A simple and rapid decalcification procedure of skeletal tissues for pathology using an ultrasonic cleaner with D-mannitol and formic acid

Decalcification procedures are required in order to prepare histopathological preparations of hard tissues such as bone and teeth. Decalcification is usually performed by immersing the hard tissue in different decalcification fluids with various properties. These decalcification fluids typically include inorganic and organic acids, a neutral fluid containing a chelating agent, or a mixture of solutions. Unfortunately, there is no universal decalcification fluid that satisfies all the requirements of pathologists such as rapid decalcification, easy handling, and minimal tissue damage. Techniques involving use of microwaves (MW) or ultrasonic apparatus (US) have been shown to be useful for shortening the time for decalcification procedures. In the present study, we investigated a unique decalcification procedure that uses a common commercial ultrasonic cleaner and a decalcification fluid (formic acid) containing a free-radical scavenger (D-mannitol). The time required to complete the procedure is approximately half of that required to complete a standard decalcification procedure. In addition, tissue morphology and antigenicity is fairly well preserved after decalcification. The procedure is quick, easy to perform, and achieves decalcification of hard tissue with minimal tissue damage.