Microbial biodiversity and its rich arsenal of natural products is an important and complex biological concept. We propose the use of Streptomyces, one of the most diverse bacterial genera and a major inhabitant of soil, as a model to explain the relevance and importance of this concept. Students will perform experiments ranging from isolation and selection of Streptomyces species, to performing fungal and bacterial challenge assays to evaluate their biocontrol capacity, to screening for specialized metabolic properties such as the production of lipases, amylases, and cellulases. Accompanied by active discussions on the experimental process and results, and integrated into the general microbiology curriculum, this real-life discovery-based lab exercise engages students in current topics concerning natural product discovery and reinforces their understanding of several important concepts in microbiology and biotechnology.

Introduction

Bringing a real-life scientific project into the classroom enables students to connect textbook study material to current field and laboratory research and demonstrates the true nature of science – a process of observation, discovery, and hypothesis testing. The field of microbiology lends itself very well to that perspective, yet traditional microbiology lab exercises often fail to make that connection because the exploratory component is missing. Most lab exercises include familiar bacterial species, presented as prepared pure cultures, and instruct students to re-culture the bacteria on various media to observe their metabolic traits. Although these exercises are valuable, with the following activity we aim to integrate core microbiological concepts with hands-on exploration of bacterial diversity and metabolism. Hence, this exercise reinforces the understanding of several important concepts and current topics in microbiology and biotechnology, including (1) the ubiquity of microorganisms, (2) inter-organismal interactions, (3) the origin and discovery of antimicrobial compounds, (4) continued natural product research urged by the rising threat of antibiotic resistance, and (5) the extensive metabolic diversity of microbes that can be exploited for our use. Additionally, students will develop essential microbiological skills such as aseptic technique, bacterial purification, and culturing.

Soil is one of the richest and most densely populated microbial environments. It houses most representative types of bacteria, including Actinobacteria, and a large number of microenvironments in which microbes interact with each other. Actinobacteria are a very diverse group of Gram-positive bacteria with a preference for G and C nucleotides over A and T nucleotides in their DNA and include Streptomyces, one of the most dominant inhabitants of soil. This large bacterial genus, encompassing several hundred species, is characterized by a complex life cycle that at first glance resembles that of filamentous fungi (Hopwood, 2007). Spores germinate to form branched hyphae that constitute the vegetative mycelium, with the primary function of nutrient acquisition. Upon depletion of these nutrients, growth is directed upward, forming nonbranching aerial hyphae, the individual cells of which develop a strong hydrophobic cell wall that is often pigmented (Barka et al., 2016). Apart from an unusual developmental process, Streptomyces are able to produce an exceptional arsenal of natural products with important agricultural, medical, and biotechnological applications, such as antibiotics, antifungals, antivirals, immunosuppressants, insecticides, catabolic enzymes able to degrade complex polymers, toxins, and pigments (Chater et al., 2009). Although the fungus Penicillium is probably the best-known antibiotic-producing microorganism, Streptomyces species alone are responsible for the production of over two-thirds of the antibiotics known to date (Barka et al., 2016). One of the most remarkable examples is the discovery and impact of ivermectin and related avermectins, natural products derived from S. avermitilis that are widely used as antiparasitic drugs and have saved thousands of lives (Crump & Omura, 2011). Other examples reflecting the wide variety of natural products produced by Streptomyces species include the tetracyclines, with broad-spectrum antibacterial activity (Nelson & Levy, 2011); the plant toxins thaxtomin (King & Lawrence, 2001) and desmethylmensacarcin (Lapaz et al., 2018); and the volatile compound geosmin, responsible for the characteristic odor of moist soil (Jiang et al., 2007).

Despite these remarkable characteristics, Streptomyces species are given little attention in traditional microbiology textbooks. They are often overlooked during lab exercises as well, probably due to the misconception that they are difficult to grow and handle. Yet the exploration of Streptomyces diversity and properties lends itself very well to a discovery-based lab exercise that engages students in current topics concerning natural product discovery by literally allowing them to explore what is going on in their backyard.

Concept Explanation

The proposed activity captures several topics that form the foundation of a microbiology course and provides students with real-life research experience. The practical component starts by introducing students to the rich bacterial diversity by plating extracts of soil samples collected from diverse locations. Streptomyces isolates are selected for further analysis of their antifungal and antibacterial abilities and their production of extracellular catabolic enzymes. This remarkably simple activity reflects the study and exploration of inter-organismal interactions beyond the more known host–pathogen interactions, in combination with the ongoing search for new and useful natural products. It also shows how modern advancements in medicine, agriculture, and multiple other aspects of everyday life rely on the discovery and application of natural processes.

Materials for the Practical Component

Materials were purchased from Genesee Scientific, Fisher Scientific, or Sigma Aldrich, unless otherwise indicated.

Soil Sampling & Isolation of Streptomyces

  • Zip lock bags

  • Clean spoons

  • Beakers

  • Balance

  • Sterile microcentrifuge tubes or glass tubes to perform a dilution series

  • Micropipettes or transfer pipettes with gradations

  • Cell spreaders

  • Sterile tooth picks

  • Inoculation loops

  • Agar

  • Optional: Corn meal agar (CMA), International Streptomyces Project Medium No. 4 (ISP-4; e.g., HiMedia), or potato mash agar (PMA: 50 g instant potato flakes and 20 g agar per liter)

  • Antimicrobial solution (10 mL for 1 L medium) (Loria et al., 2010): nystatin (optional; 50 mg), penicillin G (1 mg), polymyxin B (5 mg), and cycloheximide (50 mg) (e.g., GoldBio)

  • International Streptomyces Project Medium No. 2 (ISP-2); this medium can be purchased (e.g., HiMedia) or prepared by dissolving 4 g yeast extract, 10 g malt extract, and 4 g dextrose in 900 mL DI water. Adjust the pH to 7.2 before bringing the volume to 1 L. Pour the solution into a bottle containing the appropriate amount of agar (20 g/L) and autoclave.

  • Autoclave

  • Petri dishes

  • Optional: plate incubator at 28–30°C

Challenge Assays

  • One of the following media: CMA, Sabouraud dextrose agar (SabDex), or potato dextrose agar (PDA)

  • Tryptic soy agar (TSA)

  • Petri dishes

  • Inoculation loops

  • Inoculation needle or sterile toothpicks

  • Bunsen burner or Bacti-Cinerator (e.g., Carolina Biological Supply)

  • Bacterial and fungal cultures of choice

  • Optional: plate incubator at 28–30°C

Enzymatic Tests

  • Petri dishes

  • TSA starch: add soluble starch (2 g/L) to the TSA mixture before autoclaving

  • TSA yolk: mix equal parts (50 mL) of yolk and physiological serum (0.9% NaCl), autoclave, and combine with separately autoclaved TSA at 10 mL/L. To accommodate the addition of the yolk mixture, the TSA should be dissolved in less DI water.

  • Basal medium-carboxymethylcellulose (CMC): dissolve 0.75 g MgSO4.7H2O, 2.5 g NaNO3, 1.75 g CaCl2.H2O, and 10 g CMC in 900 mL DI water. Pour the solution into a bottle containing the appropriate amount of agar (15 g/L) and autoclave (Arotupin, 2007)

  • Lugol's iodine solution and Congo Red (e.g., Carolina Biological Supply)

  • 0.1 M NaCl

  • Optional: plate incubator at 28–30°C

Experimental Setup

Students are asked to collect soil from a site of choice, preferably from an “interesting” site that they might encounter on their way to school. For example, California State University Bakersfield serves students in a wide area of the southern San Joaquin Valley, and many students commute to campus on a daily basis. They pass a variety of soil ecotypes, including well-irrigated lawns, agricultural fields containing a large variety of crops and trees, desert soils, and sites exploited for oil extraction. Students collect soil in a clean zip lock bag from 5–10 cm depth or from the rhizosphere of a plant. They are also asked to record information about the sampling site, such as the soil type, the vegetation, and the location, through pictures and a description. To avoid contamination from other origins than the sampling site, bags must be kept closed as much as possible.

In the lab, 10 g of soil is transferred to a beaker with 100 mL of sterile DI water. After 10 minutes incubation under frequent mixing, a sample is taken to start a tenfold dilution series up to 10−4. Then 100 µL of each dilution is plated on an agar medium of choice. We recommend one of the following media, depending on availability and/or budget: CMA, ISP-4, PMA, or simply 1.5% agar in water (Figure 1A). Antimicrobials are added after autoclaving, when the medium is allowed to cool to 50°C. Plates are incubated in the dark for at least a week at a temperature ranging from room temperature to preferably 28–30°C.

Figure 1.

Visual inspection and isolation of Streptomyces soil isolates. (A) Bacterial diversity from a single soil sample on various media recommended for the isolation of Streptomyces: CMA (corn meal agar), ISP-4 (International Streptomyces Project Medium No. 4), PMA (potato mash agar), and 1.5% agar-water. (B) Quadrant streak of a selected Streptomyces isolate on ISP-4.

Figure 1.

Visual inspection and isolation of Streptomyces soil isolates. (A) Bacterial diversity from a single soil sample on various media recommended for the isolation of Streptomyces: CMA (corn meal agar), ISP-4 (International Streptomyces Project Medium No. 4), PMA (potato mash agar), and 1.5% agar-water. (B) Quadrant streak of a selected Streptomyces isolate on ISP-4.

During the next lab session, students visually inspect the plates to get a sense of the bacterial diversity of their soil sample. Although the plates, especially those with rich media, will show growth of a variety of species (Figure 1A), students are reminded that only a small percentage of the actual soil-inhabiting bacteria can be cultured in the lab. Potentially, inter-organismal interactions can be observed between neighboring colonies. This can be in the form of antibiosis (growth inhibition inflicted by one of the interacting partners), the production of pigmented compounds, or differences in growth patterns where two colonies meet.

Based on their knowledge of the exceptional developmental life cycle of Streptomyces, students will identify potential Streptomyces isolates (Figure 1A). A sterile toothpick is used to isolate selected colonies by initiating a quadrant streak on a fresh plate of the isolation medium (Figure 1B). If agar-water was used for the isolation process, ISP-2 medium is recommended to ensure good growth and sporulation of the Streptomyces strains. Plates are incubated as described above.

The obtained pure cultures can now be examined to uncover their antifungal, antibacterial, and enzymatic properties. Considering the slower growth rate of Streptomyces species in comparison to most other bacteria and fungi, the challenge assays need to be performed in two steps. Streptomyces are inoculated on the plates at least one week before the selected target bacteria and fungi.

To test for antifungal activity of the isolated Streptomyces, CMA, SabDex, or PDA plates are inoculated as shown in Figure 2, with the fungal isolate in the middle of the plate surrounded by up to three different Streptomyces isolates. Fungal spores are easily dispersed through the air, which can lead to contamination of the entire plate. To avoid this, we recommend one of the following procedures for the inoculation of fungi. A loop or a micropipette can be used to carefully transfer a small amount (2–5 µL) of a spore suspension (made in sterile DI water) to the middle of the plate without dripping. Alternatively, with an inoculation needle or a sterile toothpick, fungal spores can be inoculated on a plate that is held upside down. Inoculated plates are incubated in the dark at room temperature, which is optimal for fungal growth.

Figure 2.

Challenge assay of Streptomyces isolates against the fungus Aspergillus niger on corn meal agar (CMA).

Figure 2.

Challenge assay of Streptomyces isolates against the fungus Aspergillus niger on corn meal agar (CMA).

Each Streptomyces isolate can be tested against up to six different bacteria on a single TSA plate. The target bacteria are inoculated in close proximity to, but without touching, the Streptomyces isolate (Figure 3). Students inspect the plates daily to evaluate potential fungal or bacterial growth inhibition.

Figure 3.

Challenge assay of a Streptomyces isolate (middle) against the bacteria Citrobacter freundii (Cf), Escherichia coli (Ec), Enterococcus faecalis (Ef), Pseudomonas aeruginosa (Pa), Staphylococcus aureus (Sa), and Salmonella typhimurium (St) on TSA.

Figure 3.

Challenge assay of a Streptomyces isolate (middle) against the bacteria Citrobacter freundii (Cf), Escherichia coli (Ec), Enterococcus faecalis (Ef), Pseudomonas aeruginosa (Pa), Staphylococcus aureus (Sa), and Salmonella typhimurium (St) on TSA.

To screen the different Streptomyces isolates for the production of various biotechnologically important enzymes such as amylases, lipases, and cellulases, the isolates are inoculated on TSA starch, TSA yolk, and CMC (Arotupin, 2007), respectively. Up to four different Streptomyces isolates can be inoculated on a single plate. Plates are incubated, preferably at 28–30°C, for at least one week. Production of the respective enzymes will lead to a clearing of the medium that can be seen as a halo around the growth (Figure 4A). For visualization of the cleared zone due to amylase activity, the TSA starch plates are flooded with Lugol's iodine (pour off excess solution) and scored after 10 minutes (Arotupin, 2007; Figure 4B). Streptomyces species that produce one or more cellulase enzymes will be able to grow on the CMC-containing plates (Figure 4C, left panel). The loss of cellulose in the plate due to extracellular digestion is visualized by flooding the CMC-containing plates with 0.1% Congo Red for 5–10 minutes and washing several times with 0.1 M NaCl (Arotupin, 2007), leaving a clear orange zone around cellulase-positive isolates (Figure 4C, right panel).

Figure 4.

Screen for enzymatic properties of Streptomyces isolates. (A) Lipase activity shown by the cleared zone around the growth of certain Streptomyces species on TSA yolk. (B) Amylase activity revealed by clearing of the dark color after treatment of the TSA starch plate with Lugol's iodine (right panel). (C) Cellulase activity enables Streptomyces isolates to grow on Basal medium-CMC (left panel) and can be visualized as a clear orange zone around the growth after treatment with Congo Red (right panel).

Figure 4.

Screen for enzymatic properties of Streptomyces isolates. (A) Lipase activity shown by the cleared zone around the growth of certain Streptomyces species on TSA yolk. (B) Amylase activity revealed by clearing of the dark color after treatment of the TSA starch plate with Lugol's iodine (right panel). (C) Cellulase activity enables Streptomyces isolates to grow on Basal medium-CMC (left panel) and can be visualized as a clear orange zone around the growth after treatment with Congo Red (right panel).

Instruction

The activity is well suited for AP Biology and college-level undergraduate microbiology courses. For students to fully benefit from this experiential learning activity, it is critical that the practical exercise is supported by a solid theoretical background and guided discussions. The level and depth of the accompanying lectures can be adapted to align with existing course objectives and the academic level of the students. The isolation and exploration of soil Streptomyces can be connected to several lecture topics in the general microbiology curriculum, including microbial diversity, metabolism, growth, development and differentiation, ecology, environmental microbiology, and antimicrobial drug discovery, applications, and the growing threat of antibiotic resistance. For advanced classes, topics might also include the biotechnological optimization of enzyme activity, enzymatic transformation of plant biomass for biodiesel production, and biological pest management for agriculture. Nevertheless, the timely topic of natural product discovery and inter-organismal interactions is significant to students with different backgrounds and career goals and can be adjusted to suit general education classes.

We have outlined a schedule in Table 1 comprising two lectures (75 minutes each) and six lab sessions (2.5 hours each) to capture the essential components and learning outcomes of this activity. We recommend at least two lectures to provide students with the necessary background for the exercise. To design these lectures, teachers can rely on any of the traditional microbiology textbooks, supplemented with primary literature for more specific information on Streptomyces (suggestions include Chater, 2006; Hopwood, 2007; Chater et al., 2009).

Table 1.
Suggested outline of the exercise, capturing the essential components and learning outcomes of the lecture (75 minutes) and lab sessions (2.5 hours).
ContentMain Learning Outcomes
Lecture 1 Introduction to microbiology and the ubiquity of microbes
Bacterial vs. basic fungal growth
Unique morphology and development of Streptomyces bacteria 
Understanding the main differences in morphology and growth of bacteria and fungi (molds)
Understanding that nature is not always black and white with the demonstration of Streptomyces, bacteria with growth and development resembling fungi (molds) 
Lecture 2 Rich microbial diversity of the soil and their role in nature
Natural inter-organismal interactions
  • Symbiosis

  • Antibiosis


Antibiotics
  • Discovery and origin

  • Streptomyces as the main antibiotic-producing bacterial genus

  • Potential cellular targets

  • Applications (medicine, biocontrol)


Nutritional diversity
  • Dependent on enzymatic properties

  • Applications (industry)

 
Recognition that microbes are everywhere
Understanding of the role of microbes in nature and how they interact with others
Understanding that antibiotics are natural compounds produced by microorganisms (role in nature)
Understanding the potential applications of antibiotics and antibiotic-producing organisms for our benefit
Understanding of the link between nutritional range and enzymatic properties
Understanding the potential applications of unique microbial enzymes in our daily lives 
Lab 1 Exploration of macroscopic differences between bacteria and fungi
Introduction of Streptomyces with non-typical macroscopic bacterial characteristics
Formulation of hypotheses based on
  • Differences in sampling sites and soil properties

  • Properties of the agar medium (nutrients, antimicrobial compounds)

 
Recognition that microbes are everywhere
Being able to macroscopically differentiate between bacteria and fungi (molds)
Becoming familiar with the macroscopic characteristics of Streptomyces colonies
Being able to formulate scientific hypotheses 
Lab 2 Isolation of Streptomyces from soil
  • Aseptic technique

  • Dilution series

  • Plating bacteria on agar medium

 
Becoming familiar with basic tools, materials, and requirements for microbial cultivation
Acquiring the skills to isolate and grow bacteria 
Lab 3 Evaluation of tested hypotheses
Selection and purification of obtained Streptomyces isolates
  • Visual inspection of obtained colonies

  • Quadrant streaking

 
Being able to evaluate and correctly interpret the results of the tested hypotheses
Acquiring skills to handle bacteria
Acquiring the quadrant streak technique 
Lab 4 Challenge assays (part 1)
Inoculation of Streptomyces isolates for challenge assays 
Acquiring skills to handle bacteria
Understanding of the different growth rates and patterns of different microorganisms 
Lab 5 Challenge assays (part 2) and enzymatic tests
  • Inoculation of fungi and bacteria to be challenged by the Streptomyces isolates

  • Inoculation of Streptomyces isolates on plates to test their different metabolic properties

 
Acquiring skills to handle and inoculate fungi (molds)
Understanding of the enzymatic properties to be tested 
Lab 6 Completion of the enzymatic tests
Evaluation and discussion of all results (tested hypotheses, challenge tests, and enzymatic tests)
How to report scientific data 
Being able to correctly interpret the challenge assays and the enzymatic tests
Being able to efficiently summarize the research data
Being able to write scientifically 
ContentMain Learning Outcomes
Lecture 1 Introduction to microbiology and the ubiquity of microbes
Bacterial vs. basic fungal growth
Unique morphology and development of Streptomyces bacteria 
Understanding the main differences in morphology and growth of bacteria and fungi (molds)
Understanding that nature is not always black and white with the demonstration of Streptomyces, bacteria with growth and development resembling fungi (molds) 
Lecture 2 Rich microbial diversity of the soil and their role in nature
Natural inter-organismal interactions
  • Symbiosis

  • Antibiosis


Antibiotics
  • Discovery and origin

  • Streptomyces as the main antibiotic-producing bacterial genus

  • Potential cellular targets

  • Applications (medicine, biocontrol)


Nutritional diversity
  • Dependent on enzymatic properties

  • Applications (industry)

 
Recognition that microbes are everywhere
Understanding of the role of microbes in nature and how they interact with others
Understanding that antibiotics are natural compounds produced by microorganisms (role in nature)
Understanding the potential applications of antibiotics and antibiotic-producing organisms for our benefit
Understanding of the link between nutritional range and enzymatic properties
Understanding the potential applications of unique microbial enzymes in our daily lives 
Lab 1 Exploration of macroscopic differences between bacteria and fungi
Introduction of Streptomyces with non-typical macroscopic bacterial characteristics
Formulation of hypotheses based on
  • Differences in sampling sites and soil properties

  • Properties of the agar medium (nutrients, antimicrobial compounds)

 
Recognition that microbes are everywhere
Being able to macroscopically differentiate between bacteria and fungi (molds)
Becoming familiar with the macroscopic characteristics of Streptomyces colonies
Being able to formulate scientific hypotheses 
Lab 2 Isolation of Streptomyces from soil
  • Aseptic technique

  • Dilution series

  • Plating bacteria on agar medium

 
Becoming familiar with basic tools, materials, and requirements for microbial cultivation
Acquiring the skills to isolate and grow bacteria 
Lab 3 Evaluation of tested hypotheses
Selection and purification of obtained Streptomyces isolates
  • Visual inspection of obtained colonies

  • Quadrant streaking

 
Being able to evaluate and correctly interpret the results of the tested hypotheses
Acquiring skills to handle bacteria
Acquiring the quadrant streak technique 
Lab 4 Challenge assays (part 1)
Inoculation of Streptomyces isolates for challenge assays 
Acquiring skills to handle bacteria
Understanding of the different growth rates and patterns of different microorganisms 
Lab 5 Challenge assays (part 2) and enzymatic tests
  • Inoculation of fungi and bacteria to be challenged by the Streptomyces isolates

  • Inoculation of Streptomyces isolates on plates to test their different metabolic properties

 
Acquiring skills to handle and inoculate fungi (molds)
Understanding of the enzymatic properties to be tested 
Lab 6 Completion of the enzymatic tests
Evaluation and discussion of all results (tested hypotheses, challenge tests, and enzymatic tests)
How to report scientific data 
Being able to correctly interpret the challenge assays and the enzymatic tests
Being able to efficiently summarize the research data
Being able to write scientifically 

During the first lab, students will become familiar with the different groups of microbes by observing and discussing the different microbial morphologies of bacteria, fungi, and the atypical bacterial genus Streptomyces grown on solid media. To illustrate diversity, a TSA and a SabDex plate opened to the outside air (~10 minutes) and incubated for about seven days at room temperature can be used, as these plates will display a wide variety of bacterial and fungal growth, respectively. Subsequently, students are introduced in detail to the project, and teachers initiate and moderate discussions that will guide students to formulate hypotheses and predictions that could possibly be tested in relation to the project. Meaningful topics for discussion include soil features such as moisture, organic matter, the presence or absence of plants, and their effects on microbial diversity; metabolic properties; and the level of occurrence of interactions between different microbial species. Here are two examples of potential hypotheses:

  • Microbial diversity is a reflection of the amount of organic matter contained in the soil; therefore, soil sampled in the vicinity of plant roots or soil containing decaying leaves and roots will contain a greater variety of microbes than soil with a low amount of organic matter.

  • Isolation of Streptomyces species on PMA will favor bacteria with a preference for starch; therefore, more amylase-positive Streptomyces isolates will be obtained when using PMA as the isolation medium.

In addition, the discussion should include potential technical limitations such as the inability to culture most microbes in the lab, the diverse nutritional preferences of different microorganisms and their effect on the choice of culture medium, the differences in growth rate and pattern, and the use of certain antimicrobials in the agar medium.

Upon completion of the project, students will be instructed on data evaluation, presentation of results, and how to write a lab report. Students discuss in small groups the obtained results in relation to the methods used and the hypotheses tested.

In summary, the overall outcome of the exercise is to broaden students' ideas and knowledge on the following main topics: (1) the rich microbial diversity of the soil, (2) the main differences between bacteria and fungi, (3) the origin and discovery of antibiotics, (4) the versatility of microbial enzymatic properties, and (5) the applications and impact of these natural products in and on our daily lives.

Student Assessment

Before the start of the research component of this exercise, we recommend that students are assessed regarding their understanding of the topic and upcoming experimental design in two quizzes. The first quiz contains questions about the theoretical lecture material as outlined in Table 1. The second quiz is aimed to evaluate whether the students understand and are ready for the practical inquiry part of the exercise and will focus on the project goal and experimental design. Upon completion of all the tests, students will discuss and summarize their results in a table. This table will include all tested Streptomyces species accompanied by a description and a picture to show their colony morphology, color, and other macroscopic features, the results of the bacterial and fungal challenge assays, and their particular metabolic properties. This table will then serve as part of the results section of a lab report alongside a description of the goal of the project, a materials and methods section, and a conclusion. The materials and methods section is subdivided into a description of the sampling site and soil, the isolation and purification methods, and the procedures for the challenge assays and the enzymatic tests.

We thank Dr. Rodrigo Achigar for sharing the composition of the media for the amylase and lipase assays, Yves Denert for photographically documenting the obtained results, and Dr. L. Maynard Moe for critical reading of the manuscript. We acknowledge financial support from the National Institute of Food and Agriculture, U.S. Department of Agriculture, under award nos. 2016-67032-25008, for lab supplies and student support.

References

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