histology introduction

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

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



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


Lift out section on the slide

Dissolve paraffin wax

Stain with Hematoxylin - blue

Wash

Stain with eosin - red

Nuclei - blue

Cytoplasm- red

Wash


Dissolve paraffin wax

Stain with Hematoxylin - blue

Wash

Stain with eosin - red

Nuclei - blue

Cytoplasm- red

Wash


Potassium+ eosinate- stain + charged amine, etc, groups

on proteins bind -eosin “Acidophilic staining”

“Basophilic”

SLIDE PREPARATION III Steps


Remove the water & replace with wax-solvent







Images versus REALITY Artifacts are appearances not true to the original state of the tissue

SLIDE PREPARATION IV Artifacts








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


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


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

histology introduction

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