Name: Victor Brown

ID#: 620044335
Course: Applied and Environmental Microbiology
Lab: 4
Topic: Interrelation of Microbes

Aim: To isolate pure colonies of Actinomycetales from EMB streak plates then identify them based on
colony morphology and test them for antibiotic activity on plates inoculated with E. coli and B. Subtilis.

Abstract: Pure cultures of bacteria and other microorganisms are usually being studied for their microbial
activities. In nature however these organisms live in environments and communities populated with many
microorganism that will affect their overall survival. The relationship between these organisms can be
positive or negative depending on the effect of one organism’s activities on another. In this experiment the
effect of antibiotic producers in soil such as the Actinomycetales is being examined. The Actinomycetales
are isolated from soil samples by streaking soil samples on EMB plates then inoculating plates with isolates
from streak plate to obtain pure colonies. After pure colonies have been attained 2 emulsion plates are made
one of E. coli and the other B. Subtilis. Plugs are taken from the pure colonies and inverted into the
emulsion plates to observe for inhibition of growth. Clear regions were observed in both emulsion plate
which indicated antibiosis.

Method: Experiment was carried out as outlined on page 7-11 of the lab manual with the following
adjustments being made.
 B. Subtilis was used in the Emulsion Agar instead of S. aureus
 Plates were divided 4 section

Results:

Table Showing Observation of Colonies Isolated from Streak and Spread Plate of Sample D
Sample Type of Plate Observation
Spread Plate Actinomycete colonies are dark grey in colour with convex surfaces,
D edges are rough.
Streak Plate Pure powdery white colonies. The colonies were circular with convex
elevation. Colonies grew closely together for the uppermost streak but
grew more isolated towards the end of the streak which might be an
indicative of the colonies needing more time for development.

Groups
C.W. & T.N.
Sash & Rash
S. Facey, S. Clarke & S. Gourbong
S. Prendagast & R. Higgins
D. Perry & S. McKenzie
T. Orgill, V. Brown & R. Shallow
L. Gabbidon, D. Duncan & M. Thomas
S. Taylor, Y. Linton
M. Blair, S. Benjamin & K. Baily
Table Showing Class Results for
Soil Sample Observations
E Round colonies, red in colour, entire edge, convex elevation and rough,
dull, dry and wrinkled surface
E Cream pigmentation, Rough, dull & powdery surface with round, lobate
margin and flat elevation.
A Orange red, dry & powdery, round with scalloped margins, thread like,
hilly elevation
C Red pigmentation, wrinkled conformation, umbonate elevation, rough
surface, dry/ powdery surface.
A Wrinkled, hilly elevation, wavy margin, rough surface, grey pigmentation
with powdery surface
D 1. Grey round Colonies with raised margins and convex
elevation.
2. Red in appearance, circular margin and flat elevation. Has a dry
and powdery appearance
3. Grey with convex elevation colonies had a dry or powdery
appearance.
F 1. White powdery colonies with elevated surfaces, circular with
smooth surfaces.
2. Small Orange colonies circular in shape with raised surfaces.
3. Large cream colonies, raised elevated surfaces and powdery in
appearance.
4. Red colonies, raised elevated surface, circular shape and smooth
surface.
A 1. Colonies were pinkish orange, concentric configuration in colour,
smooth entire margin, crateriform elevated, rough dry powdery
surface.
2. Complex configuration, raised elevation, irregular margin,
wrinkled surface, red pigmentation
3. Wrinkled surface, hilly, wavy margin with rough grey
pigmentation.
E 1. Circular in shape, margin scalloped, red pigmentation and
powdery surface with raised elevation. Smooth margin, hard in
 structure.
2. Flat, lobate margin, powdery margin creamy pigmentation.
3. Grey pigmentation, rough, dull powdery surface, raised elevation,
smooth margin and round configuration.
A. Foster & S. Williams D 1. Filiform configuration, wavy margin and umbonate elevation.
Smooth and powdery surface with white pigmentation.
2. L-form configuration, thread like margin, umbonate elevation,
smooth, dull and powdery surface with grey pigmentation.
3. Round with radiating margin configuration, ciliate margin, hilly
elevation, dry and rough surface with pink pigmentation.
F
M. Martell & C. Powell F 1. Concentric, wavy margins, elevation is convex, powdery surface
with red pigmentation.
2. Concentric, wavy margins, elevation is convex, powdery surface
with grey pigmentation.
C. Haynes & J. George E 1. Red inner colour with white colour on outter margin, hard dry
surface, round scalloped margin, umbonate elevation
2. White colour, smooth and dry surface, lobate margin, L-form
colonies, drop like elevation.
C. Wilson & C. Mears C 1. Round with raised edge, smooth, smooth margin, convex
elevation, powdery appearance with grey pigmentation.
2. Complex raised elevation, irregular margin, red pigmentation.
3. Radiating margin, umbonate elevation, wrinkled surface with
powdery appearance and pink pigmentation.
K. Miller & Dane McBean D 1. Round smooth edge, umbonate elevation with rough surface, dull
appearance with grey colour with white colour around the edges
2. Round, wavy edges, raised elevation; surface has a dry, rough dull
appearance with pink pigmentation.
B
S. Benjamin & H. Pullen E 1. L-form configuration, orange in colour with a pink elevated centre.
Colonies have rough surface and wavy edges.
2. Colonies are raised with wavy margins and filiform configuration
D and red pigmentation
3. The colonies were irregular and spread with rough configuration,
smooth surface and grey pigmentation
S. Noble & O. Levy E 1. Round with scalloped margin with ring, rough crateriform
2. Round with raised margin, smooth with flat elevation.
 W. Hare & C. Antonio D 1. White and powdery, small and flat elevation
2. Black and powdery, shiny with convex elevation, small colonies.

Key
Soil Sample Site
A Taylor Hall
B Aqueduct Field
C Aqueduct Tree
D Old Harbour
E Mandeville
F May Pen

Table Showing the Zone of Inhibition and Satellite colonies for each compound tested
Sample D Diameter of Zone of Inhibition (mm)
E. coli B. Subtilis
E1 E1 Inhibition Zone E1 Inhibition Zone
Clear zone in the media are an indication Radius= 4cm Radius= 5cm
of inhibition by the actinomycetes. In Diameter=8cm Diameter= 12cm
some areas there is still bacterial growth E1 Satellite Zone E1 Satellite Zone
even though there is inhibition of growth. Radius= 5cm Radius= 5cm
These areas contain resistant strains of E. Diameter= 10cm Diameter= 10cm
coli and B. subtilis
E2 No Zone of Inhibition E2 Inhibition Zone
There was no inhibition of E. coli which Radius= 7cm
was indicated by cloudy spots around the Diameter=14cm
bacterial plug. There was however E2 Satellite Zone
inhibition of B. subtilis. Radius= 1cm
Diameter= 2cm
Discussion: Actinomycetales is an order of bacteria to which the actinomycete belong. This group of
bacteria are responsible for most of the antibacterial compounds presently being used in the treatment of
bacterial infection. The antibacterial compounds isolated from these microbes are sold as prescription drugs
and antibacterial therapy treatment. An example of this is the use of tetracycline to treat acne and respiratory
infections. The effect of the antibiotic on different types of bacteria is of particular importance since these
antibacterial compounds inhibit the growth of bacteria they can be used to treat bacterial infections
depending on whether or not they have serve side effects on infected persons. In some cases the
effectiveness of the antibiotic is assed to determine with which type of bacterial infection this will be
effective. An example of this lies in the case of gram positive vs gram negative bacteria. The use of certain
antibiotics won’t have any effect and so should not be used in the treatment of the bacteria that it is
ineffective against. In the case of the gram negative bacteria despite their cell wall being thinner than the
gram positive bacteria these bacteria have near impermeable lipid membranes.
It was expected that antibiotic producing bacteria would be isolated from the actinomycetals which
was correlated based on the inhibition of growth for both E. coli and B. subtilis. Based on the morphology of
the bacteria isolated from the soil it has characteristics of actinomycete. The powdery appearance of the
bacteria is a result of the aerial hyphae that grows from the bacteria similar to that of fungi. From the class
results other genus of Actinomycetales were isolated from the soil which includes Streptomyces. In the test
for antibiotic production there were areas in the inhibition zone that had colony growing this implies that
there were resistant strains of bacteria present in the E. coli and B. subtilis for the particular antibiotic agent
being produced. For E2 in the group sample there was no inhibition of E. coli which suggest that the
antibiotic produce by E2 was not effective against gram negative bacteria.

Conclusion: The actinomycete isolated from the soil sample produced an antibiotic that was effective
against gram positive bacteria as well as gram negative for the E1 while E2 was not effective against gram
negative bacteria.

Questions:
1. Why are there so many antibiotic-producing organisms in soils?
There is competition for the resources available to the microorganisms in the soil. Those organisms that are
able to produce antibiotic compounds have more resources at their disposal as there is less completion for
the resources due to the inhibitory effect of the antibiotic compounds on the growth of other organisms.

2. What are the characteristics of Actinomycetales? How do you recognize an actinomycete colony?
How do you recognize Actinomycetales on a microscope slide?
Actinomycetales are gram positive, aerobic as well as facultative anareobic, filamentous bacteria that form
mycelium which is a tubular filamentous structure usually found in fungi. The mycelium is the vegetative
part of a fungus. Actinomycetales have characteristics of both bacteria and fungus which include being
single celled, they do not have distinct cell walls and their cell walls are not composed of chitin and
cellulose like fungi. Like fungi Actinomycetales have mycelium they also produce hyphae and conidia /
sporangia like fungi. These organisms can be identified on culture media by their slow growth unlike
bacteria which have a more rapid growth rate, they also have a powdery consistency and stick firmly to agar
surface. Streptomyces produce a well-developed mycelium. The diameter of the hyphae seldom exceeds 1.0
μ and is usually only 0.7-0.8 μ.

3. Can you name one well-known antibiotic produced by an actinomycete and the organism that
produces it?
Tetracycline is a popular antibiotic compound produced by Streptomyces aureofaciens. This compound is
used to treat bacterial infections, including pneumonia and other respiratory tract infections; acne; infections
of skin, genital and urinary systems; and the infection that causes stomach ulcers. Tetracycline works by
binding specifically to the 30S ribosome of the bacteria, preventing attachment of the aminoacyl tRNA to
the RNA-ribosome complex. It simultaneously inhibits other steps of the protein biosynthesis. Tetracycline
can also alter the cytoplasmic membrane and this in turn causes leakage of nucleotides and other compounds
out of the cell. This does not directly kill the bacteria but instead inhibit it.

References:

My Agriculture Information Bank. Soil Microorganism – Actinomycetes. http://agriinfo.in/?page

=topic&superid=5&topicid=148

H. Burge. Actinomycetes. June 2008. https://www.emlab.com/s/sampling/env-report-06-2008.html.

(Accessed 17/11/2014)

S. A. Waksman, H. A. Lechevalier. Guide to the Classification and Identification of the Actinomycete and
their Antibiotics; Williams and Wailkins: Baltimore, 1953.