Difference Between Buffered Unbuffered and Neutralized Formalin

The key difference between buffered unbuffered and neutralized formalin is that buffered formalin acts as the best grade formalin to preserve tissues, whereas unbuffered formalin and neutralized formalin show poor preservation of tissues.

Formalin is a colourless solution of formaldehyde in water. It is an organic compound that occurs naturally, and it has the chemical formula CH2O-(H-CHO). Pure formaldehyde has a pungent odour, and it mainly occurs as a smelling colourless gas that can undergo polymerization spontaneously, forming paraformaldehyde.

Formalin can be found in three major forms: buffered, unbuffered and neutralized forms. These three groups are divided as such based on the buffering capacity of these solutions. These three grades of formalin can be used for studies regarding formalin fixatives. Formalin fixatives are chemical solutions we can use to preserve parts from living things such as animal or plant tissues. The most common formalin fixative used in laboratories is buffered formalin fixative.

1. Overview and Key Difference
2. What is Buffered Formalin
3. What is Unbuffered Formalin
4. What is Neutralized Formalin
5. Side by Side Comparison – Buffered Unbuffered vs Neutralized Formalin in Tabular Form
6. Summary

What is Buffered Formalin?

Buffered formalin is the standard and preferred formalin fixative for tissue preservation. This solution is mostly purchased as a prepared solution, and it avoids the additional risks of handling needed to mix the buffered formalin solution from a stock solution. Generally, this buffered formalin solution is prepared by mixing one part of stock formalin with 9 parts of distilled water. In order to get the buffering capacity, we can add reagents such as monobasic sodium hypophosphate and dibasic or anhydrous sodium hyper phosphate. However, there is another method that involves the addition of sodium chloride and dibasic sodium hyper phosphate to the mixture of formalin and water to the ratio of 1:9.

When ultrastructures of tissues are stored in buffered formalin, we can observe that tissues are well preserved, showing the least damage to these tissue parts. This type of solution is very important in long-term storage needs and large throughput laboratories.

What is Unbuffered Formalin?

Unbuffered formalin is a solution of formalin in water. This type of solutions is formed when one part of formalin is mixed with 9 parts of water. This solution mixture has a pH of about 3-4 that can vary based on the concentration of the formalin stock we are using for this purpose.

Since the unbuffered formalin solution has an acidic nature (pH ranges from 3 to 4), the acidity can cause the reactions of haemoglobin in the tissue parts we are going to preserve, and it can produce a dark brown acid formaldehyde haematin precipitate that can complicate the histological interpretation.

What is Neutralized Formalin?

Neutralized formalin is a solution of formalin in water, having a neutral pH value. Therefore, the pH of this type of solution should be 7.0. When formalin stock solution is mixed with water, it gives an acidic solution, so we need to fix the pH using a base such as sodium hydroxide.

What is the Difference Between Buffered Unbuffered and Neutralized Formalin?

Formalin can be found in three major forms: buffered, unbuffered and neutralized forms. The key difference between buffered unbuffered and neutralized formalin is that buffered formalin acts as the best grade formalin to preserve tissues, whereas unbuffered formalin and neutralized formalin show poor preservation of tissues.

The below infographic shows more details of the differences between buffered unbuffered and neutralized formalin in tabular form for side by side comparison.

Summary – Buffered vs Unbuffered vs Neutralized Formalin

Formalin fixatives are preservative agents we can use to preserve tissue parts, etc., in laboratories. There are three types of formalin fixatives that we can use in our studies: buffered, unbuffered and neutralized formalin. The key difference between buffered unbuffered and neutralized formalin is that buffered formalin acts as the best grade formalin to preserve tissues, whereas unbuffered formalin and neutralized formalin show poor preservation of tissues.