Dear Dr. Ruzauskas;

Biofilm forming bacteria can adhere and form biofilm layer on glass surfaces. Due to hydrophilic nature of glass, bacteria can attach. Surface hydrophobicity of bacteria is also important. After incubating biofilm forming bacteria with glass, it is washed and can be stained with crystal violet. This is easy way. Some fluorescent techniques are also present in literature.

I have observed a lot of biofilms (with SEM) grown on plastic (Thermanox) coverslips with glass-like smooth surface.

Hello Modestas, The answer to your question is a definite Yes.

Biofilms can develop on any surface and glass is not an exception.

There are many factors  that influence biofilm formation including type of nutrient and component concentrations, shear rate and temperature. Glass surfaces are hydrophilic, so biofilm formation will not be as easy as on other surfaces, though.

Thank you for the clear answers. Maybe some of you have description of methods how to make an experiment for the study of microbial adhesion on the microscopic glass surface and to prepare this for SEM?

As our microbiology lab and SEM is in different places of the city what is the stage during sample preparation (for example after fixation or after dehydratation...) were the samples are stable and might be transported to SEM premises in one week or any other period of time after bacteriological preparation of the sample?

Nice response by above researchers

The easiest way - to transport specimens in glutaraldehyde fixative (specimens can be stored in the fixative at refrigerator for a week or even a month).

The safest way (in terms of biofilm detachment) - to transport specimens in fully dehydrated state. Care should be taken not to disturb bacteria during liquid changes; for a final step I prefer HMDS to CPD. After dehydration specimens should be stored in a desiccator, but not for too long.

Biofilms can form on both glass & polymer surfaces (coverslips, slide, chambered slides). I'm not sure about individual cell counting after staining biofilms (with crystal violet) as the biofilm layer is usually dense after prolonged incubation (more than 12 hours) and the matrix components may obscure visualisation or hide cells, making counts inaccurate. Fixation of biofilms on glass can also be achieved using methanol (99%) for safe transport.

Thank you. How to make glutaraldehyde fixative? Are any other steps after incubation of slides and placing them into fixative? For example is it necessary to wash the samples in ultrasonic bath?

Biofilms can form on glass only if the material (glass) has the ability to allow the deposit of residues which serve as substrate for microbial proliferation.

I think you'd better contact your SEM lab. They should have fixative, their preferred method of specimen preparation, etc. And no ultrasonic bath. Washing culture in buffer before fixation is a must.

Dear Modestas,

I am very surprised with your question. However, the same question was asked here several years ago by Regiane R. Santos (look for it). Such is live, Hegel's philosophical concept of the development within the circle is valid also in the investigation

If you browse the web, you can find, e.g.,

https://biology.mit.edu/wp-content/uploads/2017/12/BiofilmLesson-1.pdf

Be sure, different bacteria are able (or not able) to form a biofilm on the glass slides, it has been well document in the past. However, variables / parameters of your experiment are also important (including nutrient limitation and others) . See, e.g.

https://academic.oup.com/femsle/article/283/1/83/547875

Generally, no special treatment of slides is needed even though bacteria must pass the surface potential barrier and the first step (it is not really lag phase) will depend on the experimental conditions.

You should admit, too, there is some literature from the half of the last century, still valid. Before the boom of biofilm literature (generally, from the medical point of view) in 90', waste water biotechnologists / engineers were studying all the aspects of biofilm formation. During 60'-80', tons of literature were produced on biofilm formation in waste water treatment. Please, do not filter in your browser such publications (my waste water biofilm Master, where I was using such technique dates to 81').

Maybe, you should read also

https://link.springer.com/article/10.1007%2FBF00381140

that refer to the complex formation of all aufwuchs (periphyton) organisms.

Also in many articles by Sládečková and Vymazal you will be able to find reasonable data about the colonization (biofilm formation) in the environment. Even though, plastics was used in other studies, maybe, glass is better due to the minimum electrical potential charge and/or Zeta potential.

Much research has been already done. If you repeat it, you must give the credits and show that you have applied much more advanced analysis methods. Yes, we were working with Gram staining, Ziehl-Neelsen (for mycobacteria), Delafield's haematoxylin was apllied for ciliates and amoebae… it is already over. Be sure, also SEM and Confocal microscopy have been used many years ago

https://pdfs.semanticscholar.org/8a53/e0f819e6ced71363aa4e697781ce54169378.pdf

FISH is governing now

https://elifesciences.org/articles/00230

Good luck with your up-to-date research, however, try to return to the past wihin you literature seeking.

Best wishes

Miroslav Macek

Thank you Miroslav. I never had anything with biofilms. During one week I have obtained a lot of information. Thank's for all!

Yes. Some bacteria can form biofilm in any surface such as glass.

Please go through the following PDF attachment.