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Multimethod approach: Characterization of unknown bacteria

Multimethod approach to isolate and characterize potential Streptomyces from a soil sample
Travis Rempel (100348953)
BIOL 3330 S01
April 11, 2022
Professor: Monica De Boer
Lab Instructor: Gurneet Bola Shah
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Multimethod approach: Characterization of unknown bacteria

Introduction
The bacterial genus of interest Streptomyces comes from the Phylum Actinobacteria.
Streptomyces are known to be gram-positive, saprophytes that live in soil, and have a complex
morphology of vegetative and aerial hyphae which can differentiate into spores (Science Direct,
2022a). This multicellular differentiation of hyphae is similar to the hyphae of fungi, and yet
being bacteria, this differentiation and structure is unique to this group of gram-positive bacteria
(De Lima Procópio et al. 2012). It has been discovered that Streptomyces can populate anywhere
from 1-20% of all the culturable microorganisms in the soil, and that certain methods can be
used to select for their growth (Olanrewaju, & Babalola, 2019). One simple method that can be
used is simply heating the soil which reduces the number of gram-negative bacteria and has been
shown to increase the growth of actinomycetes which are gram-positive (Abidin et al., 2015).
Streptomyces are known to produce numerous secondary metabolites which can function
as signaling molecules, as well antimicrobials which have been extremely useful in treating
infections. After the overuse of penicillin during the 1930s, the need for other antibiotics rose. A
new source arose from Streptomyces with the discovery of streptothricin and streptomycin in the
early 1940s (De Lima Procópio et al. 2012). It is estimated that of all the antibiotics that are
currently used, around 80% come from the genus Streptomyces (De Lima Procópio et al. 2012).
It is known that different species in the Streptomyces genus produce different antibiotics (De
Lima Procópio et al. 2012). Discovering new species requires identification which is often done
by sequencing parts or the whole 16S rDNA sequence for bacteria. However, it is known that in
the Streptomyces genus it is difficult to differentiate between Streptomyces species using this
method (Law, J. W. F., 2018). Therefore, other methods and characterization tests are often
performed to classify different Streptomyces species.
In this study, six characterization tests were performed in order to determine if the
microorganisms isolated were Streptomyces. The six tests included macroscopic characterization
of the isolate’s colonies which for Streptomyces could include assorted colours and the visual
presence of aerial hyphae structures. Next a gram stain will be performed which should result in
a gram-positive result. The structure and presence of the isolate’s hyphae will be viewed under a
microscope, as this multicellular structure is present in fungi as well as Streptomyces (De Lima
Procópio et al. 2012). Since an antifungal will be used, the presence of hyphae should be a
positive indicator that the microorganism isolated is likely to be Streptomyces. A starch
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Multimethod approach: Characterization of unknown bacteria

hydrolysis test will be performed which will indicate if the isolates are able to hydrolyze starch,
which while not true of all Streptomyces, a majority of do have this function (Kaneko et al.,
2005). Since it is known that Streptomyces often produce secondary metabolites that have
antimicrobial properties, the isolates will be tested against six bacteria, that are often human
pathogens, to see if the isolates are able to inhibit their growth. Lastly, the isolates will be
evaluated against three antibiotics to determine potential susceptibility or resistance. The other
purpose of this antibiotic test is to use resistance data to infer potential secondary metabolite
types that the isolates can produce since if they can produce an antibiotic, they would have
immunity genes that prevent them from being affected by it (Peterson & Kaur, 2018).
Overall, the main objective of this paper is to determine if microorganisms isolated from
a soil sample are Streptomyces by means of morphological, biochemical, and antibiotic tests.
Materials and Methods
This set of experiments was performed as outlined in the BIOL 3330 laboratory handouts
(Faculty of Science and Horticulture, Spring 2022).

Results
A soil sample was dried for 4 days under cheesecloth then heated at 55°C for 10 minutes
in order to promote the growth of Phylum Actinobacteria since they are more heat tolerant than
gram negative bacteria. Serial dilutions were performed, and the spread plate technique (Exercise
15) was used to plate four plates from 10-2 to 10-5 dilutions (Harley & Prescott, 2017). They were
plated onto starch casein agar plates with the addition of cycloheximide which inhibits fungi
growth (Science Direct, 2022b). The plates were incubated for 7 days to allow for growth.
Plate count methodology was used to calculate colony-forming units per millilitre (cfu/ml). The
10-3, 10-4, and 10-5 plates contained too few colonies to count while the 10-2 diluted plate
contained 82 colonies, therefore it was determined that there were 82000cfu/ml in the original
soil sample. From the 10-2 plate, four different colonies were transferred to their own half of an
ISP#2 agar plate and incubated for 7 days at 30°C. Then two of the four colonies were chosen,
and each were transferred to a new International Streptomyces Project-2 (ISP#2) agar plate using
the T-streak method and allowed to incubate for 5 days at 30°C. The colonies chosen will be
referred to as isolate #2 and isolate #3, while the colonies not chosen were denoted #1 and #4.
Six characterization tests were performed that each had to do with either the colony
morphology, biochemistry, or physiology.
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Multimethod approach: Characterization of unknown bacteria

The first of these was a macroscopic characterization of the colony. As seen in figures
1&3, the #2 colony was circular, convex, and undulate. The colonies were a darker brown the
denser they were, and a lighter reddish-brown colour the more spread out they got. They
produced a brown pigment that dissolved into the agar. These colonies were ridged,
characteristic of vegetative hyphae, and the raised centers are characteristic aerial hyphae. This
plate also contained an unknown yellow contaminant, but it was far away from the other
colonies. As seen in figures 2&4, the #3 colony was circular, convex, and undulate. The colonies
were yellow with grey lines at the edges on the top surface and when viewed from the bottom of
the plate the colonies appeared brown. The grey and yellow around the center are in lines leading
to the center, characteristic of vegetative and aerial hyphae. These colonies also produced a
brown pigment that spread into the agar. Also seen appears to be a white powder-like coating on
the top surface.

Figure 2. Prepared T-streak plate of isolate #3 on ISP#2 agar

Figure 1. Prepared T-streak plate of isolate #2 on ISP#2 agar
showing colony morphology. Isolate #3 consists of yellow/grey
showing colony morphology. Isolate #2 consists of the red/brown
colonies with visible white powdery spore-like features.
colonies, while the yellow colony on the left is a contaminant.

Figure 3. Single colony of isolate #2 showing detailed Figure 4. Single colony of isolate #2 showing detailed
colony morphology with visible aerial hyphae in dark brown. colony morphology with visible aerial hyphae yellow.

The second test was a gram-stain was performed to determine cell-wall structure as well
as cell shape and arrangement of each of the colonies. As seen in figure 5, isolate #2 was purple,
therefore gram-positive, its shape was round, therefore cocci, and its arrangement was
diplococcus as well as streptococcus. As seen in figure 6, isolate #3 was purple, therefore gram-
positive, its shape was round, therefore cocci, and its arrangement was diplococcus as well as
staphylococcus.
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Multimethod approach: Characterization of unknown bacteria

Figure 5. Microscopic image of gram-stained Figure 6. Microscopic image of gram-stained

isolate #2 showing purple gram-positive cocci. isolate #3 showing purple gram-positive cocci.

The third characterization test involved viewing the morphology of hyphae and spores for
each isolate. This used a coverslip at an angle in the agar with a streak of the isolate at the base
as to allow for hyphae to climb up the coverslip. It was stained with crystal violet to be viewed
under microscope with oil immersion. Isolate #2 consisted of rectiflexibile type hyphae and had
no spores visibly present. Isolate #3 consisted of rectiflexibile type hyphae and had no spores
visibly present.
Next was a biochemical characterization test which
utilized starch agar plates. The colonies were streaked in a line,
incubated for 5 days at 30°C, then multiple drops of Gram’s
iodine were put over the whole streak. The media around
isolate #2 turned blue, while a clearing around the streak was
observed for isolate #3 (Figure 7).
Figure 7. Starch agar plate showing starch hydrolysis
In the fifth test, each isolate was streaked down the results. A negative result (blue) for isolate #2 on the
left side of the plate. A positive result (clearing) for
center of their own plate containing Nutrient agar then they isolate #3 on the right side of the plate.

were incubated for 7 days at 30°C. For each plate, six different bacteria were streaked on one of
six lines that were perpendicular to the centre line containing the isolate, without touching the
centre line. The six bacteria were Alcaligenes faecalis (gram-negative), Enterobacter aerogenes
(gram-negative), Enterococcus faecalis (gram-positive), Escherichia coli (gram-negative),
Bacillus subtilis (gram-positive), and Staphylococcus aureus (gram-positive) (Science Direct,
2022c; Moehario et al., 2009). The plate was incubated at 37°C overnight to allow for potential
growth. This test was to determine the antimicrobial activity of each isolate against certain
bacteria. Results of growth inhibition due to antimicrobial activity of the isolate were compared
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Multimethod approach: Characterization of unknown bacteria

in a 4-point scale where “+++” was good inhibition, “++” was moderate inhibition, “+” was
weak inhibition, and “-” was no inhibition. For isolate #2 as seen in figure 8, E. coli was in the
good inhibition category, E. faecalis was in the moderate inhibition category, B. subtilis, A.
faecalis, and E. aerogenes were in the weak inhibition category, and S. aureus was in the no
inhibition category. For isolate #3 as seen in figure 9, E. coli was in the good inhibition category,
E. faecalis was in the moderate inhibition category, B. subtilis, A. faecalis, and E. aerogenes
were in the weak inhibition category, and S. aureus was in the no inhibition category.

Figure 8. Antimicrobial activity of isolate #2 on Nutrient agar Figure 9. Antimicrobial activity of isolate #3 on Nutrient agar
against six bacterial species. On the left side from top to bottom is against six bacterial species. On the left side from top to bottom
E. coli, B. subtilis, and S. aureus. On the right side from top to is E. coli, B. subtilis, and S. aureus. On the right side from top to
bottom is A. faecalis, E. aerogenes, and E faecalis. bottom is A. faecalis, E. aerogenes, and E faecalis.

For the last test, each isolate was inoculated in a tube with starch casein medium in order
to increase the growth to be used with the Kirby-Bauer method (Exercise 43) (Harley & Prescott,
2017). A lawn was prepared on starch casein agar for each isolate, and three antibiotic disks were
placed on the surface of the agar. The antibiotic disks were streptomycin, penicillin, and
chloramphenicol. For isolate #2, no lawn growth and therefore also no inhibition was observed
(Figure 10). For isolate #3, a complete lawn was not observed, but it could be seen that

Figure 10. Antibiotic sensitivity test on Starch Casein agar for Figure 11. Antibiotic sensitivity test on Starch Casein agar for
isolate #2 showing no lawn growth and no inhibition. The isolate #3 showing growth and inhibition for the Streptomycin
antibiotic disks from left to right are Penicillin 10ug, 10ug disk. The antibiotic disks from left to right are Penicillin
Chloramphenicol 30ug, and Streptomycin 10ug. 10ug, Chloramphenicol 30ug, and Streptomycin 10ug.
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Multimethod approach: Characterization of unknown bacteria

streptomycin greatly inhibited the growth with an inhibition radius from the disk of 20mm, while
no inhibition was seen for either penicillin or chloramphenicol (Figure 11).

Discussion
Soil samples were chosen since Streptomyces are known to make up around 1-20% of all
the soil microbes that are culturable (Olanrewaju, & Babalola, 2019). Streptomyces fall under
phylum Actinobacteria which is the third largest phyla of bacteria that are found in the soil,
behind Acidobacteria and Proteobacteria (Janssen, P. H., 2006). Since Acidobacteria and
Proteobacteria are both gram-negative, and Actinobacteria are gram-positive, the soil sample was
heated in order to inhibit the growth of gram-negative bacteria and promote the growth of gram-
positive bacteria, especially Streptomyces (Abidin et al., 2015). Cycloheximide was used during
the plating process since it inhibits fungi growth by stopping ribosomal function of the
eukaryotic 60S large subunit (Science Direct, 2022d). Four different colonies were chosen, and
afterwards two out of the four that were the most distinct were chosen, and this was done in
order to choose potential streptomyces that might produce different secondary metabolites
(Faculty of Science and Horticulture, Spring 2022). They were plated onto International
Streptomyces Project-2 (ISP#2) agar since this media is often used in the cultivation of
Streptomyces (ActinoBase, 2019).
Table 1. Summary of results of morphological, biochemical, and physiological tests for isolate #2 and isolate #3
Colony/Cellular properties Isolate #2 Isolate #3
Colony growth properties Dark brown, ridged Yellow & grey, ridged, white powdery
coating
Form, Elevation & Margin Circular, convex, undulate Circular, convex, undulate
Substrate mycelium colour Brown Yellow & grey
Aerial mycelium Raised, brown Raised, yellow, white powdery coated
Production of soluble pigment +, Brown +, Brown
Gram stain Gram-positive Gram-positive
Shape/Arrangement Diplococci & Streptococci Diplococci & Staphylococci
Hyphae Morphology (spores) Rectiflexibile type, (-) Rectiflexibile type, (-)
Starch hydrolysis (+/-) Negative Positive
Antimicrobial activity against six bacteria
“+++” Good inhibition E. coli E. coli
“++” Moderate inhibition E. faecalis E. faecalis
“+” Weak inhibition B. subtilis, A. faecalis, & E. aerogenes B. subtilis, A. faecalis, & E. aerogenes
“-” No inhibition S. aureus S. aureus
Antibiotic sensitivity
Streptomycin N/A 20mm, Sensitive
Penicillin N/A Resistant
Chloramphenicol N/A Resistant
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Multimethod approach: Characterization of unknown bacteria

Six characterization tests were performed that each had to do with either the colony
morphology, biochemistry, or physiology, and a summary of the results can be seen on table 1
above. The six tests were: macroscopic characterization, gram stain and cellular shape
identification, hyphae morphology using the coverslip culture technique, a starch hydrolysis
biochemical test, isolate antimicrobial activity using cross streak method, and an antibiotic
susceptibility test.
Through macroscopic observation of the colonies, it could be seen that the two isolates
were quite different from each other in colour as isolate #2 was a brown colour while isolate #3
was mainly yellow with stripes of grey. However, they were similar in distribution of vegetative
hyphae below and around the edges with a central column of aerial hyphae that was raised.
Isolate #3 also had a white powdery coating that is indicative of spores produced by aerial
hyphae. This hyphae cellular differentiation is characteristic in Actinobacteria (Science Direct,
2022a). Another similarity is that both isolates produced a brown soluble pigment that could be
seen in the agar which could be indicative of production of a secondary metabolite.
Gram staining resulted purple, and therefore gram-positive cells, for both isolates which
is a characteristic of streptomyces. The cellular shape seen for isolate #2 was cocci which could
be spores. Isolate #3 also consisted of cocci shaped cells.
Next the coverslip culture technique was used in order to allow for growth of aerial hyphae onto
the coverslip in order to preserve the shape and arrangement. Both isolates consisted of aerial
hyphae that did not show any spores and were classified under rectiflexibile type chains since
they were mostly straight chains that were sometimes in groups (Li et al., 2016).
To characterize the isolates by biochemical means, they were evaluated for the ability to
hydrolyze starch (Exercise 22) with the α-amylase enzyme (Harley & Prescott, 2017). Gram’s
iodine is used since it binds to starch and produces a blue colour, while hydrolyzed starch does
not bind with gram’s iodine. Isolate #2 turned blue, indicating that no starch had been
hydrolyzed, while isolate #3 had a clearing around the inoculation streak therefore showing that
the starch around the isolate had been hydrolyzed. Although not all Streptomyces have the ability
to hydrolyze starch, there are a large amount of Streptomyces that do have this ability (Kaneko et
al., 2005).
Each isolate was then assessed for its ability to produce secondary metabolites that would
inhibit the growth of other bacteria. Both the isolates had the same inhibitory effects against the
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Multimethod approach: Characterization of unknown bacteria

six test organisms. They both inhibited Escherichia coli the most, had no inhibitory effects
against Staphylococcus aureus, and had moderate or weak inhibitory effects against the other
four. Although the two isolates are morphologically different, the similarity of both producing a
brown soluble pigment could be indicative of producing similar or the same secondary
metabolites, which would be the reason for similarly inhibiting the six bacteria at the levels
mentioned. The secondary metabolites produced seemed to have no preferential for inhibiting the
growth of either gram-positive or gram-negative bacteria.
Using the Kirby-Bauer method (Exercise 43), each isolate was assessed for growth
potential on agar containing antibiotic disks of streptomycin, penicillin, and chloramphenicol
(Harley & Prescott, 2017). Isolate #2 had no lawn growth and therefore no inhibition potentially
due to an error when the growth tube was inoculated. Therefore, isolate #2 has unknown
antibiotic sensitivity to the three antibiotics. While there was not a complete lawn of growth for
isolate #3s plate, there was enough to determine sensitivity levels. Isolate #3 was largely
sensitive to streptomycin while showing no signs on inhibition to either penicillin or
chloramphenicol. It is known that bacteria like Streptomyces that produce antibiotics must also
have a way to not be affected by the same antibiotics they are secreting, and this is due to
immunity genes in their genome (De Boer, 2022). It has been found that the genes that produce
the antibiotic are often close in proximity to genes that resist the antibiotic and that these two sets
of genes often get transcribed simultaneously to ensure the cell’s survival (Peterson & Kaur,
2018). Since isolate #3 was highly sensitive to streptomycin, it can be assumed that this isolate
does not contain any aminoglycoside modification enzymes which are used to inactivate
streptomycin and other aminoglycoside antibiotics (Peterson & Kaur, 2018). Although penicillin
targets gram-positive bacteria, it is known that Streptomyces that produce antibiotics can have
elevated levels of resistance to penicillin through target modification and due to the fact that
these bacteria produce β-lactam antibiotics of which penicillin is a part of (Peterson & Kaur,
2018). Therefore, it is possible that isolate #3 has these mechanisms and may be able to produce
β-lactam antibiotics. It is also known that many Streptomyces produce β-lactamases which
hydrolyze β-lactam antibiotics (Peterson & Kaur, 2018). Lastly, isolate #3 was able to resist the
antibiotic chloramphenicol which could be due to possessing chloramphenicol acetyl transferase
enzymes which inhibit the antibiotic. The resistance ability of isolate #3 to penicillin as well as
chloramphenicol are highly characteristic of Streptomyces (Peterson & Kaur, 2018).
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Multimethod approach: Characterization of unknown bacteria

Conclusion
Although this lab is unable to certainly verify the identity of each isolate, the combination
of the results of each characterization test can be inferred as to whether the isolates were
Streptomyces. The two isolates were chosen due to having the characteristic look of
Streptomyces, which included being “small, hard and friable” along with being dull in colour,
although colour may vary (Faculty of Science and Horticulture, Spring 2022). These isolates
match those descriptions along with showing characteristic structure of hyphae on agar and
under microscope. Both were gram-positive which is known of Streptomyces. One difference
between the two was the starch hydrolysis test. While a positive starch hydrolysis test is
indicative of Streptomyces, not all Streptomyces have this ability, and other bacteria have this
ability as well (Kaneko et al., 2005). The similarity in both the isolates producing a brown
pigment could be the reason they had similar antimicrobial activity to the six test bacteria. It is
known that many Streptomyces are able to produce a vast number of secondary metabolites
which can function as antimicrobial agents against certain other bacteria (De Boer, 2022). Lastly,
unfortunately there is no comparison with regards to antibiotic sensitivity between the two
isolates since no lawn formed for isolate #2. In order to avoid this in future experiments, multiple
replicates should have been used. Isolate #3 possesses mechanisms to inhibit penicillin as well as
chloramphenicol, but it is extremely sensitive to streptomycin. Resistance to certain antibiotics
could mean that the bacteria happen to have mechanisms to resist the effects, and it is highly
possible that they could be self-resistance genes which are accompanied with the ability to
produce these or similar antibiotics which is a known feature of Streptomyces (Peterson & Kaur,
2018). Based on all these aforementioned characteristics and methods to select for
Actinobacteria growth, it is highly plausible that both isolate #2 and isolate #3 are Streptomyces.
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Multimethod approach: Characterization of unknown bacteria

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