The researchers were able to observe compact growth of bacterial colonies in three dimensions. Credit: Neil Adelantar/Princeton University
The researchers found that colonies of bacteria formed in three dimensions in rough, crystal-like shapes.
Bacterial colonies are often grown as streaks in Petri dishes in laboratories, but no one has understood how the colonies settle in more realistic three-dimensional (3-D) environments, such as tissues and gels in the human body or soil and sediments in the environment. , until now. This knowledge can be important for the development of environmental and medical research.
A Princeton University the team has now developed a method to observe bacteria in 3D environments. They found that when the bacteria grew, their colonies consistently formed fascinatingly rough shapes that resembled a head of branching broccoli, more complex than what appeared in a Petri dish.
“Since the discovery of bacteria more than 300 years ago, most laboratory research has studied them in test tubes or Petri dishes,” said Sujit Datta, an associate professor of chemical and biological engineering at Princeton and lead author of the study. This was not a result of lack of interest, but of practical limitations. “If you try to watch bacteria grow in tissue or soil, they’re opaque and you can’t see what the colony is doing. That was really a problem.”
The researchers are Sujit Datta, associate professor of chemical and biological engineering, postdoctoral researcher Alejandro Martinez-Calvo, and Anna Hancock, graduate student of chemical and biological engineering. Credit: David Kelly Crow for Princeton University
Datta’s research team discovered this behavior using an innovative experimental setup that allowed previously unheard-of observations of bacterial colonies in their natural, three-dimensional state. Scientists have unexpectedly discovered that the growth of wild colonies is consistently similar to other natural phenomena, such as the growth of crystals or the spreading of frost on a window pane.
“These kinds of rough, branching forms are ubiquitous in nature, but usually in the context of growing or aggregating inanimate systems,” Datta said. “What we found was that bacterial colonies growing in 3-D exhibited a very similar process, even though they were collections of living organisms.”
This new explanation of how bacterial colonies develop in three dimensions was recently published in the journal Proceedings of the National Academy of Sciences. Datta and his colleagues hope their discoveries will help a wide range of bacterial growth research, from creating more effective antimicrobials to pharmaceutical, medical and environmental research, as well as procedures that use bacteria for industrial use.
Princeton researchers in the lab. Credit: David Kelly Crow for Princeton University
“At a fundamental level, we are excited that this work reveals surprising connections between the development of form and function in biological systems and the study of inanimate growth processes in materials science and statistical physics. “However, we think this new picture of when and where cells grow in 3D will be of interest to anyone interested in bacterial growth, such as environmental, industrial and biomedical applications,” said Datta.
For several years, Datta’s research group has been developing a system that would allow them to analyze phenomena that are usually hidden in opaque conditions, such as fluids flowing through soils. The team uses specially designed hydrogels, which are water-absorbing polymers similar to those found in jello and contact lenses, as matrices to support bacterial growth in 3-D. Unlike common versions of hydrogels, Datta materials consist of extremely small hydrogel balls that are easily deformed by bacteria, allow free passage of oxygen and nutrients that support bacterial growth, and are transparent to light.
“It’s like a ball pit where each ball is an individual hydrogel. They’re microscopic, so you can’t really see them,” said Datta. The research team calibrated the makeup of the hydrogel to mimic the structure of soil or tissue. The hydrogel is strong enough to support a growing bacterial colony without enough resistance to limit growth.
“As bacterial colonies grow in the hydrogel matrix, they can easily rearrange the balls around them so they don’t get trapped,” he said. “It’s like sticking your arm in a ball pit. If you drag it, the balls arrange themselves around your arm.”
The researchers experimented with four different types of bacteria (including the one that helps create kombucha’s sour taste) to see how they grew in three dimensions.
“We changed the cell types, the nutrient conditions, the hydrogel properties,” Datta said. The researchers saw the same, rough-edge growth patterns in each condition. “We systematically varied all these parameters, but this seems to be a general phenomenon.”
Datta said two factors are responsible for the broccoli-shaped growth on the surface of the colony. First, bacteria with access to high levels of nutrients or oxygen will grow and reproduce faster than those in less abundant environments. Even the most uniform environments have uneven nutrient densities, and these changes cause spots to advance or lag on the surface of the colony. Repeated in three dimensions, this causes the bacterial colony to form bumps and nodules, as some subgroups of bacteria grow faster than their neighbors.
Second, the researchers observed that in three-dimensional growth, only bacteria near the surface of the colony grew and divided. The bacteria stuck in the center of the colony seemed to have become inactive. Because the bacteria inside were not growing and dividing, the outer surface was not subjected to the pressure that would cause it to expand evenly. Instead, its expansion is primarily driven by growth at the very edge of the colony. And growth along the edge is subject to changes in nutrients, resulting in bumpy, uneven growth.
Alejandro Martinez-Calvo, a postdoctoral researcher at Princeton and first author of the paper, said: “If the growth was uniform and there was no difference between the bacteria inside the colony and the bacteria on the periphery, it would be like filling up a balloon. “Pressure from within will fill any unrest on the periphery.”
To explain why this pressure doesn’t exist, the researchers attached a fluorescent tag to proteins that are activated in the cells when the bacteria grow. The fluorescent protein lights up when the bacteria are active and stays dark when inactive. Observing the colonies, the researchers noticed that the bacteria at the periphery of the colony were bright green, while the core remained dark.
“The colony essentially becomes a core and a shell that behaves very differently on its own,” Datta said.
Datta theorized that bacteria on the outside of the colony take up most of the nutrients and oxygen, leaving little for the bacteria inside.
“We think they’re starving,” Datta said, though he cautioned that more research is needed to find out.
Datta said experiments and mathematical models used by the researchers revealed that there is an upper limit to the bumps that form on colony surfaces. A butterfly surface is the result of random variations in oxygen and nutrients in the environment, but the randomness evens out within certain limits.
“There’s an upper limit to how big a cruciferous thing can grow — the size of a flower, if you compare it to broccoli,” he said. “We were able to predict this mathematically, and it seems to be an inevitable feature of large colonies growing in 3D.”
Because bacterial growth follows a similar pattern as crystal growth and other well-studied phenomena of inanimate materials, Datta said the researchers were able to adapt standard mathematical models to account for bacterial growth. He said future research will focus on better understanding the mechanisms behind growth, the consequences of gross growth forms for colony functioning, and applying these lessons to other areas of interest.
“Ultimately, this work gives us more tools to understand and ultimately control how bacteria grow in nature,” he said.
Reference: “Morphological Instability and Coarsening of Growing 3D Bacterial Colonies” by Alejandro Martínez-Calvo, Tapomoy Bhattacharjee, R. Kōnane Bay, Hao Nghi Luu, Anna M. Hancock, Ned S. Wingreen, and Sujit S. Datta, October 2012 Proceedings of the National Academy of Sciences.
DOI: 10.1073/pnas.2208019119
The research was funded by the National Science Foundation, the Health Foundation of New Jersey, the National Institutes of Health, the Eric and Wendy Schmidt Foundation for Transformative Technology, the Pew Foundation for Biomedical Scientists, and the Human Frontier Science Program.
