histology introduction
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HISTOLOGY: INTRODUCTION WABeresford
Regions Organs
Molecules
Tissues
Connections Cells
Parts
OrganellesDevelopment
Functions
Systems
“What is going on ?”
Pulling it together
Noon talks for Internal-Medicine residents’ Board prep
Two recurring themes --
Is it what it appears to be ?
Does the treatment/procedure do what is claimed for it ?
What is the evidence?
MEDICINE: Some aspects
Regions
Molecules
Tissues
Connections Cells
Parts
OrganellesDevelopment
Functions
What gives with this patient?
Regions
SystemsSystems abnormal
Parts
Connections
Development
Tissues
Cells
Organelles
MoleculesFunctions
Microbes
Medicines
AgePopulations
Costs
?
GenderOrgansOrgans Abnormal
Abnormal variants for all the earlier fields of knowledge
Developing judgment - weighing various contributions for relevance & quality of evidence
Foretaste of the ‘pulling it together’ in the PBL experiences, but much omitted, e.g., therapy, follow-up, cost; likewise for clinical correlations
This doubling, plus more fields, e.g. microbes, is why medical training takes several years
Any twit can lay hands on an LCD projector, and push images at you reminds one that the story may be faulty; it is one of many; and there are omissions
?
Feel for the aspects that yield valid risk factors in this particular diagnosis
PORNOGRAPHY & “THE REAL THING”
Images versus REALITY
What is the evidence for the real?
Images versus REALITY - Functional Anatomy
REALITY is the living person, often via images
Surface anatomy Palpation Endoscopy+ Radiology PET scans Ultrasound Doppler flows Gait & Reflexes etc
Biopsies Fine-Needle Aspiration Cervical, Blood, etc Smears Flow cytometry & cell sorting Cell culture & grafting etc
(Bits cut or sucked out for microscopy)
REALITY is the dead person
DISSECTION [Surface anatomy Endoscopy Palpation Radiology Ultrasound are sometimes useful as adjuncts to autopsy & histology correlations]
Organs and large pieces cut out, examined, & prepared for MICROSCOPY- histology & histopathology (normal & altered side-by-side)
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
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
Bed-rock
MICROSCOPIC SLIDE Side view of slide
Glass coverslip
Glass slide 1”X3”
Tissue Section
Mounting medium
Mounting medium: permeates section; fastens coverslip to slide; is clear; has refractive index as for glass
Label
SLIDE USE - Cautions
GLASS IS FRAGILE ! Take care with individual slides & especially with the boxes of slides
Label
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
The label may have been put on the non-coverslip side, as shown
~
Slides & Microscope remain in the teaching Lab, always!
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
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
50 % ethanol 70 %
ethanol95 % ethanol
100 % ethanol
benzene/xylene
Dehydrating series
paraffinwax
Remove the water & replace with wax-solventImbed the oriented specimen in molten wax
Miscible with ethanol; dissolves wax
Fresh tissue
10% Formalin fixative
label
MICROTOME - a fancy meat-slicer - holds the wax block, & cuts off thin slices, as the block is slowly advanced mechanically
Block
Knife
Section
Glass slide
Water-bath
After it is solid, hold the wax block & cut slices
Mount the thin slices (sections) on slides
Lift out floating section on the slide
FREEZING MICROTOME holds the frozen tissue, & cuts off thin slices, as the block is slowly advanced mechanically
Block is the tissue
Knife
Section
Water-bath
Glass slide
For fast biopsy, imbedding is omitted - frozen sections
Mount the thin slices (sections) on slides
Lift out section on the slide
Dissolve paraffin wax
Stain with Hematoxylin - blue
Wash
Stain with eosin - red
Nuclei - blue
Cytoplasm- red
Wash
When dry, remove the wax, & stain the section
Dissolve paraffin wax
Stain with Hematoxylin - blue
Wash
Stain with eosin - red
Nuclei - blue
Cytoplasm- red
Wash
When dry, remove the wax, & stain the section
Potassium+ eosinate- stain + charged amine, etc, groups
on proteins bind -eosin “Acidophilic staining”
“Basophilic”
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
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
Images versus REALITY Artifacts are appearances not true to the original state of the tissue
SLIDE PREPARATION IV Artifacts
Excise & Fix (preserve) the tissue in fixative
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
Knife scores, chatter
Bruising/splitting from cutting; Poor preservation, e.g., gut lining, enzymes, lost fat
Wrinkles, section not flat, splits
Weak/unbalanced staining
Dirt, hair, bubbles
Dirt on lenses, bad illumination
Misleading orientation, Shrinkage & distortion, Mislabeled
CLASS LIGHT MICROSCOPE
Max MAGNIFICATION
Eyepiece (10X) times
‘Oil’ Objective (100X)
= 1000XBase
Eyepiece/Ocular
StageSlide
Light source
Body
Objective lenses
Condenser
CLASS LIGHT MICROSCOPE Controls I
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
left rear
CLASS LIGHT MICROSCOPE Controls II
Base
Condenser
Eyepiece/Ocular
Slide
Light
Body
Stage clip for slide Condenser
focusing
Condensercentering
Ocular focusing
left-side
OPERATION I
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
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
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
Base
Condenser
Eyepiece/Ocular
Slide
Light
Body
Inter-ocular distance
Moving stage
Iris diaphragm
Coarse & Fine focus
Light intensity
On/Off
Objective selection
SMEAR - another method of preparationDrop of blood
Slide 1Slide 2
On contact, slide 2 extends the drop along its 1” side
Slide 2
Slide 2
Pushing angled slide 2 along #1 smears the line of blood across slide 1
Lift away slide 2; dry #1 ; stain; coverslip
SmearA few cells are damaged; smear is not evenly thick; & staining is uneven. Same apply to SPREADS
TEASING - a method of preparation
Lumbo-sacral cord
Roots
Terminal thread
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
(Filum terminale)
Cut across BONE shaft twice
Saw out a sector
Lay sector flat & grind thin
Wash ground section
Dry ; place unstained on slide
Coverslip for viewing
GROUND PREPARARTION
HISTOLOGY SOURCES
303 Human Structure Syllabus next to last section p.8 Powerpoints - Comments & Standing assignment
Histo Powerpoints Histology Full-text* & Histology Lab Guide http://wberesford.hsc.wvu.edu http://www.geocities.com/Athens/Academy/1575 *
Recommendation - catch it while you can: download the above this week. We’re talking about 30 megabytes, and some of the above items could fit on floppies.
It is never too soon to attune yourself to examiners’ thinking. Syllabus p. 56 (lower-right #) presents the formats in which Histo lab exam questions will be framed
SBLC computers have “Histology Lab Assistant”
WebBoard at Course 303 on Anatomy Dept site
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)
TODAY’s LAB PROCEDURES 1
Take only your book-bag (& computer, if you have it with you) into 4023. 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. Open the envelope , remove the slip with the number to your Gross locker in the hallway. Write down this number in two other places.
Take the small key out of the envelope and attach it to a key ring or something. If you are worried about your coat, or if this is already too much “structure”, take a break to try your locker combination and put your coat and other surplus stuff away.
Once settled back at your place, 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. 1 for slide numbering. Check your slide boxes against the inventory. Place forms in the wall folder .
Start the exercise on p.3, but with slide A-1 blood smear LAST of the four on examples of preparation methods. 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.
TODAY’s LAB PROCEDURES 2
The next exercise - Artifacts - should be straightforward.
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. Skip C-1. The cells are poor examples of neurons.
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.
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.
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
collecting duct
osteoclast
CELL DESCRIPTION What is one looking for?
Cell Size?
Cell Shape?Nuclei - #?
Nucleus - size, shape, density?
Nucleoli -prominence , #?
Nucleus -position?Cells’ relations?
Cytoplasm - granular?
Cytoplasm -philia?
Cell membrane - visible?Cell surface
specializations?
Basal lamina
neuron
eosinophil
airway lining
GO GRANULAR
Cerebellar Granule layer packed, small neurons- granule cells (& granulosa cells in ovary)
Melanin granules in melanocytes & keratinocytes
BasEosPMN
Blood Granulocytes from their very granular cytoplasm
Layer
Cell
Granule
Some differences between light and electron microscopy I
Light microscopy Electron microscopy----------------------------------------------------------------------Section thickness (1-30 m) gives Very thin sections provide noa little depth of focus for depth of focus, but 3-D informationappreciation of the third dimension. can be had from: (a) thicker sectionsSerial sections can be cut, viewed by high-voltage EM; (b) shadowedand 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 morebe 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 evenfor mucus counterstained to show visible bodies can be improved bycell nuclei. varying the technique.
Specimen can be large and Specimen is in vacuo. Its small sizeeven alive. creates more problems with sampling and orientation.
Some differences between light and electron microscopy II
Light microscopy Electron microscopy--------------------------------------------------------------------------
Image is presented directly to the Image is in shades of green oneye. 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 2,000,000but a wider field of view and easier thus the range of magnificationorientation is greater
Resolving power to 0.25m. Resolving power to 1 nm (0.001 m.)
Frozen sections can yield an image Processing of tissue takes a day atwithin 20 minutes. least.
Crude techniques of preparation High resolution and magnificationintroduce many artefacts. demand good fixation (e.g. by(Histochemical methods are better.) vascular perfusion), cleanliness and careful cutting, adding up to fewer artefacts.
****
Did I choose the right medical school?
****Complete Ameba Medicine 10 4 ed. Pp 29
“Please take your zillion+ cells elsewhere. I’m an Ameba doctor.”
Test Pattern for the Apocalypse