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.
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.
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).
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).
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
Figure 2. Challenge assay of Streptomyces isolates against the
fungus Aspergillus niger on corn meal agar (CMA).
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.