6: Culturing and Isolating Microbes

Antibiotic Discovery

Amazingly, most antibiotics on the market today were discovered as byproducts of other bacteria. This is because in complex environments (including soil and aquatic environments) bacteria must compete for limited resources. As a result one species of bacteria makes weapons that help it fight off neighboring bacteria. There is so much to be discovered about how and why and the optimal conditions for these bacteria produce antibiotics, but we know that a small fraction of the bacteria in the soil do produce antibiotics under laboratory conditions. The key, and our goal this semester is to find some!

Can Students Make a Difference?

We certainly can!! Although the path of an antibiotic from soil bacteria to the pharmacy shelf takes much longer than a semester, we can start this process running and find some potential antibiotics -- and learn about the process of scientific research in the process. In my experience doing this project with students, approximately 50% of the research groups are successful in finding antibiotics. Allof the other groups also have successful projects finding bacteria that are fascinating for their own reasons. Additionally, I save and store each antibiotic producer for further analysis and we contribute to the Tiny Earth database which helps identify trends in antibiotic production worldwide.

Through this research, we Citrus College students are participating in a global community of scientists helping to address this international antibiotic crisis.

Definitions

Microbe refers to all organisms that cannot be seen without a microscope. These microbes include bacteria and archaea (prokaryotes), and microscopic eukaryotes, such as yeasts, molds, and protists. Viruses are also generally classified as microbes, although they are generally considered nonliving. As you know, prokaryotes and eukaryotes differ in their level of complexity and cellular organization. Eukaryotes package their genetic material inside a compartment called the nucleus, whereas prokaryotes do not. Prokaryotes do not contain any intracellular compartments and have their genetic material in the cytoplasm. The number of individual prokaryotic cells in soil across the planet has been estimated to be 26 × 1028 (260 billion billion billion) (Whitman, Coleman, & Wiebe, 1998).

Our research focuses on bacteria because:

• Bacteria are found in all environments on the planet

• Bacteria are simple but are able to make a diverse array of chemical compounds, many of which have human applications;

• Bacteria greatly impact our health—some may cause infectious disease and others protect our bodies from invasion by harmful bacteria.

Getting Started

In our Tiny Earth research project, we will design and execute experiments to answer questions related to microbial ecology and bacterial interactions focusing in particular on soil microbes abilities to create antibiotics and inhibit the growth of neighboring microbes. As microbiologists, we will use many techniques from biological fields including cell biology, biochemistry, and genetics. In this way, our research will tie in quite nicely with the topics that we are discussing in lecture.

Soil

Soil covers the earth's surface, supports plant life, and is an ecosystem for some of the most numerous, diverse, and dynamic organisms on earth—bacteria. The main characteristics defining soil are its physical structure, its chemical composition, and its association with plant roots or other resident organisms. The association of soil with plant roots creates a distinction between soil types. The rhizosphere is soil that is directly in contact with plant roots and bulk soil is everything else. The root systems in the rhizosphere create microenvironments that may favor the growth of specific bacteria, so although soil looks the same throughout, it is far more complicated and diverse.

Soil can be made of three major sediment types—sand, slit, and clay. These three components provide the structure for the soil and influence how permeable the soil is to water, how well it is aerated, and how well nutrients are maintained. Each of those features influence which microbes thrive in each environment. Courser soils (like sand) hold air well, but not water resulting in them drying out rapidly and losing nutrients. Finer soils (like silt) and clay can pack densely limiting O₂ availability, but stay moist longer. To tell whether your soil is sand, silt, or clay, grab a small handful, make it moist and try to squeeze it together. If it holds, then your soil has a clay component. To look for sand vs silt, rub a small amount of the soil between your fingers. If it feels course and rough like sandpaper, you have sandy soil while a slick smooth feeling is silt instead.

In addition to the sediment types influencing the microbes present in the soil, the animal and plant life drastically impact the types and number of microbes in the environment. Environments with greater biodiversity—more animals and plants present—tend to have greater microbial diversity as well. The particular types of plant roots in the soil as well as soil pH and decaying organic matter create different mini ecosystems for the bacteria to inhabit.

Deeper in the soil, the physical, biological, and chemical properties of the soil changes. The soil is divided into distinct layers called horizons (Figure 1). Horizons are a good predictor of the nutritional characteristics of the microbes living within them. Generally, as we descend from one horizon to the next, the abundance of organic components, as well as the exposure to light and oxygen, plummets.

Figure 1. Soil Horizons: As you dig deeper into the soil, the amount or organic matter (microbes, animals, plants and decomposing material) decreases and the inorganic material (rock, minerals) increases. From Wilsonbiggs Vector: EssensStrassen, CC BY-SA 4.0 via Wikimedia Commons

The O horizon is a very thin layer at the surface and is the part of the soil that you usually see. Next is the A horizon (aka topsoil) which is quite rich in organic matter and microbial life. Here is where the microbes decompose organic material, interact with roots and other organisms. Third is the B horizon (aka subsoil) where fewer microbes live and those that are present are often anaerobes and therefore don't use oxygen for cellular respiration. Next is the C horizon where microbes are present, but few. Finally, below all of these levels is bedrock. It is important to know which layer of soil microbes are isolated from since this greatly influences microbial growth conditions.

Soil Microbes and Antibiotics

Microbes are quite prevalent in soil in terms of both number and variety. Within one gram of soil, billions of bacteria from tens of thousands of distinct species are present (Curtis, Sloan, & Scannell, 2002; Schloss & Handelsman, 2006). Soil microbes are quite diverse in their physiology and chemistry.

Some microbes decompose plant or animal matter, others cycle nutrients , and they compete with each other for food and other resources. As part of this battle, bacteria produce secondary metabolites (organic compounds that enhance an organism's survival while not being essential for life). These secondary metabolites can provide the bacteria with pigments that make them

colored, signals that encourage plant growth in the area, and antibiotics to kill or limit the growth of competing microbes. In their natural habitat, microbes produce antibiotics to ward off competitors. Scientists and doctors have used this bacteria vs. bacteria weapon to save the lives of those who have an infectious disease.

Most antibiotics used to fight disease today are derived from soil bacteria. The most common bacterial species making these antibiotics are Actinomycetes, which are quite common in soil. Since such a small fraction of soil bacteria have been thoroughly studied, there are probably many more antibiotics produced by soil bacteria just waiting to be discovered. Because this environment is so poorly understood and easily accessed, our research project will begin by collecting soil samples so

that we can study it further and hopefully find antibiotic producing bacteria of our own. As you do this, keep in mind that your soil sample is a mini ecosystem and pay attention to the inorganic as well as biological features of this ecosystem so that we can better understand the microbes within it.

Our worldwide research collaborations through the Tiny Earth ranging from Malaysia to Kenya to Massachusetts, USA can result in a huge database of soil and microbial information. The data that you gather here from soil type, to microbial number, to number of antibiotic producing bacteria will help future students and scientists worldwide find new sources of antibiotics.

Bacterial Growth in the Lab

To culture (grow) bacteria in the laboratory, scientists have developed standard media (bacterial food sources) which are a mixture of nutrients. Each particular bacterial species has its own preferences for the types of nutrients that it prefers, and, in our research, you will have the chance to select the media that you wish to use for your microbe. This media can come in either a liquid or solid form, though we will primarily be using solid media for our research. There is no one media choice that is best for all bacteria, and each week you will have choices to make related to the type of media that you use. The solid media that we are using is referred to as an agar plate. The agar is the solidifying agent used to make the nutrients solid in the Petri dish (Figure 3). As we work to culture our bacteria, you will carefully control and record the culture conditions—media type, temperature, humidity, oxygen levels, etc.

Figure 3. (A) A sterile, empty agar plate which contains nutrients, water, and the solidifying agent agar. (B) The same type of plate, with the bacteria Pseudomonas pudita.

CFUs and Bacterial Colonies

It is important to note that most soil bacteria cannot be cultured in the lab. Most of these bacteria require growth conditions that are not easy to replicate in the lab. Therefore, although we will isolate and identify many different bacteria in the course of our project, these bacteria that we identify are a tiny fraction of the soil bacteria. It is estimated that only 0.3 percent of all bacteria in soil can be cultured in the lab (Amann, Ludwig, & Schleifer, 1995).

To culture the bacteria in the soil, we will spread a diluted soil sample on a plate and look for the appearance of colonies. Just as you'll see spots of mold on an old piece of bread, you can see a bacterial colony on an agar plates. A colony is a pile of genetically identical bacteria that arise from a single cell. Single cells that give rise to a colony are referred to as CFUs (colony forming units). As a this single cell reproduces, the mass of dividing cells eventually becomes large enough such that a macroscopic colony becomes visible to the naked eye. By the time you can see a colony on a plate it contains approximately 1 million cells. Colonies are our way of seeing into the microscopic world, and each bacterial type has particular colony features that can be recognized by experienced microbiologists.

Our goal this week is to isolate bacteria from the soil to obtain single colonies. These single colonies are distinct and not touching other colonies. They are also uniform in color and texture throughout indicating that they came from just one single bacterial cell. If we are successful in isolating single colonies, all of the bacteria within the colony are the same species. The trick to be able to do this successfully is to dilute them and spread out the CFUs so that they grow into non- overlapping colonies.

To do this, microbiologists dilute the soil samples repeatedly in a solvent to decrease the number of cells in any sample. We will then spread a small volume (100μL) on an agar plate to separate and spread out the bacteria all over the surface of the plate where they will grow into single colonies (Figure 4). It is absolutely essential, while examining these bacteria, that we ensure that these single colonies are not contaminated with other species. To help determine this, first do not use plates where bacteria have spread all over the surface creating a lawn, also be sure to select only colonies that are not touching any other colonies. Our goal is to obtain pure cultures that contain only one single species of bacteria. We will use these pure cultures in our subsequent experiments during the semester.

Figure 4. (A) A serial dilution plate of a rich soil sample. Bacteria in the soil sample were suspended in sterile water and diluted 10,000-fold (10–4) to spread the bacteria out well. After incubating this plate, single colonies can be picked from the dilution plate and smeared on a new plate to make the master plate. (B) The master plate is the collection of each of the distinct bacteria you isolated from the soil sample. Each of these patches was transferred from a dilution plate and then the plate was incubated to grow these patches. We can now test each of these patches. (C) The unique isolates on the master plate are “streaked” onto a new plate to isolate single colonies.

References

Protocol 1: Obtaining a Soil Sample

Soil harbors an incredible number and diversity of bacteria. The goal of this experiment is for you to select a local ecosystem, study it, and bring back a soil sample to study in the laboratory. Try to select an ecosystem that will have great bacterial diversity.

SOIL SAMPLE DATA COLLECTION SHEET

Authored by: Kristen Butela—Seton Hill University

Additional Documentation:

• Photo of sample site

• Identify any plant species present near site

• Photo of soil in collection tube