Biofilms are aggregates of microorganisms on either living or inert surfaces. They are embedded in secreted extracellular polymeric substances constituted by extracellular polysaccharides, proteins, DNA and lipids. Structural and physiological complexity of biofilms is such, that some researchers refer to them as “cities” of microorganisms.

This multicellular behavior allows bacteria to prolong survival in different niches, such as in hospital settings, where they become a special problem as they can spread amongst patients.

In fact, 60-80% of microbial infections are caused by bacteria growing in biofilms with a role being attributed to them in a wide variety of chronic infections and diseases, from cystic fibrosis, to kidney stones, periodontal disease or atherosclerosis in coronary heart disease.

A major problem in biofilm observation is caused by the low contrast and high transparency of the samples. The 3D Cell Explorer allows for biofilm visualization in a novel, marker-free fashion.

Analysis of 3D colonies of bacteria

This image E.coli bacteria embedded in alginate beads (generated by Encapsulation Unit – Var J30, 40-70 microns diameter). The beads were mounted on a slide into Minimum Media (MM) diluted 1:4 with distilled water (plus 1% v/v Fumarate 100mM). The time-lapse imaging experiment was conducted using the 3D Cell Explorer with a standard top-stage incubator set to 37°C and 90% humidity for 3 hours, capturing images every 10 minutes.

This image and video demonstrates an E.coli bacteria colony growing overnight (MM medium, 90% humidity, 37°C, no agitation) imaged with the 3D Cell Explorer.

Bacteria in tissues and cell samples

Rehydrated paraffin embedded bee gut sections (5 µm) imaged with the 3D Cell Explorer. The panoramic view of the whole tissue section (more than 400 micrometers) was obtained by stitching multiple acquisitions using a free online software. Like human intestine, bee gut is colonized by bacteria population. Most of the gut microbial community resides in the ileum.

Pap-smear analysis

Exfoliative cytopathology is an easily performed, noninvasive, and inexpensive procedure used in screening for preinvasive and invasive cancer cells. The incidence of cervical cancer has decreased by more than 50% in the past 30 years. That is mainly due to the increased use of a cytopathology screening called Papanicolaou test, better known as a Pap test. It involves exfoliating cells from cervix and smearing them on a glass slide. In order to examine them under a common microscope, the smeared biological sample must be fixed and stained. If not performed well, these procedures will compromise the test results. Thanks to Nanolive’s technology, the Pap-test screening can be done in real time with no need for fixation and chemical staining. The waiting time for Pap-test results can be reduced from two weeks to a couple of minutes!

A microscopic view of label-free and living Listeria bacteria

What is Listeria and where does it come from?

Infection from Listeria monocytogenes is a food borne bacterial illness that can be very serious for pregnant women and people with impaired immune systems. Listeria infection can be contracted by eating badly preserved meat and unpasteurized milk products (including soft cheese, ice cream, and yogurt).

Healthy individuals are normally resistant to listeria infection, but the disease can be fatal to unborn babies and very young babies. Immunodepressed people also are at higher risk. Listeria bacteria is very resistant and can survive refrigeration and even freezing. This is why people at risk should just avoid consuming products at risk.

Mouse macrophages infected with Listeria

In this video we observe mouse macrophages that have been infected with Listeria monocytogenes.

At the beginning of the video, the cell at the top of the field of view is going through mitosis. The chromosomes’ condensation is clearly observable in the center of the cell. Listeria bacteria are observable in this same host cell: they are either free to move around in the cytoplasm or inside cytoplasmic vacuoles, replicating. When the host cell divides, we can observe the transmission of these pathogenic bacteria to the daughter cells.

In the second part of the video, we move the attention to the cell below. At the end of its reproductive cell cycle, the Listeria bacterium is released through the host cell’s plasma membrane and is free to infect other cells. Here we clearly see the vacuole localization of Listeria followed by membrane destruction and cell death during its release.

Macrophages phagocytosing E.coli

Macrophages are present in almost all tissues. They are contributing to various processes in the healthy organism, such as development, wound healing, infection and tissue homeostasis. They can rapidly change their phenotype in response to variations in their environment. Macrophages are known for their classical function as antimicrobial phagocytes but support immune function as well by the presentation of antigens. Their research applications are vast, and in vitro assays are increasingly used in a wide range of research areas, including immunology, bacteriology and parasitology, as well as in biomedical and transplantation studies. Two advantages of macrophages in cell culture are that they are relatively easy to generate and to cultivate.

E.coli being engulfed through phagocytosis

Phagocytosis Assay Kits by PromoCell were used to test the viability and cellular functioning of the macrophages (video 2 & 3). E.coli particles, visible as small ellipsoid particles, are trapped by the cells, transported and lysed. This system can be used to provide a robust screening system for activators and/or inhibitors of phagocytosis and Toll-like Receptor (TLR) ligands.

Scientific Publications

The 3D Cell Explorer allows for:

  • Label-free 3D infection process monitoring
  • 3D localization of bacteria inside living cells  
  • Detection of mammalian cell infections 
  • Volume measurements of intra-cellular parasites 
  • Visualization of microorganism internalization process
Scientific Publication in Biofilm formation:

Impact of the Food Additive Titanium Dioxide (E171) on Gut Microbiota-Host Interaction

Authors: Gabriela Pinget, Jian Tan, Bartlomiej Janac, Nadeem O. Kaakoush, Alexandra Sophie Angelatos, John O’Sullivan6, Yen Chin Koay, Frederic Sierro, Joel Davis, Shiva Kamini Divakarla, Dipesh Khanal, Robert J. Moore, Dragana Stanley, Wojciech Chrzanowski and Laurence Macia

Published in frontiers in Nutrition

The University of Sydney & University of New South Wales have recently published their work on the “Impact of the Food Additive Titanium Dioxide (E171) on Gut Microbiota-Host Interaction”.

They have investigated the impact that Titanium Dioxide (E171) – mostly found in processed foods such as mayonnaise or candies – has on gut microbiota of mice. The study shows that the food additive E171 can induce gut microbiome changes associated with the development of diseases, including colorectal cancer and inflammatory bowel disease.

Visualizing bacteria and biofilms has been complicated with traditional microscopy due to low-contrast and high transparency of these samples. Thanks to the 3D Cell Explorer it has been possible to visualize E. coli and E. faecalis to reveal the clustering effect of TiO2 on both E. coli and E. faecalis in vitro in this study. Please see Figure 1 for reference.

TiO2 triggers biofilm formation by commensal bacteria. (A,B) The clustering effect of TiO2 on (A) E. coli and (B) E. faecalis in vitro was visualized by Nanolive imaging in the presence of 0, 0.5, 1, or 50 μg/ml TiO2 after 24 h incubation. Black represents the refractive index of TiO2 and green represents bacteria. From Gabriela Pinget et al., Frontiers in Nutrition., 2019

More Scientific Publications

Scientific publication covering the topic “An alternative method to determine the integrity of cells“:

Diagnosing water treatment critical control points for cyanobacterial removal: Exploring benefits of combined microscopy, next-generation sequencing, and cell integrity methods

Authors: Arash Zamyadi, Caitlin Romanis, Toby Mills, Brett Neilan, Florence Choo, Lucila A., Coral, Deb Gale, Gayle Newcombe, Nick Crosbie, Richard Stuetz, Rita K. Henderson

Published in Science Direct. Find the publication here: Download.

Scientific Publication in Biofilm dispersion:

Nitric Oxide-Loaded Antimicrobial Polymer for the Synergistic Eradication of Bacterial Biofilm

Authors: Rashin Namivandi-Zangeneh, Zahra Sadrearhami, Ali Bagheri, Maeva Sauvage-Nguyen, Kitty Ka Kit Ho,Naresh Kumar, Edgar H. H. Wong, and Cyrille Boyer

Published in: ASC Macro Letter.Find the publication here: Download.

Scientific Publication in Bacteria killing assessment & Biofilm dispersion assessment:

Enhancing the antimicrobial and antibiofilm effectiveness of silver nanoparticles prepared by green synthesis

Authors: Susan Oliver,  Harsha Wagh, Yuanli Liang, Shuang Yang  and  Cyrille Boyer

Published in:Journal of Materials Chemistry B. Find the publication here: Download.

Scientific Publication in Bacteria killing: 

The Effects of Polymer Topology and Chain Length on the Antimicrobial Activity and Hemocompatibility of Amphiphilic Ternary Copolymers

Authors: Rashin Namivandi Zangeneh, Rebecca J. Kwan, Thuy-Khanh Nguyen, Jonathan Yeow, Frances L. Byrne, Stefan H. Oehlers, Edgar Wong and Cyrille Boyer 

Published in: Polymer Chemistry Journal. Find the publication here: Download.