A laboratory guide forhistologyA laboratory guide forhistology
刘尚明 武玉玲
IntroductionIntroduction
As other medical courses, the study ofhistology consists of two parts: lecturesand laboratory work. The purpose oflaboratory work is mainly to integratetheoretical knowledge with practical workand to train the ability of observing thestructures of cells, tissues and organs withmicroscope. As other medical courses, the study ofhistology consists of two parts: lecturesand laboratory work. The purpose oflaboratory work is mainly to integratetheoretical knowledge with practical workand to train the ability of observing thestructures of cells, tissues and organs withmicroscope.
Preparation and rules of laboratory workPreparation and rules of laboratory work
Preview the relevant text well before thelaboratory work and know the contents andobjects of each laboratory work.Preview the relevant text well before thelaboratory work and know the contents andobjects of each laboratory work.
Have the tools ready. These include microscope,slides, paper, pencils, rubber, ruler and so on;Have the tools ready. These include microscope,slides, paper, pencils, rubber, ruler and so on;
Keep the laboratory clean and quiet. Raise yourhand before asking the teacher. No spiting, nosmoking and no eating in laboratory.Keep the laboratory clean and quiet. Raise yourhand before asking the teacher. No spiting, nosmoking and no eating in laboratory.
Take good care of specimens and instruments.Take good care of specimens and instruments.
At the end of each laboratory work return theslides in order. Put the slide box and microscopeback where they belong. Clean the room and turnoff the light. Be sure that the windows are closedbefore you leave the laboratory.At the end of each laboratory work return theslides in order. Put the slide box and microscopeback where they belong. Clean the room and turnoff the light. Be sure that the windows are closedbefore you leave the laboratory.
The structure of the microscopeThe structure of the microscope
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Mechanical part:
1. Base
2. Stand
3. specimenholder
4. Observationtube
5. Nosepiece
6. Coarseadjustmentring
7. Fineadjustmentring
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Optical
components:
1. The sourcesof light
2. Condenserlens
3. Objectivelenses
4. Eyepiece
A. low power objectiveA. low power objective
1. Turn the power switch on, openthe iris diaphragm and center thelow power objective by turningthe nosepiece;1. Turn the power switch on, openthe iris diaphragm and center thelow power objective by turningthe nosepiece;
2. place a slide on the stage thencenter the object by adjusting themechanical stage；2. place a slide on the stage thencenter the object by adjusting themechanical stage；
3.Turn the coarse adjustment toraise the stage until the objectiveis about 0.5cm form the specimen,look through eyepieces and turnthe coarse adjustment slowly tolower the stage until thespecimen is seen；3.Turn the coarse adjustment toraise the stage until the objectiveis about 0.5cm form the specimen,look through eyepieces and turnthe coarse adjustment slowly tolower the stage until thespecimen is seen；
The use of microscopeThe use of microscope
The use of microscopeThe use of microscope
4. Adjust the interpupillary distance.Hold the 2 eyepiece tubes with yourboth hands to push the tubestogether or pull them apart, untilyou can see the same image withboth eyes；4. Adjust the interpupillary distance.Hold the 2 eyepiece tubes with yourboth hands to push the tubestogether or pull them apart, untilyou can see the same image withboth eyes；
5. Look at the image through the righteyepiece with your right eye andmake the specimen in sharp focusby turning the fine adjustment. Lookat the image through the lefteyepiece with your left eye androtate the tube length adjustmentring to focus on the specimen.5. Look at the image through the righteyepiece with your right eye andmake the specimen in sharp focusby turning the fine adjustment. Lookat the image through the lefteyepiece with your left eye androtate the tube length adjustmentring to focus on the specimen.
High power objectiveHigh power objective
After observing with lowpower objective enter thehigh power one. A slightturn of the fineadjustment will bring theimage into sharp focus.Don’t turn the coarseadjustment to avoiddamaging the specimenor the objective.After observing with lowpower objective enter thehigh power one. A slightturn of the fineadjustment will bring theimage into sharp focus.Don’t turn the coarseadjustment to avoiddamaging the specimenor the objective.
Oil immersion objectiveOil immersion objective
It requires more careful usethan the others.It requires more careful usethan the others.
First place a small drop ofcedar oil on the slide, thencenter the lens slowly. Turnthe coarse adjustment untilthe lens touches the oil andthen turn the fineadjustment upward ordownward to focus.First place a small drop ofcedar oil on the slide, thencenter the lens slowly. Turnthe coarse adjustment untilthe lens touches the oil andthen turn the fineadjustment upward ordownward to focus.
Note that the cedar oilmust be wiped off with lenspaper after using the oilimmerse lens.Note that the cedar oilmust be wiped off with lenspaper after using the oilimmerse lens.
The care of microscopeThe care of microscope
Check the microscope assigned to you. If youfind anything wrong, inform the teacherimmediately.Check the microscope assigned to you. If youfind anything wrong, inform the teacherimmediately.
Don not take apart of the microscope.Don not take apart of the microscope.
Keep the microscope clean. Clean lens with lenspaper only. Avoid blowing or wiping it with finger,rough paper or cloth.Keep the microscope clean. Clean lens with lenspaper only. Avoid blowing or wiping it with finger,rough paper or cloth.
Before replacing your microscope, be sure thatlenses or stage and condenser are at the lowestposition. Then cover the microscope with softsilk.Before replacing your microscope, be sure thatlenses or stage and condenser are at the lowestposition. Then cover the microscope with softsilk.
Procedure of observationProcedure of observation
First observe the shape, color and the featuresof the specimen with naked eyes.First observe the shape, color and the featuresof the specimen with naked eyes.
Observe the general structure and itscomponents with low power objective. Thenselect a representative area and observe it indetail with high power objective then with the oilimmersion lens when necessary.Observe the general structure and itscomponents with low power objective. Thenselect a representative area and observe it indetail with high power objective then with the oilimmersion lens when necessary.
Make a drawing of the representative area andlabel it clearly.Make a drawing of the representative area andlabel it clearly.
A drawing of the representative areaA drawing of the representative area
Interpret your sampleInterpret your sample
Must think in 3 dimensionsMust think in 3 dimensions
For best interpretation, must have serialsectioning and reconstruction.For best interpretation, must have serialsectioning and reconstruction.
Preparing a Biological Specimenfor Light MicroscopyPreparing a Biological Specimenfor Light Microscopy
1. Obtain sample and cut intoworkable pieces (1cm cube)1. Obtain sample and cut intoworkable pieces (1cm cube)
Biopsy, surgical excision,postmortem examinationBiopsy, surgical excision,postmortem examination
From animalsFrom animals
Taken quickly, use sharpinstrumentsTaken quickly, use sharpinstruments
2. Fixation2. Fixation
generally in reactive aldehydes likeformaldehyde.generally in reactive aldehydes likeformaldehyde.
This cross links macromolecules,particularly proteins This cross links macromolecules,particularly proteins
Has hardening effect on soft tissuesHas hardening effect on soft tissues
Done rapidly to prevent enzymes from livecells degrading the tissueDone rapidly to prevent enzymes from livecells degrading the tissue
3.Dehydrate the sample by runningthrough a series of increasingconcentrations of ethanol.3.Dehydrate the sample by runningthrough a series of increasingconcentrations of ethanol.
Paraffin (embedding material) is notwater soluble, so water is removedParaffin (embedding material) is notwater soluble, so water is removed
4. Clear the sample of ethanol andpass through several changes ofxylene4. Clear the sample of ethanol andpass through several changes ofxylene
Paraffin is also insoluble in ethanolParaffin is also insoluble in ethanol
5. Embed the sample in asupporting medium like wax(paraffin) or resin5. Embed the sample in asupporting medium like wax(paraffin) or resin
Tissues are soft and fragileTissues are soft and fragile
Pass through several changes ofwarm paraffin waxPass through several changes ofwarm paraffin wax
Melted wax occupies space formerlyfilled with waterMelted wax occupies space formerlyfilled with water
Cooled wax hardens, easier to cutCooled wax hardens, easier to cut
6. Section on amicrotome, whichslices like a mini meatslicer, into sections 1-10m thick.6. Section on amicrotome, whichslices like a mini meatslicer, into sections 1-10m thick.
7. Mount the sectionson microscope slides.7. Mount the sectionson microscope slides.
Unstained section
8. Stain the tissue sections with stainof your choice.8. Stain the tissue sections with stainof your choice.
stains are aqueous so need to reversexylene, ethanol seriesstains are aqueous so need to reversexylene, ethanol series
Automatic stainer
9. Dehydrating and clearing : Pass slidethrough ascending grades of alcohol todehydrate. Clear the tissue with xylol.9. Dehydrating and clearing : Pass slidethrough ascending grades of alcohol todehydrate. Clear the tissue with xylol.
10. mounting: put a drop of canadabalsam on the center of slide and cover itwith a glass coverslip.10. mounting: put a drop of canadabalsam on the center of slide and cover itwith a glass coverslip.
