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The isolation of the antibiotic Streptomyces bacteria from the soil
Colleen Dunford and Willow Malick
Microbiology 342 Lab
Abstract
Streptomyces sp. are gram positive bacteria of the order Actinobacteria known for their production of bioactive metabilites that often have antibiotic properties. Strepomyces sp. were cultured from soil samples collected during the winter months in Anchorage Alaska. The bacteria were isolated through the use of specialized media and isolation streak procedures. The resulting pure cultures were tested for antibiotic properties through the use of streak plating with Bacillus cereus and Micrococcus luteus. A Streptomyces sp. Was isolated that produced a zone of inhibition when tested against M. luteus, while B. cereus was not inhibited by the Streptomyces cultures.
Introduction
Streptomyces is a genus of Actinobacteria, a group of Gram-positive bacteria and are found predominantly in soil and in decaying vegetation1,3. Commonly producing spores, the bacterium is noted for their "earthy" odor. They are also unique amongst bacteria in their mycelial, sporulating life cycle, which involves complex regulation of gene expression in space and time3,5.
None of the Streptomycetes are pathogenic but many are medically significant because of the antibiotics that they produce. To date the majority of these antibiotics are applied in human and veterinary medicine and agriculture, as well as anti-parasitic agents, herbicides, pharmacologically active metabolites (e.g. immuno-suppressants) and several enzymes important in the food and other industries1,2.
The purpose of this investigation is to isolate antibiotic-producing bacteria (genus Streptomyces) from local soil samples. This experiment replicates the first step in the search and screen research that is currently underway in many labs worldwide4.
Materials
Two Alaskan soil samples were obtained (approximately ½ cup each), for the isolation of Streptomyces. Each was from a residential garden but of different locations. Frozen, wet samples were air dried and then kept in refrigeration until they were required for use.
Isolation media required to isolate the organism consisted of agar, dextrose, soybean meal, NaCL, yeast extract, CaCO3, glycerol, and cycloheximide5. Exact measurements for all media can be found at the end of the materials section. The dry ingredients measured, distilled water was added (to bring total volume to 500ml) and heated to a rapid boil5. Once cooled the mixture was autoclaved and then poured into petri plates. Transfer (growth) medium consisting of dextrose, yeast extract, potassium nitrate, potassium monohydrogen phosphate, agar and cycloheximide5, were prepared and poured into petri plates as previously outlined.
Both Serial dilutions and dilution plating each required 1gram (g) of sample soil and each set of serial dilutions required four standard test tubes labeled 1-4. Test tube blanks were prepared with 4.5ml of distilled water.
Gram staining of select colonies employed the use of standard gram stain chemicals and dyes3, 4. Test bacteria, Bacillus.cereus and Micrococcus luteus were pure broth cultures (non-pathogenic) used in the Assay.
Isolation Media
Agar 7.5g
Dextrose 15.0g
Soybean meal 7.5g
NaCl 2.5g
Yeast extract 0.5g
CaCO3 0.5g
Glycerol 1.25g
Cycloheximide 0.5ml
Transfer (Growth) Media
Agar 7.5g
Yeast extract 0.5g
Potassium nitrate 0.5g
Potassium monhydrogen phosphate 0.05g
Cycloheximide 0.5ml
Dextrose 5.0g
Methods
Soil samples were allowed to dry for a week at room temperature. By drying the soil, other competing bacteria were killed off, and the remaining spore -forming Streptomyces were enabled to grow on the provided media.
In the preparation of the isolation and growth media, the addition of cycloheximide was important to inhibit fungal growth. The medias were both adjusted to a pH of 6.8 +/- 0.2 @ 25oC before being autoclaved.
The medias prepared, two serial dilutions, (one for each soil sample) were performed as follows. Labeling four test tubes 1-4, 1 gram of soil was transferred into test tube #1 and enough distilled water added to bring the volume to 5ml. Test tubes 2-4 were established as blanks by delivering 4.5ml of distilled water to each. The contents of test tube #1 mixed, 0.5ml of the soil solution was transferred via pipette to test tube #2 making a dilution factor of 10-1. The procedure carried out to test tube #4 to a dilution factor 10-3. Individual aseptic 0.1ml transfers of diluted soil solutions from test tubes #2, #3 and #4 were plated to three petri plates of isolation media resulting in the respective dilution plates of 10-2, 10-3 and 10-4.
Immediately after the transfers, each soil solution was spread aseptically with a glass spreader to completely cover the plate. Labeled with the appropriate dilution factor, the plates were incubated at 35oC for 4-7 days. Plates were incubated in the dark to mimic natural conditions of the soil.
After the incubation period, plates were observed and colonies counted. Viable colonies of Streptomyces identified (30-300), colonies were then aseptically transferred with a loop and an isolation streak made onto a plate of transfer media. Incubated once again in the dark at 35oC for 3-5 days, plates were then qualitatively observed and noted.
A gram stain of selected colonies was made. The organism viewed under the oil immersion lens of a microscope, observations were noted and recorded.
To test for the production of the secondary metabolite, a single streak of Streptomyces (from the isolation plate) was made down the middle of a plate of transfer media. Two plates were prepared in this manner and incubated at 35oC for 3-5 days. After such time, a single streak of each test organism (Bacillus.cereus and Micrococcus luteus) were made perpendicular to the Streptomyces streak plates and incubated 2-3 days at 35oC . Observation of inhibition by Streptomyces was then made.
Results
’er 300) for either soil sample. However, isolation plates of df 10-4 had the respective counts; soil sample A = 36, and soil sample B = 32. See table one for calculated CFU’s of the original soil samples.
Table one: Calculated CFU’s
Plate count Final Dilution Viable no. in original culture (CFU/ml)
Soil sample A = 36 1: 104 36 x 104 = 3.6 x 105
Soil sample B = 32 1: 104 32 x 104 = 3.2 x 105
Distinct colonies were observed on both dilution plates of df 10-4. Different species were observed as white, orangish pink and brown, where as the Streptomyces were round, chalky cream- white, colored colonies with a “fuzzy” edge. Once pure colonies were grown (3 days) on streak isolation plates, staining revealed gram positive, branching, filamentous arrangement of cells (mycelium) from exhibits A and B. when examining the organism under the oil immersion lens, spores were observed from the organism stained from soil sample A, but were not observed from sample B. Important to note was the distinct “earthy” smell from both isolation plates of the soil bacteria.
The filamentous mycelium of Streptomyces.
From: Natural Resources Conservation Service
Isolation plates were returned to the incubator and observed daily for three days, at which time secondary metabolites were observed. What was noted as the antibiotic product, appeared as a “dew drop” formed on top of the Streptomyces colonies.
From soil sample plate A, the performed assay test with test organism Micrococcus luteus revealed a zone of inhibition after only 5 days for but not for Bacillus.cereus. The zone of inhibition appeared as a slight cleared ring where the organism was streaked.
Isolation plate of Streptomyces with
secondary metabolite production.
Summary of Observations
Soil Sample A
Dilution plate Chalky texture color elevation Surface appearance Soil odor filaments spores Antibiotic production
10-5 yes creamy convex dull yes yes yes yes
Zone of clearing
M luteus yes
B cereus no
Soil Sample B
Dilution plate Chalky texture color elevation Surface appearance Soil odor filaments spores Antibiotic production
10-5 yes creamy convex dull yes yes no no
Zone of clearing
M luteus no
B cereus no
Discussion
The medias used in this experiment did contain cycloheximide to inhibit fungal growth. However, it would have been especially advantageous to have used an antibiotic while making the isolation media. To do so could have encouraged a variety of isolated species of Streptomyces. Although, we are convinced that we did successfully isolate one species of this organism from soil sample A.
The chalky-white appearance of colonies, and the production of the liquid (secondary metabolite), helped to validate the characteristic colony morphology of the soil bacteria. Other observations of a filamentous mycelium, spores (conidia) and a gram positive reaction of the organism were also factors leading to our conclusion. The strong earthy odor from the isolated colonies and prevalent zone of inhibition from the Assay test helped to confirm that we had isolated the antibiotic producing bacteria.
Bibliography
1. Booser, D.J. and Hortobagyi, D.N. (1994). Anthracycline Antibiotics in Cancer Therapy. Focus on Drug Resistance. Drugs 47, 223-258
2. Brockmann, H. and Bauer. K. (1950). Rhodomycin, ein rotes Antibioticum aus Actinomyceten. Naturwiss. 37, 492-493
3. Fujiwara, A. and Hoshino, T. (1986). Anthracycline antibiotics. CRC Crit Rev Biotechnol 3, 133-157
4. Hutchinson, C.R. (1988). Prospects for the discovery of new (hybrid) antibiotics by genetic engineering of antibiotic-producing bacteria. Med Res Rev 8, 557-567
5. Motamedi, H., Wendt-Pienkowski, E. and Hutchinson, C.R. (1986). Isolation of tetracenomycin C-nonproducing Streptomyces glaucescens mutants. J Bacteriol 167, 575 - 580
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