Histology is a branch of biology which focuses on the anatomy of microscopic cells and tissues. The study of histology is commonly applied to medical biopsies, forensics, pathology, and biology for a number of applications. Whether diagnosing disease or determining cause of death, there are a number of uses for histology.
While histology can be used for an array of applications, the steps to prepare a sample typically follow the same protocol. The sample preparation for histology preserves the sample for future analysis and ensures the cell biology can be observed by utilizing stains and dyes. The classic steps for sample preparation are relatively simple and universal to the field of histology:
- Fixation
- Processing
- Embedding
- Sectioning and Staining
1. Fixation
Following sample extraction, the decomposition process begins to occur at a rapid pace. This process, if not properly stopped, will result in sample decay, cell autolysis, and putrefaction.
Formalin accomplishes the preservation of samples by penetrating fluid cells forming a semi-solid gel. To accomplish fixation without damaging the samples, a number of variables must be controlled.
The pH is a key consideration in the fixation process. A low pH can cause autolysis and allow a reaction in the solution causing precipitation of formaladehyde-hemoglobin pigment in the sample. To keep pH relatively constant, a buffered solution is used to prevent unwanted pH drops.
Concentration of formalin in these buffer solutions is typically 10% concentrated. This is generally the accepted concentration used due to its cost, effectiveness, and solubility in water. While formaldehyde can be dissolved up to 40% under ambient conditions, 10% provides enough to adequately preserve the sample without driving up the fixation cost.
2. Processing
Once the tissue has been exposed to a fixation agent, sample decomposition is prevented. The next step is removing water from the sample to prepare it for penetration via paraffin wax.
This process is carried out using increasing concentrations of alcohol over a number of steps. Gradual steps help prevent a rapid exchange across the cell membranes which could shrivel or damage the cells.
Following dehydration using alcohol, a clearing agent is used to act as an intermediate between the paraffin. While xylene has traditionally been used as an intermediate, health concerns have pushed the industry towards safer substitutes, such as naphtha. These clearing agents purge the sample of alcohol and are soluble with paraffin.
Embedding
The sample, now clear of alcohol, is ready to begin the embedding process. Embedding media are typically wax or other solid resins which provide the sample with structural support.
Paraffin, being the most common, has properties making it safe and reliable for embedding. With a melting point typically between 102°- 155°F, the wax can be easily held in a liquid state for long periods of time to allow it to infiltrate the sample. Then, the sample can be cooled rapidly and is ready for sectioning.
4. Sectioning and Staining
The ridged sample can then be cut by hand or by using a microtome. The microtome gives the advantage of reproducibility and speed, cutting micron-sized sections tunable by adjusting the cutting blade’s angle.
Samples are then transferred to microscope slides and stained. The two most common stains are hematoxylin and eosin. To stain acid groups, such as cell nuclei, hematoxylin is used providing the blue colors commonly seen throughout histology. Cytoplasm and other basic groups are stained with eosin, which is expressed in pink and maroon colors.
After the stains are used, a coverslip is attached to protect the sample. Now it is ready to be analyzed via light microscopy. The rigorous procedure allows the sample to be stored and reanalyzed as needed.
Potential Hazards
With such a variety of solvents utilized throughout sample preparation, there are a number of exposure risks. Formaldehyde, used in fixation, is a known carcinogen causing coughing, wheezing, and nausea at levels exceeding 0.1 ppm. Alcohols, such as methanol, can cause poor coordination, vomiting, and abdominal pain.
Many of the compounds used within histology are volatile and strong smelling, making them undesired even at a below hazardous threshold. Formaldehyde is detectable by the human nose at even 100 parts per billion. Protect yourself and others in your lab by utilizing proven carbon filtration.
AirClean Systems Solutions
We offer steps for each part of your tissue processing and slide staining procedure. Our activated carbon filters are bonded in layers, allowing for multiple steps of your tissue fixation procedure to be conducted in a single unit.
To protect your microscope and finalized histology slides from dust, AirClean Systems offers an array of microscope enclosures which use laminar air flow and HEPA filtration to keep your lenses free from particulate and minimize the need for frequent cleaning.