A laboratory guide for histology 刘尚明 武玉玲

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

Preparation and rules of laboratory work  Preview the relevant text well before the laboratory work and know the contents and objects of each laboratory work.  Have the tools ready. These include microscope, slides, paper, pencils, rubber, ruler and so on;  Keep the laboratory clean and quiet. Raise your hand before asking the teacher. No spiting, no smoking and no eating in laboratory.  Take good care of specimens and instruments.  At the end of each laboratory work return the slides in order. Put the slide box and microscope back where they belong. Clean the room and turn off the light. Be sure that the windows are closed before you leave the laboratory.

The structure of the microscope

Mechanical part: 1. Base 2. Stand 3. specimen holder 4. Observation tube 5. Nosepiece 6. Coarse adjustment ring 7. Fine adjustment ring

Optical components: 1. The sources of light 2. Condenser lens 3. Objective lenses 4. Eyepiece

A. low power objective 1. Turn the power switch on, open the iris diaphragm and center the low power objective by turning the nosepiece; 2. place a slide on the stage then center the object by adjusting the mechanical stage ； 3.Turn the coarse adjustment to raise the stage until the objective is about 0.5cm form the specimen, look through eyepieces and turn the coarse adjustment slowly to lower the stage until the specimen is seen ； The use of microscope

4. Adjust the interpupillary distance. Hold the 2 eyepiece tubes with your both hands to push the tubes together or pull them apart, until you can see the same image with both eyes ； 5. Look at the image through the right eyepiece with your right eye and make the specimen in sharp focus by turning the fine adjustment. Look at the image through the left eyepiece with your left eye and rotate the tube length adjustment ring to focus on the specimen.

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

Oil immersion objective  It requires more careful use than the others.  First place a small drop of cedar oil on the slide, then center the lens slowly. Turn the coarse adjustment until the lens touches the oil and then turn the fine adjustment upward or downward to focus.  Note that the cedar oil must be wiped off with lens paper after using the oil immerse lens.

The care of microscope  Check the microscope assigned to you. If you find anything wrong, inform the teacher immediately.  Don not take apart of the microscope.  Keep the microscope clean. Clean lens with lens paper only. Avoid blowing or wiping it with finger, rough paper or cloth.  Before replacing your microscope, be sure that lenses or stage and condenser are at the lowest position. Then cover the microscope with soft silk.

Procedure of observation  First observe the shape, color and the features of the specimen with naked eyes.  Observe the general structure and its components with low power objective. Then select a representative area and observe it in detail with high power objective then with the oil immersion lens when necessary.  Make a drawing of the representative area and label it clearly.

A drawing of the representative area

Interpret your sample  Must think in 3 dimensions  For best interpretation, must have serial sectioning and reconstruction.

Preparing a Biological Specimen for Light Microscopy  1. Obtain sample and cut into workable pieces (1cm cube) Biopsy, surgical excision, postmortem examination Biopsy, surgical excision, postmortem examination From animals From animals Taken quickly, use sharp instruments Taken quickly, use sharp instruments

 2. Fixation generally in reactive aldehydes like formaldehyde. generally in reactive aldehydes like formaldehyde. This cross links macromolecules, particularly proteins This cross links macromolecules, particularly proteins Has hardening effect on soft tissues Has hardening effect on soft tissues Done rapidly to prevent enzymes from live cells degrading the tissue Done rapidly to prevent enzymes from live cells degrading the tissue

 3.Dehydrate the sample by running through a series of increasing concentrations of ethanol. Paraffin (embedding material) is not water soluble, so water is removed Paraffin (embedding material) is not water soluble, so water is removed  4. Clear the sample of ethanol and pass through several changes of xylene Paraffin is also insoluble in ethanol Paraffin is also insoluble in ethanol

 5. Embed the sample in a supporting medium like wax (paraffin) or resin Tissues are soft and fragile Tissues are soft and fragile Pass through several changes of warm paraffin wax Pass through several changes of warm paraffin wax Melted wax occupies space formerly filled with water Melted wax occupies space formerly filled with water Cooled wax hardens, easier to cut Cooled wax hardens, easier to cut

 6. Section on a microtome, which slices like a mini meat slicer, into sections  m thick.  7. Mount the sections on microscope slides. Unstained section

 8. Stain the tissue sections with stain of your choice. stains are aqueous so need to reverse xylene, ethanol series stains are aqueous so need to reverse xylene, ethanol series Automatic stainer

 9. Dehydrating and clearing : Pass slide through ascending grades of alcohol to dehydrate. Clear the tissue with xylol.  10. mounting: put a drop of canada balsam on the center of slide and cover it with a glass coverslip.