This is a grant application that I (Thomas Crouzier) submitted in January of 2016 as an “Urgent Grant” to the Swedish Research Council FORMAS. The idea revolves around the idea that culture substrates used in microbiology could be made much better in several respects. And that a recent world shortage in agar could be a good opportunity to conduct the needed research to develop such alternatives. The grant was rejected, probably in part because it did not really correspond to the type of urgency this grant scheme is aimed at.

If you, reader, are interested in working on this project with us. Please do not hesitate to contact us. If you wish to conduct the research on your own, perfect! I would love to know about it though 🙂

I – Project relevance and objectives.

Agar is a mixture of the agarose polysaccharide and smaller polysaccharides called agaropectins. It can be extracted from the cell walls of certain seaweeds and is commonly used in the food and pharmaceutical industries as thickeners and in research and development as a substrate for cultivating microorganisms. Since Robert Koch’s introduction of agar substrate for microbiology in the late 19th century, microbiologist are dissolving agar in a mixture of nutrients by heating the solution, then casting the agar solution in Petri dishes. Microorganisms such as bacteria or fungi grow well on these relatively stiff hydrogels, extracting the moisture and the nutrients from the gels. Agar gels are now an essential part of the microbiologist’s toolbox. They are used to identify new strains, quantify the number microorganism number, assess their susceptibility to certain chemicals or measure their ability to migrate.

Unfortunately, there is now a severe worldwide shortage of agar [1–3]. A dramatic decrease in the production volume from the world’s main producer, Morocco, has had direct repercussions on the availability of research-grade agar. At the same time, there is a strong demand of agar from other sectors such as the food industry. Consequently, the price of agar has now tripled its price since the beginning of the shortage, reaching around $45 per kg. Several large agar suppliers have suspended their sales of certain agar products, including Thermo Fisher and Millipore Sigma. This could soon have a profound effect on microbiology labs, but could also affect the routine biomedical analysis performed in hospitals throughout the world.

The shortage is due in large part to environmental constraints, as the Gelidium Seaweed from which agar is extracted has been overharvested in Morocco for years, leading to its near-disappearance in certain region of the country[4]. New environmental laws in Morocco have had cut agar production from 14,000 ton to 6,000 tons, with only 1,200 tons allowed for exportation. Morocco was in overproduction given its environmental constraints, and it will take years before the production of agar reaches the level seen before the shortage.

However, this troubling shortage is also an opportunities to develop alternative materials to agar for use in culturing microorganisms such a bacteria. Synthetic polymers including polyacrylamide gels have been investigated before[5], but are not ideal because of their relative toxicity. Other natural materials extracted from the biomass could complement or even surpass agar if introduced rapidly. A limited number of these materials have been investigated as an alternative to agar to culture bacteria. This includes gellan (Gelrite), a heterosaccharide derived from Pseudomonas[6], and eladium, a polysaccharide produced by Rhizobium[7]. These were mostly investigated as specialized surfaces to grow bacteria that would not grown on the more conventional agar surface.

We hypothesize that cellulose-based materials could be a well adapted response to the agar shortage. Sweden’s forest industry is constantly re-inventing itself as it is seeking alternative markets to paper. This project could result in an unique opportunity for the Swedish industry to seize this $200 million dollar market[8]. It is hopeful that as materials proves adapted for microbial cultures since bacterial cellulose has already been tested with success[9]. However the preparation of bacterial cellulose gels is not readily scalable for industrial production. We offer to use methylcellulose, a chemically modified cellulose that spontaneously gels when above gelation temperature, typically between 25 and 60 degrees (Figure 1).

Figure 1

Figure 1. A) Chemical structure of methylcellulose. B) Hydrophobic section of the molecule provides by the methyl groups substituted on the cellulose backbone, tend to assemble at higher temperature, crosslinking the polymers into a stiffer network. Figure from Nasatto et al. [10]

Justification of the urgency.

The agar shortage might not be indefinite. As producers change their source of seaweed, global productions could recover in a few years. During that time, some laboratory could be affected by the shortage, and will be looking for alternative. This is now a unique opportunity for cellulose to enter this multi-million market. The shortage has been a reality for a few months only, and the press as only recently taken this information public (Figure 2). We need to act fast, to provide rigorous proof that cellulose derivatives are highly suitable, if not superior to agar for this purpose. If done fast enough, the shortage will draw attention and funds to further develop this innovation. Efforts to connect with major agar retailers and producers will be undertaken over the course of this project so that this project continues beyond this FORMAS Urgent grant.

Figure 2

Figure 2. Article titles from and Nature in December 2015.

Why methylcellulose?

Methylcellulose, a derivative of cellulose, is in many ways similar to agar and could thus prove to be suitable substitute for bacteria culture. Once introduced to the market, microbiology-grade methylcellulose could even maintain itself as a new standard given a number of additional benefits it offers compared to agar.

  • Like agar, methylcellulose forms a hydrated gel, in which nutrients can be included and can diffuse to support the growth of bacteria on its surface.
  • Methylcellulose is liquid at low temperature and gels at higher temperature. Meaning no heating is required to form the gels. This limits to risks of burns, not uncommon in manipulation of hot agar. One only needs to pore the cold liquid in a mold then place it in an incubator for it to spontaneously gelate.
  • Over twenty years of intense study of cellulose and methylcellulose chemistry makes it a robust material to use. The industrial processes to obtain the material as well as the chemistry to modify it are well understood.
  • Methylcellulose is already used for mammalian cell culture. For instance it is used to determine the concentration of infectious viruses (phage)[11]. This is a first hint, suggesting good biocompatibility of the material.
  • Methylcellulose is rather resistant to biodegradation by bacteria[12]. Which makes it an ideal support for cultivation.
  • The price of methylcellulose is on par with agar or even cheaper depending on the type of methylcellulose used. Sigma-Aldrich sells 1 kg of Methylcellulose (Methocel A15, ref#64605) at 2,222 sek, while 1kg of bacterial agar (ref#A5306) is sold at 10,737 sek. Even if only equally suitable for microbial cultures, this would make methylcellulose a valid competitor to agar.
  • Agar has shortcomings that other materials such as methylcellulose could overcome. Agar does not support the growth of all bacteria and can be degraded by some others[13]. It is a heterogeneous with poorly controlled chemistry.

Several companies already produce methylcellulose, some located in Sweden (such as Caldic Sweden AB and Apotek Produktion & Laboratorier AB). The Dow Chemical Company, based in the United States offers a range of methylcelluloses with well-defined chemistries. The gelation temperatures of their different products have been well characterized, as well as the effect of additives (Figure 3)

Figure 3

Figure 3. Technical information extracted from the Dow Methocel technical handbook. Methocel A is a methylcellulose, while F and E are hydroxypropyl methylcellulose. A) Methocel A forms a gel at lower concentrations than any other product offered. B) Additives can slower the gelation point significantly.

The objective of this urgent project is to bring a reasonable answer to the shortage in agar by developing a methylcellulose substrate for microbial culture. We hypothesize that methylcellulose will prove to be more convenient to use, safer, and more flexible in its application that agar for this purpose. Methylcellulose could then become a new standard for cultivating bacteria. This one-year project will be separated into three aims.

Aim I. Selecting an optimal methylcellulose formulation.
We will select a methylcellulose formulation that gels at or above 25°C with the mechanical properties and porosity that support microbial growth.

Aim II. Cultivating bacteria on methylcellulose.
We will test cultivating bacteria gram positive and gram negative at both 30°C and 37°C on the selected formulation

Aim III. Common microbial assays using methylcellulose.
We will test a couple assays usually performed with agar beyond cultivation. This will include test antibiotic diffusion assay and migration assay.

II – Project description.

The project will be executed linearly, divided into its three separate aims. A timeline for the project in presented in table 1.

Table 1

Table 1. This one-year project will be executed in a linear fashion. With aim I and aim II taking 5 months, aim 3 taking 3 months.

Aim I. Selecting an optimal methylcellulose formulation.

We will first use Methocel A provided by Dow Chemical Company, as it is the methylcellulose with the lowest gelation point that they offer. By using plate on plate rheology, we will measure the gelation point of Methocel A as a function of temperature. We will aim for a gelation point around 25°C, which ensures that methylcellulose is in the form of a stable and insoluble gel at typical culture temperature of 30°C or 37°C. This gelation point will vary when salts and other additives are present in the solution. Thus we will measure the effect on gelation temperature of growth medium typically used for microbial culture, including Luria Broth (LB) or Brain Heart Infusion (BHI) broth.

The gels also need to be robust enough to be manipulated. For instance it should resist streaking of bacteria. Typical agar gel used for bacteria culture gave a tensile strength of a few tens of kPa. We will measure the mechanical properties of the methylcellulose gels by using uniaxial compressive test and extracting the material’s young’s modulus. The results will be compared with agar gels at various concentrations typically used in microbiology: from 0.5% to 2%. To reach satisfactory mechanical properties, we will vary the concentration of Methocel A we use. The optimal concentration will be the lowest concentration at which satisfactory mechanical properties are maintained at 25°C. If the mechanical properties depend on the type of broth used, then several optimal formulations will be selected (one for each broth).

The diffusion of nutrients through the gel is of utmost importance to support the culture of bacteria. We first test the Methocel A formulations previously selected for their optimum gelation and mechanical properties. We will compare them between themselves and with an agar gel reference. We will cut small fragments of gels then embed them in a solution of fluorescently-labeled dextran molecules or fluorescent dye such as fluorescein. We will follow their diffusion over time using time-lapse fluorescence confocal microscopy. This will provide us with information about the diffusion of molecules with sizes representative of the various constituents of complex culture broth. We will select the gels with diffusion speeds close or superior to those of agar.

Aim II. Cultivating bacteria.

Once a formulation that present suitable mechanical properties and diffusion properties is found, we will follow the growth of a number of bacteria on methylcellulose hydrogels, while comparing them to agar gels. We have pre-selected both gram positive and gram-negative bacteria, some grown at 30°C, other at 37°C, in different growth medium. The intention is to test a number of condition typically used by microbiologists while cultivating common types of bacteria (Table 2).

Table 2

Table 2. Selection of bacteria that will be grown on the new methylcellulose surfaces.

The bacteria culture on the methylcellulose gels will be done following already published procedures[9]. Bacteria will be first inoculated on the surface of the hydrogel then incubated at the optimum temperature for each bacterium. The growth of bacteria at the surface will be estimated in two ways. First, the methylcellulose gels will be formed in the well of a 96 well plate, and the optical density at 600 nm followed over 24 hours using a microplate-reader. Second, we will collect the bacteria of the methylcellulose gel surface at various time points, dilute the solution, then plate the solution on agar surfaces. By counting Colony Forming Units (CFU), we will have an estimation of the number of viable cells at each time point. Growth curves and double time will be obtained from these experiments and compared to the agar standard. The color, appearance and morphology of the bacterial colonies will also be observed and quantified and compared to standard agar surfaces.

Aim III. Common microbial assays using methylcellulose.

To test whether the new chosen material can support common assays performed with agar, we will perform Kirby–Bauer antibiotic testing (or disc diffusion antibiotic sensitivity testing) using our methylcellulose materials and compare it to agar. In this test, a paper filter disk will be embedded with antimicrobial and deposited on the gel (Figure 4A). Diffusion of the antibiotic through the gel will create an area of no growth, which is typically visible to the naked eye. In particular we will try Staphylococcus epidermidis and its inhibition by neomycin, Escherichia coli and ampicillin, and Pseudomonas aeruginosa and tetracycline.

Figure 4Figure 4. A) Kirby–Bauer antibiotic testing obtained by exposing Staphylococcus epidermidis to neomycin. Image taken from B) Swarming phenomenon observed on gar plates with Pseudomonas aeruginosa. Image from Wikipedia[14]

Second, we will perform swarming assays with our new material. Swarming is a type of migration where bacteria move in a coordinated fashion across a solid surface (Figure 3B). Obtaining swarming behavior in the laboratory can be difficult since it is highly dependent on the type of bacteria, the type and quantity of nutrient present, and on the properties of the surface[15]. This assay is thus representative of the need of microbiologists for substrates with tunable properties (mechanical properties, growth media type and concentration). Typically, soft agar gels are prepared for swarming assays using around 0.5 %w/vol agar (while standard is 0.75 to 1.5%). We will test various concentrations of methylcellulose with the goal to obtain swarming from both Pseudomonas aeruginosa and Bacillus subtilis.


I have experience with both the development of hydrogel materials and the study of cell/material interactions[16,17]. This project thus first perfectly my area of expertise. Indeed I have worked with both with the formation and characterization of biopolymer-based hydrogels, and well as the study of microbial interactions with such materials. However, I will also collaborate closely with Dr. Qi Zhou from the division of glycoscience, an expert in the chemical modification and assembly of cellulose-based materials. His group will bring valuable insights when selecting and characterizing the methylcellulose formulations and hydrogels.

III – Societal relevance and communication plan.

Progress in microbiology has allowed the most transformative inventions of human history: our master of microbial pathogens. But with multi-resistant strains becoming more common, and more recent discovery of thousands of unknown bacteria in various environment because uncultivable before, and the growing evidence that our microbiome dictates many aspect of our health, microbiology is more relevant field of science than ever. Agar, a heritage of the past, has allowed microbiology to cultivate many (but not all) bacteria in the laboratory. The recent shortage in agar comes as an opportunity to establish a new material as standard. It will allow microbiologist to continue to conduct their experiment undisturbed by the shortage and at a reasonable price, but could also allow new bacteria to be cultured more easily and faster.

This new standard will originate from one of the most abundant natural resource in the world (and Sweden): cellulose. The Swedish forest industry would then see a multimillion-dollar market open for its resources, which would contribute to the well being of our economy and our society.

Good communication of the objectives, the progress and the accomplishments of this project is of utmost importance in the mind of the leading researcher. Frequent updates will be posted on the website of the research group (accessible at, especially in the blog section. Active communication through social media will also help publicize the project. The goal is to attract public awareness, but also engage with microbiologists to gather their inputs, as well as the reagent industry that are foreseen as an important partners to continue develop the research after 1 year of FORMAS urgent gent.

To assure commercialization of any innovation this grant will produce, we will prioritize patenting our results. However the results of the study will be published in open access as soon as patent application is deposited.


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  16. Co JY, Crouzier T, Ribbeck K. Probing the Role of Mucin-Bound Glycans in Bacterial Repulsion by Mucin Coatings. Advanced Materials Interfaces. Wiley Online Library; 2015; Available:
  17. Duffy CV, David L, Crouzier T. Covalently-crosslinked mucin biopolymer hydrogels for sustained drug delivery. Acta Biomater. 2015;20: 51–59.

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