Six soil microorganisms were screened for antibacterial activity against Staphylococcus aureus, using the paper disc method. Three isolates (YC 1 C2, BC] C2, and GC1 C2) were taken from the damp grassy area, two (BC2 A2 and WC2 A2) from the compost pit, and one (YGC1 BI) from the lagoon area. DOST broth was used to grow the organisms and produce the antimicrobial substances. Shake flask cultures were grown for eight (8) days at 30°C at 125 revolutions per minute (rpm). Isolate (BC2 A2) exhibited the highest antibiotic activity against S. aureus at an antimicrobial activity index (A 1 ) of 0.143 for the first replicate and 0.286 for the other. Isolate WC2 A2 showed signs of inhibition only on the eight days of incubation with an Al of 0.071. Only isolate BC2 A2 exhibited antibacterial activity throughout the eight days of incubation although this was not shown from the first replicate since earlier samples were contaminated. Among the isolates screened, only two showed inhibitory activities against S. aureus although the two weren’t much better than Penicillium. sp.
Many think that bacteria are all pathogenic but certain types of these microorganisms are discovered to inhibit growth of other disease-causing microorganisms. Such are filamentous, branching bacteria which are widely distributed in nature called actinomycetes. Canestan. an actinomycete has been found to have antifungal activities. Since these soil microorganisms are widely distributed in nature and yet few are utilized as antibiotic, isolates of chosen microorganisms are screened against S. aureus, gram-positive bacteria. S. aureus is a common cause of many pathogenic conditions such as boils, pimples, and pneumonia. This research focuses on possible sources of antibiotics against .certain diseases, caused by pathogens specifically S. aureus. Utilizing antibiotics from such soil Microorganisms may be an important contribution to the field of medicine.
The research deals only with what isolates have antibiotic properties against S. aureus and not on how they inhibit such pathogen’s growth.
REVIEW OF RELATED LITERATURE
The bodies’ responses to infection may seem to suggest that nothing could harm us — the immune system can conquer all. Unfortunately, this is not true because the infection often becomes a race between the invading microbes and the immune responses. So, in some instances, when the infection is massive or the microbes produce particularly toxic products, the victim may die before the full activation of the immune response can take place (Audesirk, 1986). The introduction of antibiotics has greatly improved the rate of survival from numerous diseases.
Commercially useful antibiotics are produced primarily by filamentous, branching bacteria of the actinomycetes group. While others are parasitic, most actinomycetes isolated from soil produce antibiotics which are of medical importance these days (Lechavalier, 1974). Isolates of soil microorganisms were screened for antibacterial activity against S. aureus (Erwealor and Hjolu-Ohi, 1990).
MATERIALS AND METHOD
A. Preparation of MYEA Media One hundred forty mL of sterile (sterilized at 115°C, 20 mins.) malt yeast extract agar (MYEA) was placed in six test tubes as slants and 10 mL MYEA in petri dishes for screening of the cultures.
B. Collection of Soil Samples Soil samples were collected from the compost pit and damp grassy area inside the campus and also one from the lagoon area inside the park.
C. Preparation of Soil Suspension
1. Preparation of 0.1% peptone A hundred mL of 0.1% peptone was prepared, 10 mL was transferred to each of 6 test tubes, and 9 mL to each of the 3 test tubes.
2. Preparation of Different Concentrations Using Serial Dilution
A gram of each of the soil samples (3 types of samples) was suspended in the 10 mL peptone water and mixed by using a vortex mixer. The suspensions were allowed to stand for a week for the particles to settle. Only the supernatants of this set of soil suspension were used for the preparation of the other two less-concentrated suspensions of each soil sample. One mL each from 3 suspensions was transferred to the remaining three 10 mL peptone water then mixed by vortex to serve as the second set having different concentration. Again, 1 mL each from the previous set was transferred to the three 9 mi. peptone water. It is then mixed to serve as the last set with the least concentration. These suspensions of soil containing microorganisms, about 2 mL each, were transferred to the 9 MYEA plates. The suspensions were swirled to spread them out evenly. The plates were incubated for 3 days at 30°C.
A. Isolation of Soil Microorganism after 3 days, there was isolation of different colonies found in the plates of the prepared MYEA slants. The subculture isolates with identification codes were incubated for 7 days at 30°C.
B. Preparation of Shake Cultures for Screening
1. Preparation of DOST Broth While the isolates were growing, 330 mL of DOST broth was prepared.
2. Preparation of DOST Suspensions for Shake-Flask Cultures
Five rnL of DOST broth was transferred to each of the 1-day old isolate in the MYEA slants. The surface of the culture slants were scraped using a wire loop to let the isolate spore suspend in the DOST broth. From these suspensions, 0.5 rnL each were transferred to flasks with 50 mL DOST broth. The flasks were then incubated at the shaker for 8 days at 30DC and 125 rpm. Sampling was after every 4 days.
3. Preparation of Antibiotic Discs From the previous samples, 1.5 mL of each was transferred to small test tube toppered with cork. It is then centrifuged at 3000 rpm for 15 minutes so that the cells be separated from the supernatants. This was done a day before each sampling. The supernatants were separated and sterile blank antibiotic discs were dipped and allowed to dry a bit the following day before each sampling. Penicillin, a known antibiotic against S. aureus, was used as control by clipping sterile blank antibiotic discs into a suspension of about 10 million cells of Penicillin sp. C. Preparation of Test Organism 1. Preparation of Barium Sulfate Standard Barium sulfate standard was prepared for the determination of the concentration of the test organism, S. aureus, in suspension. One-tenths ml. of 1% barium chloride solution was mixed with 9.8 mL of 0.18 M sulfuric acid for the barium sulfate standard.
2. Subculture of S. aureus into NA Plates S. aureus was grown in nutrient agar (NA) slants for 24 hours at 37°C. The inoculums were prepared using the barium sulfate standard turbidity test. The BaSO4standard and the suspension for the test organism in 0.9% peptone water were placed in front of the black marker lined paper. The test organisms in agar were looped into the respective test tubes until the image of the black line had relatively the same clarity in both test tubes. The suspensions approximately contained 10 million cells/mL as compared to the prepared barium sulfate standard. From here, 0.1 mL was mixed with 5 mL melted agar which then was inoculated into the NA plates.
3. Actual Screening Using sterile forceps, the antibiotic discs were placed in equidistant points from the plates with the control at the center. The plates were incubated just before sampling at 37°C. Inhibition of S. Aureus was observed after 24 hours. The zones of inhibition were measured in remand the antimicrobial activity index was computed from the formula:
Al = (diameter of clearing diameter of disc)/ diameter of disc
RESULTS AND DISCUSSION
Six isolates were obtained from the three different soil samples, three of which were from damp grassy area, two from the compost pit, and one from the lagoon area. Five isolates were taken from plates inoculated with second preparations of the three different concentrations of soil suspension. The remaining, isolate YG1 B1, was obtained from the highest or first concentration of soil suspension. Only two outputs of the six microorganisms isolated from the soil samples showed inhibitory activity against the test organism, S. aureus. These two include isolates BC2 A2 and WC2 A2, both of which were taken from compost pit soil sample.
Four-day old samples from the first replicate were not noted for any antimicrobial activity against the test organism because the plates prepared were contaminated. The contamination of the prepared plates made it impossible for the identifieation of any antimicrobial activity from the isolates. The antimicrobial activity of the isolate BC•’A2 was at its peak among all the samples during the fourth day of incubation, with an Al of 0.571 as compared to 1.857 of Penicillum sp. also for the fourth day. However, isolate BA2 A2, depreciated antibacterial activity index (A1=0.286) as the screening continued on the succeeding days. It also showed an Al of 1.143 same as Penicillum sp. from the first replicate. Isolate WC2 A2 showed signs of inhibition (AI=0.071) on the 8′ day of incubation as shown from the second replicate. It appeared that isolate BC2 A2 was the most promising among the soil microorganism samples screened, as it showed inhibitory activity against the test organism. Although isolate BC2 A2 was not much better than the control, it was capable of maintaining its activity up to the 81h day of incubation.
The compost pit was observed to yield potential antibiotic agents. Even the damp grassy area may be a source of more microorganisms. Soil suspension of about 1% concentration yield better and more distinguishable colonies in plates for subculture or isolates to be screened. Isolate BC2 A2 appeared to be the most promising as it did not only exhibit antimicrobial activity against S. aureus but maintained its inhibition until the 8th day of incubation.
RESEARCHERS: Eleon B. DeVera Aldwin P.
Frias Rhu-Jade M.
Raguindin Runel B.
ADVISER: Miss Juanita Cruz
Philippine Science High School Quezon City