This lab report summarizes an experiment to screen soil for antimicrobial producers. Garden soil was diluted and plated on nutrient agar and potato dextrose agar. The plates were incubated for 3 days but no colonies exhibiting inhibition zones were observed. While the 10-1 dilution showed most microbial growth, the experiment was unsuccessful in isolating antimicrobial producers from the soil sample. To improve results, fewer isolated microbes should be plated or a different soil source could be tested.
This lab report summarizes an experiment to screen soil for antimicrobial producers. Garden soil was diluted and plated on nutrient agar and potato dextrose agar. The plates were incubated for 3 days but no colonies exhibiting inhibition zones were observed. While the 10-1 dilution showed most microbial growth, the experiment was unsuccessful in isolating antimicrobial producers from the soil sample. To improve results, fewer isolated microbes should be plated or a different soil source could be tested.
This lab report summarizes an experiment to screen soil for antimicrobial producers. Garden soil was diluted and plated on nutrient agar and potato dextrose agar. The plates were incubated for 3 days but no colonies exhibiting inhibition zones were observed. While the 10-1 dilution showed most microbial growth, the experiment was unsuccessful in isolating antimicrobial producers from the soil sample. To improve results, fewer isolated microbes should be plated or a different soil source could be tested.
This lab report summarizes an experiment to screen soil for antimicrobial producers. Garden soil was diluted and plated on nutrient agar and potato dextrose agar. The plates were incubated for 3 days but no colonies exhibiting inhibition zones were observed. While the 10-1 dilution showed most microbial growth, the experiment was unsuccessful in isolating antimicrobial producers from the soil sample. To improve results, fewer isolated microbes should be plated or a different soil source could be tested.
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BSB 2462- INDUSTRIAL MICROBIOLOGY LABORATORY
LAB REPORT SEMESTER 1 (2018/2019)
Name: Tuan Muhamad Asani Bin Tuan Ibrahim (SB17063)
Muhammad Irhamni Haziqi Bin Nasharudin (SB17086) Nur Awatif Athilah Binti Mohd Asri (SB17033)
Experiment: (1) Screening of antimicrobial producer from soil
Practical Date: 18/9/2018 Lecturer’s Name: Dr. Lee Chin Mei Marks Obtained:
No Content Marks 1 Introduction 2 Objective 3 Materials & Method 4 Results 5 Discussion 6 Questions 7 Conclusion 8 References TOTAL 1.0- INTRODUCTION Soil is the materials that contain enormous numbers and kinds of microorganisms. The term of soils also refers to the outer loose material of the Earth’s oceans that may be regarded as three phases system which are composes of solids, liquids and gases. Besides, soils were composed of five major components which are mineral matter, air, water, living organisms and organic matter. Living organisms in the soil includes the microorganism and the small things. The microorganisms play the most important role in the release of carbon dioxide and nutrient for plant to growth. The major group of bacteria in the soil can be rod, cocci, spirilla, but bacillus are more numerous than other bacteria. The Greek words anti, mikro and bio which are refers to all agents that act against microbial organism derived the words of antimicrobial. An antimicrobial means that any substances of semisynthetic or synthetic origin and natural that kills or inhibits the growth of microorganism but causes no or little damage to the host. The term antimicrobials include all the agents that act against all types of microorganism such as bacteria, viruses, fungi and protozoa. The constant search of soils throughout the world has yielded an abundance of antibiotics of great value for the treatment of many infectious diseases. Pharmaceutical companies are in constant search for new strains of bacteria, mold, and which is antibiotic can be produced from it. Only a small portion of new antibiotics are suitable for medical use although many organisms in soil produce antibiotics. An antibiotic is a low molecular substance produced by a microorganism that at a low concentration inhibits or kills other microorganisms. An antibiotics activity under normal growth condition was showed by the bacteria isolated from soil and were found to inhibit some gram positive and gram negative organism. Nutrient agar and potato dextrose agar were used in this experiment to screening of antimicrobial producer from soil. The number of microbes in the soil depends on the environmental condition like nutrients availability, presence of moisture in the soil, soil texture and type of vegetation cover.
2.0- OBJECTIVE 1. To demonstrate skills in isolation of antimicrobial producing microorganism from soil. 2. To explain some of the microorganisms present in the soil. 3.0- MATERIALS & METHOD 3.0.1- Materials 1. Soil 2. Nutrient agar 3. Saline water blank 4. Potato dextrose agar 5. Beaker of alcohol 6. Incubator 7. Bunsen burner 8. Micropipette 9. Inoculation loop 10. Agar plate 11. Centrifuge tube 12. Pipette tip
3.0.2- Method
1. 1g of garden soil placed in a 9 ml sterile dilution blank. The soil and water mixed thoroughly by the water-soil mixture shook vigorously for 5 minute, your elbow kept on the lab table. 2. A tenfold dilution made from tube 1 through tube 3 by 1 ml transferred from tube to tube. The mixture shook vigorously for 3 minutes. 3. A fresh pipette tip used for each transferred and the pipette-mix thoroughly before each transfer. 4. 0.1 ml for each tube transferred to different types of agar, which were nutrient agar and potato dextrose agar. Each type of agar has three petri dish respectively. 5. 0.1 ml for tube 1,2 and 3 transferred to the nutrient agar plate by using micropipette and the mixture spread with an inoculation loop. The inoculation loop sterilized by opened the flame. The loop cooled before used (Figure 1).
Figure 1: Dilution method of isolation
6. Step 5 repeated by transferred the mixture to the Potato dextrose agar plates. 7. The petri plates for nutrient agar and Potato dextrose agar inverted and the plates incubated at 37 ºC and 30 ºC respectively for 3 days. 4.0- RESULTS
THE AGAR PLATES AFTER INCUBATION FOR 72 HOURS
1. Record the number of colonies that form inhibition zone on the agar. 2. Make a drawing of colony that produce inhibition zone. 3. Record the characteristics of some colonies that formed on the agar.
*NOTE : The answers of questions above are summarized in table below.
zone: NO (ii) Drawing of inhibition zone: NO DILUTION AGAR PLATE CHARACTERISTICS NO (b) 10⁻2 Form – Circular Elevation – Raised Margin – Entire Opacity- Translucent
zone: NO (ii) Drawing of inhibition zone: NO DILUTION AGAR PLATE CHARACREISTICS NO. (c) 10⁻3 Form – Circular Elevation – Raised Margin –Entire Opacity-Translucent
(i) No. of colonies with inhibition
zone: NO (ii) Drawing of inhibition zone: NO 5.0- DISCUSSION
In this experiment, we used some soil that we got from outside of our laboratory to get the inhibition zone on the media. We used three nutrient agars and three Potato dextrose agars respectively. This method was used to compare the differences between each of serial dilution. To get the result, we left the media for about 72 hours at the incubation place at temperature of 30 °C. This temperature suitable for most of the optimal microbe’s condition. After 72 hours, we unsuccessful got the desired result. On the both of nutrient agar and Potato dextrose agar, we did not observe the inhibition zone. Based on the result obtained for both medium, the 10-1 dilution shows most grew microbes on the surface of agar than others agar plates. This is because, most of microbes contain was from the first dilution compared to the others dilution. Meanwhile, to get more the inhibition zone, number of isolated that needed to apply in this experiment must less. Inhibition zone is also called a Kirby-Bauer Test, is a qualitative method used clinically to measure antibiotic resistance and industrially to test the ability of solids and textiles to inhibit microbial growth [Chegg,2015]. The inhibition zone is an indication that the antimicrobial agent produces effective inhibition of microbial growth. The inhibition zone also used to produce an antibiotic in medicine study. For example, bacillus sp. was predominant soil bacteria to produce antibiotic. This is due to the production of vital antibiotics and they produced resistant endospore. Based on the results that we got, some microbes isolated and cultured on nutrient agar and Potato dextrose agar did not produce inhibition zone. They spread freely on the agar. As we known, nutrient agar contained beef extract, which is suitable for isolated bacteria while Potato dextrose agar contained carbohydrate, which is suitable for isolated fungi. Potato Dextrose Agar (PDA) is a general purpose medium for yeasts and molds that can be supplemented with acid or antibiotics to inhibit bacterial growth [ Aryal, S., J., A., R., Greene, R., & Singh, A.,2018]. This agar used as the detection of yeasts and molds in dairy products and prepared foods. Thus, it suitable to isolate fungi. Based on the results, there are other microorganisms isolated and cultured on the agar plate without showing the inhibition zone. They were in circular and round shape. They spread the entire of the agar plates. The opacity of the agar plates was translucent. This made us hard to identify the small and unseen killing zone. The potato dextrose sugar plates were grow by fungi which were filamentous. While doing this experiment, some precautions step needed to apply. Firstly, the aseptic technique must be strictly followed step by step. This way can help us to avoid got the contaminated results. Then, when cultured the mixture, the inoculating loop must flame and cooled first. This prevented from microbes in the mixture dead because of the temperature. After that, the tube must be well shaking before pipetting for serial dilution to ensure the microorganisms fully occupied in the solution. Thus, the chance of culturing more microorganisms will be higher. This method also can help to archive better results. 6.0- QUESTIONS
4. Why were different media used in this experiment?
In this experiment, we used 2 types of media, which are nutrient agar and Potato dextrose agar. In nutrient agar, it contains beef extract. This media used to isolate the bacteria that produce antibiotic, while Potato dextrose agar contains dextrose that suitable for the cultivation of fungi. The different media provide nutrients to different types of microorganisms and thus we can observe and culture the different microorganisms to get better results.
5. Why do you use soil as the source for isolation of antibiotic producer? We used soil as the source for isolation because in soil, the quantity of microorganisms was more than others. Then, soil was easily to find. Many microorganisms optimally live at neutral condition and the place that contains more nutrient, which is soil.
6. Why must you shake before pipetting?
The reason why we must shake before pipetting is to ensure that all microbes in the tube was fully occupied the broth. This is because, it will help to increase the chance of culturing microorganisms after serial dilution into the medium.
7. Why do you need to have 3 agar plates for each dilution?
We need to have 3 agar plates for each dilution to make sure the result obtained were almost identical. The replicate plating from a common source is no more accurate than single plating.
8. Can you distinguish bacterial colonies from fungal colonies?
Bacterial colonies grow rapidly on nutrient abundant culture media compared to fungal colonies. Besides, the different between bacterial colonies and fungal colonies is that bacterial colonies are visible masses of bacterial cells arising from single bacterial cells while fungal colonies are visible masses of fungi arising from a single spore or mycelial fragment. 7.0- CONCLUSION As the conclusion from the experiment, skills in an isolation of antimicrobials producing microorganisms from soil was demonstrated. Then, we failed to isolate and culture the antibiotic producer which is determined by absence of inhibition zone on agar plates. After that, the principle and characteristic of inhibition zone was understood and the skills of screening of antimicrobial producers are enhanced in this experiment. From the result obtained, there are a lot of microorganisms present in the soil such as actinomyces, salmonella, pseudomonas arginosa and molds. All of these are antimicrobial microorganisms which are produced from the soil. Lastly, we also learned about the method to incubate the agar at the incubator.
8.0- REFERENCES
1. C. N. (2015, December 16). Zone of Inhibition Test for Antimicrobial Activity.
Retrieved September 23, 2018, from https://microchemlab.com/test/zone-inhibition- test-antimicrobial-activity 2. Srividya, A. R., Saritha, G. S., & Suresh, B. (2008, November/December). Study of the Soil Isolates for Antimicrobial Activity. Retrieved September 23, 2018, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3040884/ 3. Differences between bacterial and fungal colonies. Post by Samanthi (July 11,2017), Retrieved from https://www.differencebetween.com/difference-between-bacterial- and-vs-fungal-colonies/ 4. Aryal, S., J., A., R., Greene, R., & Singh, A. (2018, June 11). Potato Dextrose Agar (PDA)- Principle, Uses, Composition, Procedure and Colony Characteristics. Retrieved September 24, 2018, from https://microbiologyinfo.com/potato-dextrose- agar-pda-principle-uses-composition-procedure-and-colony-characteristics/ 5. Miyaura J, Tatsumi C. Studies on the antibiotics from actinomycetes. An antibiotics pigment from Streptomyces F-23b. Bull Univ Osaka Pref Ser B 1960;1:129-37 6. Selvameenal L, Radhakrishnan M, Balaraghunathan R. Antibiotic pigment from desert soil actinomycetes; Biological activity, purification and chemical screening. Indian J Pharm Sci 2009;71(5):499-504.
