Antimicrobial Activity of Soil- Research Proposal free essay sample

Microbes produce various antibiotics and by isolating different microbes the antibodies can be tested for. Soil samples are highly rich in microorganisms. The antimicrobial  activities of microbes from 3 different soil sources (Tenby, Summerstrand, North Campus main building round-about and South Campus water pond) will be tested. Pure colonies isolated from the microbes will be tested for antimicrobial activity against known microorganisms of medical importance; these being E. coli, staphylococcus aureus, Shigella dysenteriae, Shigella sonnei and salmonella spp. Isolates of soil microbes can be categorized and  characterized based on a number of criteria ranging from gram-staining which will be done in this project, for enumeration which is a quantitative description based on the amount of microbial colonies available. The results will be recorded. Key words: soil, microbes, microbial activity, antimicrobial resistance INTRODUCTION It is vital for science today to focus on the isolation and characterisation of an unknown organism expressing interesting properties in relation to antimicrobial Activity. At the conclusion of this laboratory experience the researcher would be required to describe the isolates obtained from the soil macroscopic and microscopic appearance, perform the gram stain of the microorganisms in question and to discriminate the organisms from other microbes that could be contained in the soil and to finally make certain that the organisms have not been exposed to outside sources and be isolated in a pure culture. The researcher must first isolate the microbe then, grow the organism in a pure culture. Pure colonies will be tested for antimicrobial activity against known microorganisms of medical importance. The evaluation of the results that will be achieved are important in the understanding the development of new antimicrobial drugs. LITERATURE REVIEW Soils typically contain 109  to 1010  microorganisms per gram (dry weight), which may represent more than a million bacterial species. However, characterization of the small fraction of microbes that has been cultivated provides only a glimpse of their potential physiological capacity and influence on soil ecosystems. The absence of pure cultures or genome sequences makes it difficult to ascertain the roles of specific microbes in soil environments. Eichorst, Breznak and Schmidt (2007) Diarrhoea caused by Shigella species is estimated by WHO to cause 50% of dysentery cases. S. dysenteriae serotype 1 is particularly virulent, causing endemic and epidemic dysentery with high death rate. Salmonella organisms are endemic in many tropical and developing countries, while other salmonellas cause food poisoning and bacteraemia. It is highly infectious and resistance to common available antimicrobials is an increasing problem. Cheesbrough (2000, p. 97) Antimicrobial resistance is resistance of a microorganism to an antimicrobial medicine to which it was previously sensitive is on the rise. Resistant organisms such as bacteria are able to withstand attack by antimicrobial medicines, such as antibiotics. Infections caused by resistant microorganisms often fail to respond to conventional treatment, resulting in prolonged illness and greater risk of death. World Health Organisation (2012) discusses the problem of antimicrobial resistance Synthesis of medicinally important compounds is very difficult and thus the cost of medicine is also high because of the non availability of source materials especially aromatic compounds Gopalakrishnan (2011) AIM OF RESEARCH The main aim of this research is to determine whether or not soil bound microbes’ exhibit antibiotic activity against known bacteria of medical importance. OBJECTIVES OF STUDY * To develop a new antimicrobial agent. * To reduce the ever expanding problem of antimicrobial resistance in treatment of bacterial infections. RESEARCH METHODOLOGY * METHOD(S) TO FASCILITATE MICROBIAL IDENTIFICATION Gram stain * Gram stain will be used to identify the gram bacterial membrane reaction for easy identification. Culturing * Nutrient agar will be used for the culturing of the soil supernatant to favour growth of most bacteria. Muller-Hinton agar will be used for testing antimicrobial activity * Blood agar useful for pure culture isolation and determination of haemolysis with fastidious microorganisms. Cheesbrough (2000) Biochemical reactions Biochemical reactions will be used for the full identification of the bacteria isolated in soil and confirmation of the known enteric organisms. * API test kit Biomerieux (2009) * MATERIALS TO BE USED GRAM STAIN| GENERAL MATERIALS| Lugols’ iodine| Laboratory protective coat| Safrannin| Examination gloves| Acetone| Biohazard disposal bags| Crystal violet| Sterile sample containers | Garden hoe| CULTURE| Microscope| Culture plate| Normal saline| Wire loops| Cover slips| Bunsen burner| Glass slides| Autoclave| | Culture media| KNOWN BACTERIA FOR ANTIMICROBIAL TESTING| Incubator| Escherichia coli| Tight gas cylinders| Salmonella spp| Distilled water| Shigella dysenteriae| | Shigella flexneri| GLASS WARE| Staphylococcus aureus| Conical flasks| | Graduated beakers| BIOCHEMICAL REACTIONS| Measuring cylinders| API testing kit| Test tubes| | Cheesbrough (2000) * ETHICAL CONSIDERATIONS The experiment to be conducted does not require the use of animals. This being said, the research will not require clearance from the department or the governing organisations responsible for the regulation of experiments of that nature. The research being conducted will also be of benefit to the society. Ethical considerations in research (2002) Kanungo. R. (2006) explains the ethics in research * SAMPLE COLLECTION Designing statistically sound sampling programs for soil antimicrobial testing requires the consideration of specific monitoring questions and the state in which the soil extracts of interest will be left post extraction. Three (3) different soil sources will be used and below are the proposed sources of soil for antimicrobial activity analysis:- a. Loamy soil from Tenby, Summerstrand, b. South Campus of Nelson Mandela Metropolitan University pond c. North campus main entrance round about The soil will be collected from 5 inch depth of the surface and stored in sterile small reagent bottles. Chutia and Ahmed (2012) Wallenius (2011) describes the microbiological characteristics of soil sampling * SAMPLE ANALYSIS METHOD To address the goals of achieving a pure culture of soil microorganism and testing for antimicrobial activity, a quantitative serial dilution method will be used. Quantitative research method will measure the number of soil microorganisms with antimicrobial activity witnessed thus preferred for this particular research. Psychology Press Ltd (2004) explains Research methods and Data analysis SERIAL DILUTION PROCEDURE DAY 1 1. Mark the sterile dilution blanks in the following manner: the 100 ml dilution blank is 10-2 and the tubes sequentially are 10-3, 10-4, 10-5 and 10-6. 2. Add 1gram to the 10-2 dilution blank, centrifuge at 4000 rpm for 5 minutes and ensure the cap is securely tightened. 3. Aseptically transfer 1ml from 10-2 dilution to the 10-3 tube. Mix thoroughly. 4. Using a fresh, sterile pipette for each succeeding step, transfer 1ml from the 10-3 dilution to the 10-4 dilution blank, then from the 10-4 to the 10-5, then from the 10-5 to 10-6. Sample transferred must be thoroughly mixed with the dilution fluid before transferred to the next tube. 5. Aseptically inoculate from 10-6 dilution and incubate on nutrient agar for 24 hours at room temperature. . This procedure will apply to the soil acquired from other sources and will be done free from contamination of the other. Isolation of soil bacteria: viable titer and pure culture (n. d) Below is a demonstration of the serial dilutions:- Isolation of soil bacteria: viable titer and pure culture (n. d) DAY 2 Pure culture isolation 1. From the 10-6 dilution, choose one colony that appears to be compos ed of only one cell type. 2. Flame an inoculating loop, when cool gently touch it to the surface of the colony you will re-streak onto a Blood agar plate. Isolation of soil bacteria: viable titer and pure culture (n. d) The plate should be streaked as: Isolation of soil bacteria: viable titer and pure culture (n. d) 6. Incubate the plates at room temperature for 24 hours DAY 3 1. Examine the Blood Agar plate re-streaked for a pure colony. 2. Test the pure colony’s antimicrobial activity against a known microorganism on Muller-Hinton agar and incubate at 37 degrees Celsius for 24hours. 3. Gram-stain pure colony achieved, streak the colony onto a Nutrient Agar slant. Incubate for 1 day and place in the refrigerator (Isolates to be used for future experiments. Isolation of soil bacteria: viable titer and pure culture (n. d) DAY 4 – Expected Results 1. Antimicrobial activity will be observed by a zone of inhibition around the pure colony streak or, 2. A negative result showing growth of known bacteria. CONTROLS * Sterile water tested for antimicrobial activity must allow growth (Negative control), while a * An effective known antibiotic for particular bacteria tested for antimicrobial activity must show a zone of inhibition (positive control). Cheesbrough (2000) DISSEMINATION OF RESULTS The results achieved from the research will be documented in form of a Treatise, which will be oral presentation to the students and faculty of Health Sciences Department of Nelson Mandela Metropolitan University. Should the results of the study be of interest, there is a possibility of publishing the findings in a journal for continuous dissemination. WORK AND TIME SCHEDULE The research activity will commence early August of 2013. It will be divided into two portions; the practical component and documentation of practical findings. The practical component will be conducted on from Monday to Saturday of every week. During week days, the practical activities will be done after classes. The practical component is scheduled to run for 1 month and 1 week, after which the documentation component will commence and run for a month duration. The research is scheduled to run for a period of 2 months and a week. The write up will then be handed to the research supervisor, seeking their opinion on the outline of the Treatise. FINANCE BUDGET A list of requirements is set below, to guide the funder on the scale of requirements, amount of work and cost of the research. Below is a list of the needed materials as per category:- GENERAL REQUIREMENTS| COMMODITY PRICE| Laboratory protective coat| | Examination gloves| | Biohazard bags| | Normal saline| | Microscope| | Glass slides| | Slide cover slips| | Sterile containers for serial dilutions| | GRAM STAIN| COMMODITY PRICE| Lugols’ iodine| | Acetone| | Safrinnin | | Crystal violet| | CULTURE| | Culture plates| | Bunsen burner| | Hot plate| | Culture media| | Incubator | | Tight gas cylinders| | Distilled water | | BIOCHEMICAL REACTIONS| COMMODITY PRICE| API test kit| | Biomerieux. (2009) Cheesbrough (2000) lists the different microbiological requirements the facilitate the budgeting of a research Higdon and Topp (2004) give some guidelines on how to prepare a research proposal budget CONCLUSION The search for anti-secretory drugs continues, with real progress having been made. Soil and plants are the potential source of many drugs; but they did not yet get proper recognition. The role of antimicrobial agents in the management of infective bacterial conditions continues to be clarified with the emergence of new agents and simplified treatment regimens. Probiotics are popular with diarrhoea sufferers and have been shown to have some efficacy. However, further scrutiny is required to determine the magnitude of their effects and continued research on new development of anti diarrhoea agents in needed. Antibiotic resistance is a major clinical problem in treating infections caused by these microorganisms. One way to prevent antibiotic resistance of the pathogenic species is by using new compounds that are not based on existing synthetic antimicrobial agents. It is then vital to do more research on soil antimicrobial activity. Chanu, Chakraborty el tol (2011)