The sample preparation for cryo‐
SEM
: the real ultrastructure of microbial biofilm or just artifacts?
Authors: Hrubanova Kamila , Skoupy Radim , Nebesarova Jana , Ruzicka Filip , Krzyzanek Vladislav 
Published: December 2016

The cryo‐scanning electron microscopy (cryo‐SEM) belongs to reputable techniques in electron microscopy of hydrated samples such as biofilms. The crucial steps of the cryo‐preparation techniques are primarily the cryo‐fixation and partial sublimation of ice contamination caused during the transfer of the sample to the cryo‐high‐vacuum preparation chamber where the sublimation process is performed; optionally the freeze‐fracturing or coating by metal sputtering or carbon evaporation can be applied. In the case of cryo‐fixation, an effort is to keep the frozen biofilm in the form nearby its native state. One of the simplest cryo‐fixation techniques is a plunging of the biofilm on a substrate into a liquid cryogen. However, the plunging into a liquid nitrogen or even liquid ethane/propane is sufficient for fixation of very thin layers of biofilm (no more than a few micrometers in thickness) because it is very difficult to achieve enough cooling rates to produce amorphous ice in the sample due to the Leidenfrost effect [1]. Moreover, we show that the cryo‐fixation into liquid nitrogen can lead to significant lateral macro‐segregation of both bacteria and extracellular polymeric substances (EPS), where plunging into liquid ethane leads to micro‐segregation of EPS and macro‐segregation of bacteria (Figure 1, 2A). Substantially more effective cooling can be achieved by increasing the pressure during exposure to the liquid cryogen. This can be performed for example by the high‐pressure freezing (HPF) technique [2]. It was proved that cryo‐fixed biofilms by HPF show significantly improved preservation of bacterial ultrastructure and biofilm organization (Figure 2B).
In this study, the multi‐layered biofilms formed by microorganisms were observed by cryo‐SEM using freeze‐fracturing technique. Cryo‐fixation methods like plunging into liquid cryogen, freezing by cryo‐jet system and high pressure freezing are compared. The freeze‐fracture technique consists of fracturing a rapidly frozen biological sample; structural details exposed by the fracture plane may be then visualized by cryo‐SEM.

The well‐characterized ica operon‐positive, biofilm and slime producing
Staphylococcus epidermidis
strain CCM 7221 (Czech Collection of Microorganisms, Brno, Czech Republic) and
Candida parapsilosis
BC11 from Collection of Microbiology Institute, Masaryk University and St. Anna University Hospital (Brno, Czech Republic) was observed. The strains
Candida albicans
GDH 2346 were also included [3]. Cultures were cultivated on the sapphire discs or cover glass in the cultivation BHI medium at 37°C for two days; fractured after cryo‐fixation, then followed short sublimation of ice contamination at ‐90°C which, moreover, partially exposes interior of the biofilm. In our experiments we focused on the formation of the extracellular matrix produced during the cultivation.

The cryo‐fixation can be recognized as a sufficient way how to fix and stabilize biofilms before their examination in cryo‐SEM. The simple plunging into liquid cryogens is applicable only for very thin specimens depending on the composition and used substrate. In this case of grown biofilms which thickness is usually more than 10 µm then the inner structure of matrix and bacteria interconnections were observed applying the freeze‐fracture technique was used, the HPF technique has proved to be necessary for preserving the biofilm ultrastructure.