Specimen Preparation for Clinical Analysis
Various kinds of specimen may be collected, observed, and studied in clinical applications. These must be carefully prepared, especially if they are not visible under normal light levels, to become clearly viewable with a clinical microscope.
Steps in Specimen Preparation for Clinical Analysis
Regardless of the kind of specimen you wish to observe, the steps involved in preparing it for microscopic observation are generally the same. If the specimen will not be observed live, it must be fixated in order to preserve it in a form nearest to its original condition. If it is transparent, it must be stained to make the specimen visible. If it is too large, thin sections must be obtained with the help of a micrometer.
Fixation – This is a chemical process used most often in cell biology and pathology. Once a specimen has been fixated, all active biochemical reactions are terminated. Fixation may also cause the specimen to increase in its stability and mechanical strength.
There are two types of fixation: perfusion and immersion. The former is also known as fixation via blood flow since the fixative is transmitted directly into the heart of the specimen. From there, the fixative spreads throughout the entire system. The specimen dies once it has been completely fixed. While perfusion can achieve pristine condition for a preserved specimen, it does however require the specimen to die. Perfusion also costs a considerable amount of money because of the high quantity of fixative required.
Immersion, on the other hand, requires less fixative but it must be diffused in order to fix the specimen. Diffusion, however, takes a great deal of time and this could be a problem when you have a large specimen to observe.
Various types of agents may be used for fixation. Aldehydes are the most common. Formaldehyde and glutaraldehyde are two examples of aldehydes and they are used by people because of their ability to preserve secondary and even tertiary protein structures of the specimen. Oxidising agents like potassium permanganate and chromic acid can also be used as fixatives. Acetone and methanol are examples of precipitating fixatives and lastly, there are other less popular fixatives like mercuric chloride and picric acid.
Mounting – Slide mounts are occasionally required for certain types of specimens. There are two kinds of mounts: temporary and permanent. The former is just used for identifying a specimen based on readily distinguishable characteristics. The latter requires more preparation and lasts for a good amount of time.
Staining – This refers to a biochemical process used to make specimens or sample more visible under a microscope. At times, they are also used just to highlight specific parts of a specimen. There are different kinds of purposes and techniques for staining.
In in-vitro staining, dead cells are colored to make them distinguishable from other cells. Different dyes may be combined for staining to yield greater detail.
Gram staining is one of the oldest, simplest, and important techniques used in studying bacterial specimens. In this technique, crystal violet stain is used to make cell walls more visible. A counterstain is used to make bacteria distinguishable while iodine is used as a mordant. Those that appear dark violet or blue are considered Gram-positive bacteria while those appearing pink or red are considered Gram-negative bacteria.
In H&E staining, haematoxylin and eosin are used to make thin sections of tissue specimens more visible. Eosin makes red blood cells (RBC) even redder while increasing the visibility of connective tissues and other extra-cellular parts. Haematoxylin, on the other hand, causes cell nuclei to turn blue.
Tips on Observing a Specimen with a Clinical Microscope
As there are many types of clinical microscopes, it’s important to make sure first that you’re indeed using the right type of microscope for your specimen. If you are also using a clinical microscope that doesn’t belong to you, be sure to read the owner’s rules, terms, and conditions before proceeding.
Assuming that you’re only going to use bright field illumination on your specimen, make sure that you have already stained your specimen accordingly before placing it on the slide and setting it on the microscope’s stage. Use a mechanical stage if you think you’ll need greater control over the positioning of your sample.
Start looking for the right eyepiece and objectives for viewing the specimen. If you’re changing objectives, remember to handle only the nosepiece and not the objective lens itself! Start adjusting with the coarse adjustment knobs first and move on to the fine adjustment knobs if you still aren’t able to obtain ideal image resolution.
For more information about clinical microscopes, http://www.clinical-microscope.com is the right place.
