Evaluation of two fixation techniques for direct observation of biofilm formation of Bacillus subtilis in situ, on Congo red agar, using scanning electron microscopy

Abstract

Background and Aim: Direct observation, scanning electron microscopy (SEM) is a common method used for the observations of biofilms. N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide) (EDC) fixation method has proven to be a valuable fixation method in the observation of these biofilms. Still, it entails a method of biofilm fixation that can damage slim structures, leading to the impossible observation of biofilm development. In contrast, alcian blue and lysine (ABL) fixation technique appears more glycocalyx of biofilm, fully preserved samples, which may provide much insight into the development of B. subtilis biofilms. Materials and Methods: Here, the evaluation of the fixation of ABL technique for the study of B. subtilis biofilms was carried out in situ, on Congo red agar. In doing so, the comparison to commonly use conventional EDC technique for sample fixation, and observation was carried out. Observations were based on SEM over 30 samples. Results: Overall, ABL technique provided excellent observation of biofilms formed in situ, on Congo red agar, and revealed slime structures, which have not been observed, much in standard EDC fixation or earlier in other studies of these biofilms in B. subtilis. Conclusion: This study reported the appropriate use of ABL in the fixation technique for the preservation of biofilm of B. subtilis.

Keywords

• alcian blue
• bacillus subtilis
• biofilm
• congo red agar
• scanning electron microscopy