Preparation of Tissue for Microscopic Examination

00:01 Hello, I'm Geoff Meyer.

00:04 In this lecture, I'm going to briefly describe how tissue is prepared for microscoping viewing and secondly, a little bit about histological staining.

00:16 These are 2 areas of histology that are highly specialized.

00:21 They require very highly-trained professional technologists, so I'm only going to briefly cover the basics of these 2 processes, enough for you to understand to enable you to then, when you study histology in more detail and all the tissues in the practical classes and in these lectures, you'll have an understanding of what stains really show you and you'll have an understanding of the technology behind looking at slides and even some of the artifacts that occur during preparation of histological processing.

01:04 So it's really a lecture or a demonstration more in 2 parts.

01:11 Firstly, to briefly describe the process of preparing tissue for microscopic examination and I'm going to confine it just to light microscopic examination using paraffin embedded sections.

01:28 They're the most common slides you're going to examine in your histology classes.

01:36 And then lastly, I'm just going to briefly describe and show you some of the common histological stains you'll come across in your study of histology.

01:53 Here is a technologist with what we call a microtome.

02:00 It's a machine used to cut histological sections.

02:06 If you look at the technologist's left hand just above his fingers, you can see a bit of tissue stained a browny color embedded in a highly-stained paraffin block, and the technologist is collecting sections as he cuts them on this microtome.

02:30 He's turning the wheel with his right hand and that wheel lifts and then brings down the paraffin block of tissue onto the knife and then as the tissue section is cut from the knife, he will remove it and then float it on a water bath to flatten it out and then he'll put it on the slide.

02:55 So let me just go through some of the basic steps involved.

03:00 Firstly, what you need to do is you get the piece of tissue from either the animal or the human that you're going to examine.

03:11 You don't need a large block of the tissue.

03:14 And then you have to fix it for at least 24 hours or more in what we use as a fixative.

03:23 Fixation, to fix something, means to stabilize all its components.

03:30 A fixative is a compound such as formalin or paraformaldehyde or glutaraldehyde that is used to stabilize and fix components of cells such as proteins, phospholipids, etc.

03:49 Some fixatives won't fix all the components of a cell.

03:56 Other fixatives are better to use but more expensive to use and they're often reserved for more detailed preparation of tissue, for instance, for examining with an electron microscope.

04:11 You can fix tissue in 2 ways.

04:13 Normally, you cut a piece of tissue such in taking a biopsy from a person or you might collect the tissue by cutting it from an animal and dropping it or immersing it in the fixative, and the gently shaking that fixative around the specimen to allow the fixative to penetrate into the specimen.

04:39 That's the common way of doing it.

04:43 In a lot of animal studies, animal tissue, sometimes we perfuse the animal.

04:49 The animal is anesthetized and then a needle is put into a major vessel and saline is passed through to wash all the blood out and then fixative follows, and that allows very efficient, rapid penetration of fixative into all the body tissues, and then the tissue is removed and then examined after processing histologically.

05:17 After fixation, you then pass the tissue through a series of alcohols to dehydrate the specimen, to take all the water out of it, and that's important because what you want to do then is you want to replace that alcohol with a substance such as xylol that is miscible with paraffin because you then want to infiltrate all the spaces between the cell tissue that you have preserved or fixed with paraffin and carried through in this vehicle of xylol or chloroform, and that paraffin is then set or you imbed the block in the paraffin, you leave it for a while to set just like you would other components you might make that require a setting process like cement, and then what you do is you put it on the microtome you see here or you saw previously in more detail, and you cut very thin sections off that paraffin block.

06:29 Sections for light microscopic examination are normally about 5 µm to 10 µm thick.

06:37 This enables you using normal light microscopy and staining to get almost the limit of resolution with the light microscope to look at details of the tissue.

06:51 Once you cut that section, you then float it on a glass bath and pick it up on a glass slide, and then you have to go through the process of getting rid of that embedding medium which is going to stand in the way of the viewing of the individual components of the tissue.

07:09 So to remove that, you again pass it through alcohols to dissolve it all away and then you stain this section with whatever staining you wish to use to show details or the components of the tissue.

07:27 The normal stains we use are going to be hematoxylin and eosin.

07:32 That's the common stain used in histology and it's the common stain used for all the sections you're going to look at, or most of them anyway, you're going to look at in your histology classes.

07:46 But certainly, there are some very special stains that are used to show up very specific components of tissue that I'll show you in the latter part of this lecture.

07:59 After you stain, you've got to re-dehydrate the sections once again in alcohol and then you again clear it all away with the xylol.

08:09 Make sure all the paraffin and all the debris or all the non-tissue material is removed, and then you put a cover slip on that slide, you don't forget to label the slide so you know what the tissue is of, and then you're ready to view the slide.