A Laboratory Manual on Standard Operating Procedures (SOPs) for Laboratory Tests and Testing procedures for Trade Related Transboundary Animal Diseases INTERAFRICAN BUREAU FOR ANIMAL RESOURCES.

Histology is the study of the microanatomy of cells, tissues, and organs as seen through a microscope. It examines the correlation between structure and function.

Histology Guide teaches the visual art of recognizing the structure of cells and tissues and understanding how this is determined by their function. Rather than reproducing the information found in a histology textbook, a user is shown how to apply this knowledge to interpret cells and tissues as viewed through a microscope.

Because of the high cost of purchasing (and maintaining) microscopes and preparing (or purchasing) slide collections, histology is often taught today without laboratories. A histology atlas is frequently used as a replacement. This is unfortunate because no matter how good the few images in a textbook or histology atlas are, they cannot replace the experience of viewing a specimen through a microscope.

Histology Guide solves this problem by recreating the look and feel of a microscope in an intuitive, browser-based interface.

An Aperio slide scanner was used to obtain a high-resolution image of each slide in its entirety. Large tissues are up to 34 GB for a single, uncompressed image of 150,000 x 75,000 pixels.

The contrast, color, and sharpness of each image were adjusted to at least maintain the appearance of the tissue as seen through a microscope. In many cases, these adjustments improved upon their visual appearance.

Unlike low-resolution images, users can interactively explore these large images by zooming-and-panning in real-time. A software-based virtual microscope (Zoomify HTML5 Enterprise) allows the examination of large and small structures in the same specimen.

This approach provides a more engaging learning experience and sense of scale, proportion, and context that is not possible with a traditional histology textbook or atlas.

The Atlas of Human Histology: A Guide to Microscopic Structure of Cells, Tissues and Organs by Robert L. Sorenson and T. Clark Brelje provides a print version of the core slides from this website. Individual slides are presented as a series of images of increasing magnification to help convey a sense of scale and proportion. This atlas allows each student to have an easily accessible, printed summary of the essential slides from this website.

Histology Guide is intended to be used with - not replace - a good histology textbook.

Questions, comments or suggestions should be sent to tcbrelje@gmail.com

When preparing a sample (or multiple samples) for, there are multiple steps required. We’ve covered these steps in brief in a previous article on, but this article will focus on one particular procedure which needs to take place between tissue fixation and the embedding/sectioning of paraffin blocks: tissue processing. You simply can’t take fixed tissue and embed it!

We have and.Following fixation, tissue is transferred to a tissue cassette- see the multicolored examples below!Get your pencil outThese come in various sizes and hold and protect the tissue whilst it undergoes processing. Once the embedding stage is reached, the cassette lid is snapped off and the main part of the cassette forms a base for the paraffin wax block.

The cassettes can be labelled by hand (with pencil!) or your histology lab may have a cassette labelling machine.There are three main steps in tissue processing, namely: ‘dehydration’, ‘clearing’ and ‘infiltration’. Each of the steps of the processing method involves the diffusion of a solution into tissue and dispersion of the previous solution in the series. All the fun of the carouselIn most modern institutes and histology labs, processing will be carried out in dedicated tissue processing machines. The older design of machine is a carousel which contains a cage in which the tissue cassettes are placed. This carousel has a number of glass beakers containing solvents and solutions which ensure that the tissue is dehydrated and cleared ready for paraffin wax embedding.

The carousel vertically agitates the cage in each solution before moving on to the next solution in the dehydration/clearing method.The modern processors have a chamber in which the specimens are held and the different solutions are pumped in and out of the chamber. In general, the whole process takes around six hours and is usually set up to run overnight. First, remove the waterFirstly the tissue needs to be dehydrated to remove the water from the tissue which is present- either bound to the tissue, or free in the tissue.

Paraffin wax is hydrophobic, therefore, most of the water in the tissue must be removed before it can be infiltrated with wax. This process is carried out by immersing tissue in a series of ethanol solutions of increasing concentrations until 100%, water-free alcohol is reached. A series of increasing concentrations is used to ensure that the water in the tissue is gradually replaced by the alcohol and to avoid excessive distortion of the tissue.Various components of the cell are also removed by this process.

At the lower end of the ethanol concentrations, water soluble proteins are removed, whilst towards the 100% ethanol step, certain lipids may be dissolved. Ethanol and wax don’t mixAlthough the tissue reaches the final stage of dehydration in 100% ethanol, it’s not possible to proceed straight to wax embedding- ethanol and wax don’t mix!This is where ‘clearing’ comes in. The term ‘clearing’ refers to the property of the solvents used- they have a relatively high refractive index and when tissue is immersed in it, it becomes transparent and clear. It’s becoming clearerThe solvent used for this intermediate stage is usually xylene. The ‘clearing agent’ needs to be miscible with both ethanol and paraffin wax. Following the dehydration, the tissue is immersed in one to three different xylene immersions.

In these stages, the ethanol is gradually replaced with xylene and when the tissue is embedded, the xylene will be replaced by the molten paraffin wax.Shrinkage of tissue can occur at these final stages as the xylene also removes fat residues left in the samples. At last – a block!The final stages are called ‘infiltration’ and ‘blocking out’. Infiltration is when the final xylene is replaced with molten wax which infiltrates the tissue. Again, this is typically three different wax immersions to ensure that none of the clearing agent remains in the tissue. After the final infiltration, the tissue cassettes are transferred to an embedding station. This machine has reservoirs of molten wax, hotplates and a cold plate for setting the blocks. The infiltrated tissue is removed from the cassette and orientated within a suitably sized metal mould.

The mould is filled with molten wax, the main part of the labelled cassette is placed on top and this is also filled with wax. Excellent information. I wanted to ask a question. I’ve tried a number of different waxes for mounting my samples but I’ve had some trouble with the brittleness of the resulting paraffin block. In my country there are incredible difficulties to import specialized waxes and they are very expensive so I’ve been trying to find substitutes. I red you can add some special types of polyethylene and things like that to make a blend with improved qualities.

Do you have any information as to what additives I can put in? Thanks in advance.