Isolation of antibiotic producing microorganisms and Determination of antimicrobial spectrum of isolates

Principle:

• Soil is the prime storage of the microorganisms producing antibiotics which are able to inhibit the growth of other microorganisms. Antibiotics have been implemented in one or other forms for centuries. The screening of wild isolates from the soil has yielded the broad majority of new antibiotics. Even if the purification of several hundred naturally produced antibiotics has been performed, only a few have been proved to be successful to be used in medical practice. Those which are presently of greatest use were derived from a comparatively small group of microorganisms belonging to the genera Penicillium, Streptomyces, Cephalosporium, Micomonospora and Bacillus. In this era, the continuous efforts to develop new antibiotics are the emerging trends.
• Even if soils from various parts of the world are continually screened in industrial laboratories in order to isolate new antibiotic-producing microorganisms, industrial microbiology is directing its efforts toward chemical modification of existing antibiotic substances. This is completed by adding or replacing chemical side chains, reorganizing intramolecular bonding, or producing mutant microbial strains able to excrete a more potent form of the antibiotic. The establishment of chemical congeners accounts for the overcoming of antibiotic resistance, reducing adverse side effects in the host and increasing the effective spectrum of a given antibiotic.
• I: We will use the crowded-plate technique for the isolation of antibiotic producing microorganisms from two soil samples, one of which is seeded with Streptomyces griseus to serve as a positive control.
• II: For the determination of anti-microbial spectrum of isolates, isolates manifesting antibiotic activity will be screened against several different microorganisms to establish their effectiveness.

I. Isolation of antibiotic producing microorganisms

Requirements:

1.  Soil Suspensions:
   • – 1:500 dilution of soil sample suspension (0.1 g of soil per 50 ml of tap water) to serve as an unknown
   • – 1:500 dilution of soil sample seeded with S. griseus (0.1 g of soil per 50 ml of tap water) to serve as a positive control.
2. Media:
   • Six 15-ml Trypticase soy agar deep tubes, and two Trypticase soy agar slants.
3. Equipment:
   – 500-ml beaker
   – test tubes
   – test tube rack
   – sterile Petri dishes
   – inoculating needle
   – hot plate
   – thermometer
   – 1-ml and 5-ml pipettes
   – mechanical pipetting device
   – magnifying hand lens.

Procedure for Isolation of antibiotic producing microorganisms

• Label two sets of three sterile Petri dishes with the types of soil samples being used and dilutions (1:1000, 1:2000, and 1:4000).
• Place six Trypticase soy agar deep tubes into a beaker of water and bring to 100°C on a hot plate. Once agar is liquefied, add cool water to the water bath. Cool to 45°C, checking the temperature with a thermometer.
• Prepare a serial dilution of the unknown and positive control 1:500 soil samples as follows:
  – Label three test tubes 1, 2, and 3. With a pipette, add 5 ml of tap water to each tube.
  – Shake the provided 1:500 soil sample thoroughly for 5 minutes to effect a uniform soil-water suspension.
  – Using a 5-ml pipette, transfer 5 ml from the 1:500 dilution to Tube 1 and mix. The final dilution is 1:1000.
  – Using another pipette, transfer 5 ml from Tube 1 to Tube 2 and mix. The final dilution is 1:2000.
  – Using another pipette, transfer 5 ml from Tube 2 to Tube 3 and mix. The final dilution is 1:4000.
  – Using separate 1-ml pipettes, transfer 1 ml of the 1:1000, 1:2000, and 1:4000 dilutions to their appropriately labelled Petri dishes.
  – Pour one tube of molten Trypticase soy agar, cooled to 45°C, into each plate and mix by gentle rotation.
  – Allow all plates to solidify.
• Incubate all plates in an inverted position for 2 to 4 days at 25°C.
• Examine all crowded-plate dilutions for colonies exhibiting zones of growth inhibition. Use a hand magnifying lens if necessary. Record in the Lab Report the number of colonies showing zones of inhibition.
• Aseptically isolate one colony showing a zone 34 of growth inhibition from each soil culture
  with an inoculating needle and streak onto
  Trypticase soy agar slants labelled with the soil sample from which the isolate was obtained

• Incubate the slants for 2 to 4 days at 25°C. These will serve as stock cultures of antibiotic-producing isolates to be used in Part B.

II. Determination of antimicrobial spectrum of isolates

Requirements:

1. Cultures:
   • – 24-hour Trypticase soy broth cultures of Escherichia coli, Staphylococcus aureus, Mycobacterium smegmatis, and Pseudomonas aeruginosa.
2. Media:
   Two Trypticase soy agar plates.
3. Equipment:
   – Bunsen burner
   – inoculating loop
   – glassware marking pencil.

Procedure  for determination of antimicrobial spectrum of isolates.

• Label the Trypticase soy agar plates with the soil sample source of the isolate.
• Using aseptic technique, make a single-line streak inoculation of each isolate on the surface of an agar plate so as to divide the plate in half
• Incubate the plates in an inverted position for 3 to 5 days at 25°C.
• Following incubation, on the bottom of each plate draw four lines perpendicular to the growth of the antibiotic-producing isolate
• Aseptically make a single-line streak inoculation of each of the four test cultures following the inoculation template on each plate. Start close to, but not touching, the growth of the antibiotic-producing isolate and streak toward the edge of the plate.
• Incubate the plates in an inverted position for 24 hours at 37°C.
• Examine all plates for inhibition of test organisms, and record your observations in the Lab Report.

Observations and Results interpretations:

• I: Isolation of Antibiotic-producing micro-organisms.
  -Number of colonies showing zone of inhibition in different serial dilutions were noted and were further cultured to obtain pure cultures.
• II: Determination of anti-microbial spectrum of isolates.
  • Draw a representation of the observed antibiotic activity against the test organisms.
  • Based on your observations, record in the chart the presence (+) or absence (−) of antibiotic activity against each of the test organisms and the spectrum of antimicrobial activity (broad or narrow).