Studies on Growth Response of Fungi Using Antibiotic Ointments
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Int.J.Curr.Microbiol.App.Sci (2016) 5(3): 67-75 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 5 Number 3(2016) pp. 67-75 Journal homepage: http://www.ijcmas.com Original Research Article http://dx.doi.org/10.20546/ijcmas.2016.503.010 Studies on Growth Response of Fungi Using Antibiotic Ointments R. Sugumar and Anandha Raj* Department of Microbiology, M. R. Government Arts College, Mannargudi - 614 001, India *Corresponding author ABSTRACT K e yw or ds Xenobiotics refers to both organic and inorganic substances that mimic Xenobiotics, natural biochemicals that are essential for life. They may be persistent and Three drugs, often toxic to life. Pharmaceutical industries generate a large amount of Fungi Diversity, waste during manufacturing, housekeeping and maintenance operations. Antibiotic Hence, the present study was attempted to study the impact of these residues ointments . in the soil on fungal diversity. The three drugs that were identified were Article Info Fungitop F and Fungi top. Results show that the soil recorded very less Accepted: fungal diversity and all the species of fungi isolated showed decreased 07 February 2016 growth. Among the three drugs, fungi top inhibited the growth the most Available Online: 10, March 2016 among the different fungi. A. flavus had the most inhibitive effect. Introduction Xenobiotics are known dangerous pollutants Pharmaceutical plants also generate a large which include both inorganic elements like amount of wastes during manufacturing, heavy metals, metalloids and man-made housekeeping and maintenance operations. organic compounds such as pesticides, Worldwide detection of waste surfactants, industrial solvents, fragrances, pharmaceuticals in the environment causes flavours and pharmaceuticals as well as risks associated with their introduction into endocrine disrupters. It has been estimated wildlife habitats and is becoming a serious that over 70,000 xenobiotics may potentially issue for both regulators and the be hazardous for humans and/or ecosystems. pharmaceutical industry (Crane et al., 2006). The presence of xenobiotics in surface as Although different classes of well as underground waters have become a pharmaceuticals are used in human and problem for urban water supply as treatment veterinary medicine, only a few are of systems were originally not designed to environmental importance because of their solve xenobiotic problems (Alexander, consumption volumes, toxicity, and 1965, 1980; Skladany and Metting, 1992; persistence in the environment. Ankumah et al., 1995). 67 Int.J.Curr.Microbiol.App.Sci (2016) 5(3): 67-75 Pharmaceuticals in the aquatic environment Analysis of Physicochemical have been reported in rivers, sewage, Characteristics of the Soil streams, seawater, ground water, and drinking water. Measurable concentrations Moisture content was estimated by finding are usually low ranging from mg/l to g/l the difference in weight of known quantity (Fent et al., 2006) yet may be high close to of soil before and after drying in a hot air the point of input, particularly for veterinary oven at 60°C for 6 hours. Soil samples after medicines used in aquaculture where removing the debris were suspended in concentrations may reach the low to medium distilled water (1:2 w/v) and allowed to g/l range in surface water (Boxall et al., settle down. The pH of the suspension was 2004). Hence monitoring of pharmaceutical read using a pH meter (Systronics, India). products in the surface water and/or ground Electrical conductivity of soil was water is essential. In addition, determined in the water filtrate using pharmaceutical products reach the Conductivity Bridge as described by environment primarily through usage or Jackson (1973) while cation Exchange inappropriate disposal. Various production Capacity (CEC) of the soil was determined facilities were found to be sources of much by using 1 N ammonium acetate solution as higher environmental concentrations than described by Jackson (1973). those caused by the usage of drugs (Larsson et al., 2007). Hence in the present study, Organic carbon content was determined by some drugs that were found at the site of adopting chromic acid wet digestion method waste disposal at Thanjavur were collected (Walkley and Black, 1934), while available and studied for their impacts on soil fungi, nitrogen by alkaline permanganate method which are key players in any ecosystem for (Subbiah and Asija, 1956) and available maintaining the fertility by recycling of phosphorus by Brayl method (Bray and nutrients. Kutz, 1945). Available potassium was extracted from soil with neutral 1 N Materials and Methods ammonium acetate (1:5) and the potassium content in the extract was determined by Isolation of Fungi using flame photometer (Standfold and English, 1949); calcium (Neutral 1 N NH4 Drug spilled surface soil samples were OAC extractable 1:5) was extracted with collected aseptically from Municipal waste neutral 1 N ammonium acetate and the disposal site at Musiri and transported to the available calcium in the extract was laboratory. The samples were serially determined by Versenate method (Jackson, diluted and plated on 2% PDA Medium 1973). Available micronutrients such as Zn, (supplemented with streptopenicillin Cu and Mn were determined in the antibiotic solution 16 ml/l). The plates were diethylene triarnine pentaacetic extract of incubated at room temperature for a week. soil using Perkin-Elmer model 2280 Atomic The fungal colonies were then transferred on Absorption Spectrophotometer (Lindsay and to PDA plates for raising pure cultures. They Norvell, 1978). Other nutrients such as were identified referring standard manuals magnesium, sodium and available iron were (Raper and Thom, 1949; Von Arx, 1974; analysed following the method of Barnes Ainsworth et al., 1973; Raper and Fennell, (1959) and Muthuvel and Udayasoorian 1965; Ellis, 1976) based on the (1999). morphological characters. 68 Int.J.Curr.Microbiol.App.Sci (2016) 5(3): 67-75 Selection of Drugs By subtracting the values of B from A, the mycelial biomass (Wet weight) was The drugs that were disposed along with calculated and the values were accounted as other municipal waste were collected and a measure for the growth of fungi. identified. They were procured from druggist in required quantity to do the Combined Impact of pH and Drug on the experiments. The commercial names of Growth of Fungi these drugs are: To assay the combined impact of pH and i) Fungitop - F: A Glucocorticoid drug on the growth of fungi, single drugs derivative used in the treatment of were dissolved in 20 ml of autoclaved PD various skin disorders. Broth (later incorporated with antibiotic ii) Diprolite: It inhibits the growth of fungi solution) at 50 ml in a conical flask and the and bacteria which cause skin broth was adjusted to pH 4,7 and 9 using infections. O.1N NaOH (to increase the pH) and O.IN iii) Fungitop: It is used in the treatment of HCL (to decrease the pH). The flasks were inflammation in ear, nose and eye inoculated with the growing tip of the fungal disorders. mycelia and incubated at room temperature (27 2C) in a culture rack. After a week, Impact of Drugs on the Growth of Fungi the mycelial biomass was calculated as described earlier. The experiment was Potato Dextrose Agar broth was prepared repeated once again and the average value of and 20 ml of the broth was dispersed in each both the experiment was accounted. of 50 ml conical flasks and sterilized in an autoclave. Streptopenicillin, an antibiotic Results and Discussion solution was added to this at the concentration mentioned earlier. Single and Dilution plating of soils collected from the double doses of the drugs were allowed to Municipal waste at Musiri, resulted in the dissolve in 20 ml of the PD Broth by yielding of only 4 species of fungi. Their shaking constantly. Fungi were inoculated mean population density was 61 102 using sterile forceps to these flasks CFU/g of soil. The percentage contribution aseptically in a Laminar Air Flow (LAF) of the individual species in relation to the Cabinet. total fungal population was: Aspergillus flavus (20.5%), A. oryzae (17.6%) and A. The culture flasks were incubated at room niger (35.5%). temperature for a week, allowing the formation of complete mycelial mat on the Physiochemical Characteristics surface of the broth. After incubation, the broth was drained from the flask and the Physiochemical characteristics of the soil weights taken [weights of flasks with samples analysed from these sites are mycelium (A)]. The flasks were then presented in Table. autoclaved, the mycelial mats were discarded and again the weights were Fungitop (Table 2) measured [weights of flasks without mycelium (B)]. The drained broth were also As evident from the table, in control, the sterilised and discarded. growth of A. flavus was maximum recording 4.5 g of wet mycetial biomass. However, 69 Int.J.Curr.Microbiol.App.Sci (2016) 5(3): 67-75 with single dose drug, the growth recorded varied. Among the three, the maximum was only 33% when compared to the control affected species was again A. flavus. while in double dose concentration, there was only 15.5% of growth when compared Fungitop (Table 4) to the control. A perusal of the table reveals that in A. With regard to A. oryzae, the control flavus, the control recorded a growth of 4.5 recorded a growth of 4.9 g of wet mycelia g of wet mycelial biomass while in single biomass which decreased to 28.8% in single unit concentration growth decreased by unit and 13.0% in double unit concentration. 26.6% and in double unit concentration growth decreased to 11.1% when compared While A. niger recorded a growth of 5.3 g of to the control. wet mycelial biomass in the control, there was only a growth of 22.6% and 15.0% in While the growth of A.