The document outlines steps to isolate antibiotic-producing microorganisms from soil samples and determine their antimicrobial activity. Students will isolate Bacillus, Penicillium, and Actinomyces colonies on agar plates. Colonies will be streaked on plates seeded with Staphylococcus epidermidis or fungi to check for evidence of antibiosis. Colonies showing inhibition will be re-streaked with test pathogens to confirm antimicrobial activity through zone of inhibition assays. The goal is to isolate microbes producing antibiotics that could have clinical significance.

2. • Subject objective: Each student should be able to
• To isolate antibiotic-producing microorganisms
(Bacillus, Penicillium and Actinomycetes colonies)
from soil that may be antibiotic producers.
• To determine the spectrum of the antimicrobial activity
of the isolated antibiotic against some clinical
important bacteria (Staphylococcus epidermidis as a
Gram positive and Escherichia coli as a Gram-
negative bacteria).
• Biological assay for screening of antimicrobial activity.
• Reporter screening

3. Principle:
• The antibacterial effect of penicillin was discovered by Alexander Fleming in
1929. He noted that a fungal colony had grown as a contaminant on an agar plate
streaked with the bacterium Staphylococcus aureus, and that the bacterial colonies
around the fungus were transparent, because their cells were lysing. Fleming had
devoted much of his career to finding methods for treating wound infections, and
immediately recognised the importance of a fungal metabolite that might be used to
control bacteria. The substance was named penicillin, because the fungal
contaminant was identified as Penicillium notatum. (See Figure A) Fleming found
that it was effective against many Gram positive bacteria in laboratory conditions, and
he even used locally applied, crude preparations of this substance, from culture
filtrates, to control eye infections.
• The phenomenal success of penicillin led to the search for other antibiotic-
producing microorganisms, especially from soil environments. One of the early
successes (1943) was the discovery of streptomycin from a soil actinomycete,
Streptomyces griseus. actinomycetes are bacteria that produce branching filaments
rather like fungal hyphae, but only about 1 micrometer diameter. They also produce
large numbers of dry, powdery spores from their aerial hyphae. Actinomycetes,
especially Streptomyces species, have yielded most of the antibiotics used in clinical
medicine today. Some examples are shown in the table (1).
• Other bacteria, including Bacillus species (see Figure E: Agar plate showing inhibition
of fungal growth by a contaminating colony of Bacillus species.), have yielded few
useful antibiotics. Fungi also have yielded few useful antibiotics. Apart from penicillin,
the most important antibiotics from fungi are the cephalosporins (beta-lactams with
similar mode of action to penicillin, but with less allergenicity).

4. Figure E. The constant search of soils throughout the world has yielded an abundance of
antibiotics of great value for the treatment of many infectious diseases. Pharmaceutical
companies are in constant search for new strains of bacteria, molds, and Actinomyces that can be
used for antibiotic production. Although many organisms in soil produce antibiotics, only a small
portion of new antibiotics are suitable for medical use. In this experiment an attempt will be
made to isolate an antibiotic-producing Bacillus, Actinomyces and Penicillium from soil.
Students will work in group. Figure 1 illustrates the procedure.

5. • Antimicrobial agents: are substances that are naturally produced by a variety of
microorganisms (primarily Actinomycetes, fungi and bacteria), or have been
synthesized in the laboratory, or a combination of both.
• Antibiotic: refers only to those antimicrobial substances produced by
microorganisms, but the term is often used interchangeably with antimicrobial agent.
Antimicrobial agents have inhibitory or lethal effects on many pathogenic organisms
(especially bacteria) that cause infectious diseases.
• Antibiotic producer such as:
Antibiotics are the best known products of actinomycete. Over 5000 antibiotics have
been identified from the culture of gram positive, gram negative organisms and
filamentous fungi, but only100 antibiotics have been commercially used to treat
human, animal and plant disease. The genus Streptomycete is responsible for the
formation of more than 60% of known antibiotics. While further 15% are made by
number of related Actinomycete, Micromonospora, Actinomadura, Streptoverticillium
and Thermoactinomycetes
1. Streptomyces spp.: produce (Chloramphenicol, Erythromycin, Kanamycin,
Neomycin, Nystatin, Rifampin, Streptomycin, Tetracyclines, Vancomycin)
2. Micromonospora: produce (Gentamicin)
3. Bacillus:Produce (Bacitracin, polymxins)
4. Fungi:
• Penicillium griseofulvum: produce (Griseofulvin)
• Cephalosporium: produce Cephalosporins

7. Some clinically important antibiotics
Site or mode of
Antibiotic Producer organism Activity
action
Penicillin Penicillium chrysogenum Gram-positive bacteria Wall synthesis
Cephalosporin Cephalosporium acremonium Broad spectrum Wall synthesis
Griseofulvin Penicillium griseofulvum Dermatophytic fungi Microtubules
Bacitracin Bacillus subtilis Gram-positive bacteria Wall synthesis
Polymyxin B Bacillus polymyxa Gram-negative bacteria Cell membrane
Amphotericin B Streptomyces nodosus Fungi Cell membrane
Erythromycin Streptomyces erythreus Gram-positive bacteria Protein synthesis
Neomycin Streptomyces fradiae Broad spectrum Protein synthesis
Streptomycin Streptomyces griseus Gram-negative bacteria Protein synthesis
Tetracycline Streptomyces rimosus Broad spectrum Protein synthesis
Vancomycin Streptomyces orientalis Gram-positive bacteria Protein synthesis
Gentamicin Micromonospora purpurea Broad spectrum Protein synthesis
Rifamycin Streptomyces mediterranei Tuberculosis Protein synthesis

8. • Why the few antibiotics are clinically useful?
• Several hundreds of compounds with antibiotic activity have been
isolated from microorganisms over the years, but only a few of them are
clinically useful. The reason for this is that only compounds with selective
toxicity can be used clinically - they must be highly effective against a
microorganism but have minimal toxicity to humans. In practice, this is
expressed in terms of the therapeutic index - the ratio of the toxic dose to
the therapeutic dose. The larger the index, the better is its therapeutic value.
So the antibacterial product should be assessed by pharma and then decide
to put in the market when it passes ADME/T test
• It will be seen from the table above, that most of the antibacterial agents
act on bacterial wall synthesis or protein synthesis. Peptidoglycan is one of
the major wall targets because it is found only in bacteria. Some of the other
compounds target bacterial protein synthesis, because bacterial ribosomes
(termed 70S ribosomes) are different from the ribosomes (80S) of humans
and other eukaryotic organisms. Similarly, the one antifungal agent shown in
the table (griseofulvin) binds specifically to the tubulin proteins that make up
the microtubules of fungal cells; these tubulins are somewhat different from
the tubulins of humans.

9. • Factors affecting antibiotic production:
1. Medium Composition:
• Carbon source
• Nitrogen source
• Inorganic phosphates
• Inorganic salts
• Trace metals
• Precursors
• Inhibitors
• Inducers
2. Fermentation Conditions:
• pH
• Temperature
• Oxygen
• How can determine the target of inhibitor molecule which may inhibit one of
the biological pathways?
There are many different pathwaies can be applied such as reporter essay by using •
the reporter strains

10. Reporter screening
Reporter strain

11. This lab. Consist
of three steps
Colony selection Evidence of antibiosis
Primary isolation & &
Inoculation Confirmation

12. FIRST STEP:
• (Primary Isolation)
Unless the organisms in a soil sample are thinned out sufficiently, the isolation of
potential antibiotic producers is nearly impossible.
Materials per group of students:
1. six large test tubes, one bottle of physiological saline solution
2. Three Petri plates of glycerol yeast extract agar, Tryptic soy agar, Sabouraud
dextrose agar.
3. L-shaped glass rod, beaker of alcohol
4. six 1 ml pipettes, one 10 ml pipette
• Procedure:
1. Label six test tubes, and with a 10 ml pipette, dispense 9 ml of saline into each tube.
2. Weigh out 1 g of soil and deposit it into tube 1.
3. Vortex mix tube 1 until all soil is well dispersed throughout the tube.
4. Make a tenfold dilution from tube 1 through tube 6 by transferring 1 ml from tube to
tube. Use a fresh pipette for each transfer and be sure to pipette-mix thoroughly
before each transfer.
5. Label three Petri plates with your initials and the dilutions to be deposited into them.
6. From each of the last three tubes transfer 1 ml to a plate of glycerol yeast extract
agar.
7. Spread the organisms over the agar surfaces on each plate with an L-shaped glass
rod that has been sterilized each time in alcohol and open flame. Be sure to cool rod
before using.

13. Figure 1

14. • SECOND STEP
(Colony Selection and Inoculation)
• The objective in this laboratory period will be to select Bacillus, Penicillium and Actinomyces-
like colonies that may be antibiotic producers. The organisms Penicillium sp and Actinomyces
will be streaked on nutrient agar plates that have been seeded with Staphylococcus
epidermidis, and Bacillus will be streaked on nutrient agar plates that have been streaked
firstly by fungi, after incubation we will look for evidence of antibiosis. Students will continue to
work in groups. Figure 2 illustrates the procedure.
• Materials per group of students:
1. four trypticase soy agar pours (liquefied)
2. four sterile Petri plates
3. TSB culture of Staphylococcus epidermidis, Bacillus firmus, and Penicillium sp.
4. 1 ml pipette
5. three primary isolate plates from previous period water bath at student station (50° C)
Procedure:
1. Place four liquefied agar pours in water bath (50°C) to prevent solidification, and then inoculate
each one with 1 ml of S. epidermidis.
2. Label the Petri plates with your initials and date.
3. Pour the contents of each inoculated tube into Petri plates. Allow agar to cool and solidify.
4. Examine the three primary isolation plates for the presence of Bacillus sp. Penicillium sp. and
Actinomyces-like colonies. Actinomyces have a dusty appearance due to the presence of
spores. They may be white or colored. Your instructor will assist in the selection of colonies.
5. Using a sterile inoculating needle, scrape spores from Penicillium sp. and Actinomyces-like
colonies on the primary isolation plates to inoculate the seeded TSA plates. Use inoculums from
a different colony for each of the four plates.
6. Incubate the plates at 30° C until the next laboratory period.

15. • THIRD AND FOURTH STEPS
(Evidence of Antibiosis and Confirmation)
• Examine the four plates you streaked during the last laboratory period. If
you see evidence of antibiosis (inhibition of S. epidermidis growth and
Fungal growth), proceed as follows to confirm results.
• Materials:
1. 3 Petri plates of trypticase soy agar
2. TSB culture of S. epidermidis, PDA culture of Penicillium and Aspergillus sp.
• Procedure:
1. If antibiosis is present for each of Actinomyces, Penicillium, Bacillus, use
three TSA plates and make two streaks on each of the TSA plates as shown
in figure 2. Make a straight line streak from (antibiotic producer
microorganisms)
2. cross-streak with organisms from a culture of S. epidermidis and
Aspergillus sp.
3. Incubate at 30° C until the next period.

16. Figure 2