Dear friend Salsabil Mebarki

There are alternatives to Glutaraldehyde (GA) for preserving biofilm samples for scanning electron microscopy (SEM). Glutaraldehyde is commonly used as a fixative due to its ability to crosslink proteins and preserve the structure of the sample. However, it is known to be toxic and can be harmful to the operator and the environment. Therefore, researchers have explored alternative fixatives that are less toxic while still providing good preservation of biofilm samples.

Here are a few alternative fixatives that have been used for biofilm sample fixation in SEM studies:

1. Paraformaldehyde: Paraformaldehyde is a commonly used fixative in biological sample preparation. It is less toxic than glutaraldehyde but can still provide good preservation of biofilm structures. Several studies have successfully used paraformaldehyde for biofilm fixation in SEM, such as:

- "Characterization of the formation and structure of Aeromonas salmonicida aggregates" by D.Ø. Hjeltnes et al. (MicrobiologyOpen, 2019). doi: 10.1002/mbo3.769

2. Formalin: Formalin is a solution of formaldehyde gas in water and is widely used as a fixative in histology. It is less toxic than glutaraldehyde and can be used as an alternative fixative for biofilm samples. One study that employed formalin for SEM analysis of biofilms is:

- "Surface Structure and Biofilm Formation of a Diatom Observed by Atomic Force Microscopy and Scanning Electron Microscopy" by K. Hayashi et al. (Microscopy and Microanalysis, 2014). doi: 10.1017/S1431927614003314

3. Ethanol: Ethanol can be used as a fixative for biofilms and is relatively safe and easy to handle. It is often used as a dehydration agent in sample preparation for SEM. Ethanol fixation followed by critical point drying has been successfully used in biofilm studies, such as:

- "Three-dimensional analysis of biofilm development in a microchannel using X-ray computed tomography" by L. Yang et al. (Biofouling, 2013). doi: 10.1080/08927014.2013.775931

These references provide examples of alternative fixatives used for biofilm sample fixation in SEM studies. They highlight the successful use of these alternatives and their compatibility with SEM imaging. However, it is important to note that the choice of fixative may vary depending on the specific biofilm and research requirements. It is recommended to evaluate the suitability of the alternative fixatives for your particular biofilm system and consult the relevant literature for specific protocols and considerations.

Thank you so much for your response. It is very useful for me.

Salsabil

As Kaushik explains, there are other substitute fixatives, but none of them are quit as complete, or as life-like as glutaraldehyde. All will likely demonstrate either some level oh morphological alteration, or shrinkage, or swelling. It is unfortunate, but glutaraldehyde is recommended under normal conditions, or a double fixative to include Osmium Tetroxide *(highly toxic to the touch).