focus; then shut that eye, open the other, and focus for it with the ocular focus (turning the eyepiece knurled ring) Switch in the next higher objective, and focus, using the main focusing controls & testing for binocular fusion

22. TODAY’s LAB PROCEDURES 1
On the 4th floor, straight back from the working elevator, go to the SBLC (Rm 4005) and LEAVE your coat (in the main lab, there are papers & envelopes on the desks that can get hidden or swept off, if coats are brought in today)
Take only your book-bag (& computer, if you have it with you) into Locate your place - labeled in alphabetical order from the far end of the lab.
Find the envelope with your name & the set of inventory forms.
Take the small key out of the envelope and attach it to a key ring or something.
use the key gently to open the top drawer at your place & take out the two small boxes of slides. Place them near the middle of the bench. Open the locker, and carefully lift out the microscope.
Satisfy your curiosity - Find the microscope-use directions p.2 . Follow them with any well-stained slide. Ask us for help. [We will not issue oil for the X100 oil lens]
Switch off the scope. Complete the receipt form. Go to p. ? for convention on slide numbering. Check your slide boxes against the inventory.
Hand in the inventory. Put your scope & slides away carefully. Lock the drawer & locker. About half of you share with a dental student. Please be considerate .

23. HISTOLOGY LECTURE POWERPOINTS
WVU Dept of Neurobiology & Anatomy
HISTOLOGY LECTURE POWERPOINTS
William A Beresford
HS Spring, 2002

24. SMEAR - another method of preparation
Drop of blood
Slide 1
Slide 2
Slide 2
On contact, slide 2 extends the drop along its 1” side
Slide 2
Pushing angled slide 2 along #1 smears the line of blood across slide 1
Smear
A few cells are damaged; smear is not evenly thick; & staining is uneven. Same apply to SPREADS
Lift away slide 2; dry #1 ; stain; coverslip

25. TEASING - a method of preparation
Terminal thread
(Filum terminale)
Roots
Lumbo-sacral cord
A technique you know from using a needle to separate out the connective-tissue filum terminale from the nervous cauda equina of dorsal & ventral roots
On the MICROSCOPE SLIDE, with a needle point one can tease apart individual nerve or muscle fibers from their bundles in nerve or muscle
When tissue is already thin, it can be draped -SPREAD - over the slide like a tablecloth

26. GROUND PREPARATION Lay sector flat & grind thin
Cut across BONE shaft twice
Saw out a sector
Wash ground section
Dry ; place unstained on slide
Coverslip for viewing

27. CELL DESCRIPTION What is one looking for?
Cell Shape?
eosinophil
Nuclei - #?
Cell Size?
Nucleus - size, shape, density?
osteoclast
Cytoplasm -philia?
Cytoplasm - granular?
Cell membrane - visible?
Cell surface specializations?
collecting duct
Nucleus -position?
Cells’ relations?
neuron
Nucleoli -prominence , #?
airway lining
Basal lamina

28. GO GRANULAR
Cerebellar Granule layer packed, small neurons- granule cells (& granulosa cells in ovary)
Layer
Bas
Eos
PMN
Blood Granulocytes from their very granular cytoplasm
Cell
Melanin granules in melanocytes & keratinocytes
Granule

29. Some differences between light and electron microscopy I
LIGHT MICROSCOPY ELECTRON MICROSCOPY
Section thickness (1-30 mm) gives Very thin sections provide no
a little depth of focus for depth of focus, but 3-D information
appreciation of the third dimension can be had from: (a) thicker sections
Serial sections can be cut, viewed by high-voltage EM; (b) shadowed
and used to build a composite image replicas of fractured surfaces; (c)
or representation scanning electron microscopy (SEM).
Most materials and structures cannot Heavy metal staining gives a more
be stained and viewed at the same comprehensive picture of membranes,
time; stains are used selectively to granules, filaments, crystals, etc.;
give a partial picture, e.g. a stain but this view is incomplete and even
for mucus counterstained to show visible bodies can be improved by
cell nuclei varying the technique.
Specimen can be large and Specimen is in vacuo. Its small size
even alive creates more problems with sampling
and orientation.

30. Some differences between light and electron microscopy II
LIGHT MICROSCOPY ELECTRON MICROSCOPY
Image is presented directly to the Image is in shades of green on
eye. Image keeps the colours given the screen; photographically,
the specimen by staining only in black and white.
Modest magnification to X 1500; High magnification,up to X 2,000,000
but a wider field of view and easier thus the range of magnification
orientation is greater
Resolving power to 0.25 mm Resolving power to 1 nm (0.001 mm.)
Frozen sections can yield an image Processing of tissue takes a day at
within 20 minutes least.
Crude techniques of preparation High resolution and magnification
introduce many artefacts demand good fixation (e.g. by
(Histochemical methods are better.) vascular perfusion), cleanliness
and careful cutting, adding up to
fewer artefacts.

31. HISTOLOGY SOURCES
303 Human Structure Syllabus next to last section p. ? Powerpoints - Comments & Standing assignment
Histo Powerpoints Histology Full-text* & Histology Lab Guide
Recommendation - catch it while you can: download the above this week. We’re talking about 50 megabytes, and some of the above items could fit on floppies.
WebBoard at Course 303 on Anatomy Dept site
SBLC computers have “Histology Lab Assistant”
It is never too soon to attune yourself to examiners’ thinking. Syllabus p. # presents the formats in which Histo lab exam questions will be framed

32. FORMAT OF LAB EXAM QUESTIONS IN HISTOLOGY with nervous-system examples
By each microscope or electron micrograph will be a card with a number(s) and question(s).
How do you know to what the question on the card refers? Assume the object is at the tip of the eyepiece pointer.
However, the card may specify: at the pointer; around the pointer; filling the field (of view); filling most of the field; or
at the orange arrow (on an electron micrograph). A poorly directed question could be 'What fills the field?' (with white and gray matter in the field). If in doubt about the question, put up your hand, and we shall hold the clock while the problem is being resolved.
[Yes. Stations are limited to around 1.25 minutes each.]
Question Sample answer Location
1 Name the tissue Simple columnar epithelium Spinal ependyma
Fairly dense irregular C.T Epineurium
2 ID the structure? Mesaxon; perineurium; Meissner's
corpuscle; synaptic vesicles; etc.

33. Question Sample answer Location
3 Name the structure Nerve (myelinated)
filling most of the field Motor end-plate Endplate in TEM
Node of Ranvier Node in TEM
4 Name the organ Autonomic ganglion;
Cerebellum*
5 The material? Myelin;
Mainly RNA Nucleolus
6 What process is Myelination;
occurring Wallerian degeneration;
CSF production Choroid plexus
7 What is the cell? Purkinje cell; satellite cell;
Spinal ventral-horn motoneuron;
Schwann cell; etc.

34. The lesson is that one should read carefully the question(s) on each card and answer precisely what is asked.
* For histology, parts of the brain and organs-within-organs, such as blood vessels, parasympathetic ganglia, nerves, and lymph nodes, count as organs. Question #1 on tissues is more useful for epithelia and c.t. than nervous tissue.

35. “Please take your zillion+ cells elsewhere. I’m an Ameba doctor.”
Did I choose the right medical school?
“Please take your zillion+ cells elsewhere. I’m an Ameba doctor.”
****
****
Complete Ameba Medicine 10 4 ed. Pp 29

36. MONDAY’s LAB PROCEDURES 2
Start the exercise on p.3. The smear is difficult to focus on. It needs at least the medium-power X10 0bjective; and the glare has to be taken out of the view with the iris diaphragm on the condenser.
The last part of the Lab on “The General Structure of Cells” is illustrated on the next slide, which indicates some of the variables, but does not show much of the extracellular matrix outside cells, e.g., basal lamina, collagen fibers, reticular fibers.
The last exercise - Artifacts - should be straightforward
In this and future labs, do not get hung up on a slide. If you cannot get your question answered in a minute or so, go on to the next slide; and come back later, when the question can be answered. Remind yourself with a note by the item.
The idea behind the final exercise of this Introductory section is inexhaustible. One can go on and on about cells. Stop when you wish, and come back to it during the next two labs. .

37. Test Pattern for the Apocalypse