A laboratory guide for A laboratory guide for histologyhistology

刘尚明 武玉玲

IntroductionIntroduction

As other medical courses, the study of As other medical courses, the study of histology consists of two parts: lectures anhistology consists of two parts: lectures and laboratory work. The purpose of laboratod laboratory work. The purpose of laboratory work is mainly to integrate theoretical knry work is mainly to integrate theoretical knowledge with practical work and to train thowledge with practical work and to train the ability of observing the structures of cells,e ability of observing the structures of cells, tissues and organs with microscope. tissues and organs with microscope.

Preparation and rules of laboratory workPreparation and rules of laboratory work

Preview the relevant text well before the Preview the relevant text well before the laboratory work and know the contents and laboratory work and know the contents and objects of each laboratory work.objects of each laboratory work.

Have the tools ready. These include microscope, Have the tools ready. These include microscope, slides, paper, pencils, rubber, ruler and so on;slides, paper, pencils, rubber, ruler and so on;

Keep the laboratory clean and quiet. Raise your Keep the laboratory clean and quiet. Raise your hand before asking the teacher. No spiting, no hand before asking the teacher. No spiting, no smoking and no eating in laboratory.smoking 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 the At the end of each laboratory work return the

slides in order. Put the slide box and microscope slides in order. Put the slide box and microscope back where they belong. Clean the room and turn back where they belong. Clean the room and turn off the light. Be sure that the windows are closed off the light. Be sure that the windows are closed before you leave the laboratory.before you leave the laboratory.

The structure of the microscopeThe structure of the microscope

1

2

3

4

5

6

7

Mechanical part:

1. Base 2. Stand 3. specimen

holder4. Observation

tube5. Nosepiece6. Coarse

adjustment ring

7. Fine adjustment ring

2

3

4

1

Optical

components:

1. The sources of light

2. Condenser lens

3. Objective lenses

4. Eyepiece

A. low power objectiveA. low power objective1. Turn the power switch on, open th1. Turn the power switch on, open th

e iris diaphragm and center the loe iris diaphragm and center the low power objective by turning the w power objective by turning the nosepiece;nosepiece;

2. place a slide on the stage then ce2. place a slide on the stage then center the object by adjusting the mnter the object by adjusting the mechanical stageechanical stage ；；

3.Turn the coarse adjustment to rais3.Turn the coarse adjustment to raise the stage until the objective is ae the stage until the objective is about 0.5cm form the specimen, lobout 0.5cm form the specimen, look through eyepieces and turn thok through eyepieces and turn the coarse adjustment slowly to lowe coarse adjustment slowly to lower the stage until the specimen is er the stage until the specimen is seenseen ；；

The use of microscopeThe use of microscope

The use of microscopeThe use of microscope4. Adjust the interpupillary distance. H4. Adjust the interpupillary distance. H

old the 2 eyepiece tubes with your old the 2 eyepiece tubes with your both hands to push the tubes togetboth hands to push the tubes together or pull them apart, until you can her or pull them apart, until you can see the same image with both eyessee the same image with both eyes ；；

5. Look at the image through the right 5. Look at the image through the right eyepiece with your right eye and meyepiece with your right eye and make the specimen in sharp focus by ake the specimen in sharp focus by turning the fine adjustment. Look at turning the fine adjustment. Look at the image through the left eyepiece the image through the left eyepiece with your left eye and rotate the tubwith your left eye and rotate the tube length adjustment ring to focus on e length adjustment ring to focus on the specimen. the specimen.

High power objectiveHigh power objective

After observing with low After observing with low power objective enter the power objective enter the high power one. A slight high power one. A slight turn of the fine turn of the fine adjustment will bring the adjustment will bring the image into sharp focus. image into sharp focus. Don’t turn the coarse Don’t turn the coarse adjustment to avoid adjustment to avoid damaging the specimen damaging the specimen or the objective.or the objective.

Oil immersion objectiveOil immersion objective It requires more careful use It requires more careful use

than the others.than the others. First place a small drop of First place a small drop of

cedar oil on the slide, then cedar oil on the slide, then center the lens slowly. Turn center the lens slowly. Turn the coarse adjustment until the coarse adjustment until the lens touches the oil and the lens touches the oil and then turn the fine then turn the fine adjustment upward or adjustment upward or downward to focus.downward to focus.

Note that the cedar oil must Note that the cedar oil must be wiped off with lens paper be wiped off with lens paper after using the oil immerse after using the oil immerse lens.lens.

The care of microscopeThe care of microscope

Check the microscope assigned to you. If you Check the microscope assigned to you. If you find anything wrong, inform the teacher find anything wrong, inform the teacher immediately.immediately.

Don not take apart of the microscope.Don not take apart of the microscope. Keep the microscope clean. Clean lens with lens Keep the microscope clean. Clean lens with lens

paper only. Avoid blowing or wiping it with finger, paper only. Avoid blowing or wiping it with finger, rough paper or cloth.rough paper or cloth.

Before replacing your microscope, be sure that Before replacing your microscope, be sure that lenses or stage and condenser are at the lowest lenses or stage and condenser are at the lowest position. Then cover the microscope with soft position. Then cover the microscope with soft silk. silk.

Procedure of observationProcedure of observation

First observe the shape, color and the features First observe the shape, color and the features of the specimen with naked eyes.of the specimen with naked eyes.

Observe the general structure and its Observe the general structure and its components with low power objective. Then components with low power objective. Then select a representative area and observe it in select a representative area and observe it in detail with high power objective then with the oil detail with high power objective then with the oil immersion lens when necessary.immersion lens when necessary.

Make a drawing of the representative area and Make a drawing of the representative area and label it clearly.label it clearly.

A drawing of the representative areaA drawing of the representative area

Interpret your sampleInterpret your sample Must think in Must think in 3 dimensions3 dimensions For best interpretation, must have serial For best interpretation, must have serial

sectioning and reconstructionsectioning and reconstruction..

Preparing a Biological Specimen Preparing a Biological Specimen for Light Microscopyfor Light Microscopy

1. 1. ObtainObtain sample and cut into sample and cut into workable pieces (1cm cube)workable pieces (1cm cube) Biopsy, surgical excision, Biopsy, surgical excision,

postmortem examinationpostmortem examination From animalsFrom animals Taken quickly, use sharp Taken quickly, use sharp

instrumentsinstruments

2. 2. FixFixationation generally in reactive aldehydes like generally in reactive aldehydes like

formaldehyde. formaldehyde. This cross links macromolecules, This cross links macromolecules,

particularly proteinsparticularly proteins Has hardening effect on soft tissuesHas hardening effect on soft tissues Done rapidly to prevent enzymes from live Done rapidly to prevent enzymes from live

cells degrading the tissuecells degrading the tissue

3.3.DehydrateDehydrate the sample by running the sample by running through a series of increasing through a series of increasing concentrations of ethanol.concentrations of ethanol. Paraffin (embedding material) is not Paraffin (embedding material) is not

water soluble, so water is removedwater soluble, so water is removed

4. 4. ClearClear the sample of ethanol and the sample of ethanol and pass through several changes of pass through several changes of xylenexylene Paraffin is also insoluble in ethanolParaffin is also insoluble in ethanol

5. 5. EmbedEmbed the sample in a the sample in a supporting medium like wax supporting medium like wax (paraffin) or resin (paraffin) or resin Tissues are soft and fragileTissues are soft and fragile Pass through several changes of warm Pass through several changes of warm

paraffin waxparaffin wax Melted wax occupies space formerly Melted wax occupies space formerly

filled with waterfilled with water Cooled wax hardens, easier to cutCooled wax hardens, easier to cut

66. . SectionSection on a on a microtome, which microtome, which slices like a mini meat slices like a mini meat slicer, into sections 1-slicer, into sections 1-1010m thick.m thick.

7. 7. MountMount the sections the sections on microscope slides.on microscope slides.

Unstained section

8. 8. StainStain the tissue sections with stain the tissue sections with stain of your choice.of your choice. stains are aqueous so need to reverse stains are aqueous so need to reverse

xylene, ethanol seriesxylene, ethanol series

Automatic stainer

9. 9. Dehydrating and clearingDehydrating and clearing : Pass slide th : Pass slide through ascending grades of alcohol to dehyrough ascending grades of alcohol to dehydrate. Clear the tissue with xylol.drate. Clear the tissue with xylol.

10. 10. mountingmounting: put a drop of canada balsa: put a drop of canada balsam on the center of slide and cover it with a m on the center of slide and cover it with a glass coverslip.glass coverslip.