Lab 6

ABSTRACT/ SUMMARY

In this experiment, three samples have been used. The entire samples come from difference source. For the first sample, pond water at ‘Dataran Cendikia’ has been taken and used. The sample was used to identify the presence of microorganis microorganisms ms inside the sample. A dropper  dropper  was used to take the sample from the bottom of the bottle that has been used to keep the sample. A drop of the sample was placed in a specimen slide. The sample was covered with a covered glass slide. Then, microscopy technique was applied. The sample was being observed under the light microscope. The second sample comes from the soil. The soil at ‘Tasik Seksyen 7’ has been collected and used as the sample. Before proceed with the next step, the soil need to be dissolved inside the distilled water. After the soil has mixed with distilled water, soil solution will formed. Sterile cotton swab was used to take the sample from soil solution. By using the streaking technique, the sterile cotton swab that contains the sample was streak on the solid nutrient agar medium that have been provided. Then, the nutrient agar medium culture was sealed with parafilm tape. The medium plate was inverted and being incubates for 24 hours. The last sample comes from the dairy product. Vitagen has been used as the sample. Another sterile cotton swab was used to take the sample from the bottle. Same with the  previous step, streaking technique was used to streak the sample on the medium culture  provided. The plate was then sealed with parafilm tape. The plate was inverted and being incubates for 24 hours, same with soil sample. Observation has been made on the next day. The cultured mediums have been removed from the incubator. Inocculum loop was burned before taking the sample that has been cultured. The sample was placed on the slide and being covered with cover glass slide. The samples were observed under the microscope. All results were recorded.

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INTRODUCTION

In natural habitats, microorganisms usually in a complex mixed populations with many species. This presents a problem for microbiologists because a single type of  microorganism cannot be studied adequately in a mixed culture. Robert Koch, known as the father of medical microbiology, was one of the first to recognize that isolating a microbe (in his case, bacterium) away from other microbes was crucial for his own argument that microbes cause disease, as well as understanding characteristics of the microbe itself. His studies on Bacillus anthracis contributed too many of the laboratory techniques we still use today, including the method for isolating pure cultures of bacteria. Pure culture of  microorganisms that form discrete colonies on solid media may be most commonly obtained by plating methods such as streak plate method, pour plate method and spread  plate method. Brightfield microscope was being used to observe the microorganisms inside the sample. The sample under examination is called the specimen. Specimen preparation is an important step in light microscopic examination of a given sample. Streak plate method is used most commonly to isolate pure cultures of bacteria. A small amount of sample is  placed on the tip of an inoculation loop/needle and is s treaked across the surface of the agar  medium.

OBJECTIVES

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To identify the microorganisms inside ponds water, soil and dairy product.

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To understand the concept of microorganisms.

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To understand why even the smallest organisms is important to environment.

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To make a survey of microorganisms inside pond, soil and dairy product.

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To understand the basic function of a single cell organisms.

THEORY

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In this experiment, streak plating technique is used to isolate bacteria. Streak plating technique allows for the formation of individual colony forming units for isolation by reducing the number of bacterial. Aseptic technique refers to a procedure that maintains the sterility of the experiment in order to prevent any contamination that may occur while undergo growing and transferring of bacteria. Therefore, sterile swab is used to prevent any contamination from the environment. After inoculating culture media with bacteria, it is incubated at its optimum temperature for well growth Media flame sterilization is a frequent application for many laboratories in the life science fields of cell biology, virology, and others. Flame sterilization is a very quick  simple method of killing microorganisms on an inoculating loop. The loop is held inside a flame for a few seconds until it is red in colour and then cooled. Once the loop is cooled, it can be used for various culture manipulations. A sterile cabinet is used to keep the airborne contaminants from getting into the hood for sterile purpose. Safety and precautions steps in handling the microorganism are needed to ensure the microorganism can be grown in a culture medium (nutrient agar medium). Below are the pictures of streak plating technique and flame sterilization technique.

Figure 1: Streak plating technique

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Figure 2: Flame sterilization technique

APPARATUS AND MATERIALS

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APPARATUS •

Sterile swab

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Laminar flow chamber 

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Incubator 

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Parafilm tape

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3 Petri dishes with agar medium in it

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Universal bottle

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Dropper 

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Bunsen burner 

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Beaker 

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Glass Slides

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Cover slip

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Inoculating loop

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Microscope

MATERIALS • •

Distilled water   Nutrient agar medium

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Soil sample

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Pond water sample

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Vitagen sample

METHODOLOGY

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Part 1: Observation of pond water

Part 2: Culture and Observation of Pond Water, Soil and Vitagen Sample

RESULTS After 24 hours incubation

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Soil

Vitagen

Pond

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OBSERVATIONS

Soil

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Vitagen

Pond

DISCUSSIONS

Survey of microorganisms is an experiment that being done to investigate the type of  microorganism that living in certain rural area. In this experiment, the sample was being

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chosen by certain criteria which are based milk, soil and pond. There are different microorganisms that living in the three different places. For the soil, the microorganism that  popular among student is the nitrobacter, rhizobium where they live is soil to help the fixation of nitrogen in nitrogen cycle. In the pond, the usual microorganisms that live in that habitat are algae, protozoa and many other aquatic microorganisms. In sample that is based milk such as milk, yogurt and so on, the microorganism is inserted into the drinking material which the presence of them could gave many benefit to human being. In this experiment, the three samples was taken from pond at Dataran Cendekia, vitagen and soil near the lake of section 7. For the first sample, the sample from pond of  Dataran Cendekia is use to observe the presence of microorganism that living in there. The sample is put into universal bottle and the sample is taken from the bottom part of the  bottle. The sample taken is put under slide and cover slip is being added later. The slide is observed under brightfield microscope. From the result obtained at the end of the experiment, the microorganism that being recognised is the one that living at the top of   pond to undergo photosynthesis. The microorganism is Algae. The validity of the observation is proved as the microorganism observed has green pigment which the main component for the photosynthesis.

Image 6.1 – Microscopic view of Algae The other two samples have different procedure from the sample from pond. Algae and protozoa can directly being observed under microscope while the other two sample need to cultured first before being observed under microscope. For the sample taken from

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soil near lake of section 7, the microorganism that living in that particular area is cultured on the nutrient agar and being incubate for 24 hours. At the end experiment, the microorganism that being observed under microscope has morphology of rods shape or   bacillus that held with each other in one colony. In the based milk sample which is Vitagen, the microorganism that being observed under brightfield microscope after 24 hours incubation has the bacillus shaped bacteria. The  bacteria are well known as Lactobacillus as we all know that Vitagen has Lactobacillus as aid to human digestive system. The microorganism that living inside Vitagen has the morphology of bacillus shaped bacteria mixed with coccus shaped bacteria.

Image 6.2 – Lactobacillus

Image 6.3 – Bacillus shaped microorganism that actively living in soil. Algae can be cultured on agar medium while protozoa cannot be culture on the agar  medium and need to use other medium. Protozoa need to be cultured on blood medium which is different from agar medium. In this experiment, algae and protozoa doesn’t need

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to be culture on the agar nutrient as it visible from brightfield microscope point of view. Even though the sample from the pond is cultured, only algae would be survived on the agar nutrient and protozoa wouldn’t.

CONCLUSION

As the conclusion, the surveying of microorganism did fulfil the objective of the experiment. For the first objective which is to identify typical microorganisms that living in

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the sample from pond, the microorganism have already being identify to be Protozoa and Algae which are the most common name of microorganisms that living under aquatic environment. The second objective is referring to the taxonomy of microorganisms. In the sample taken from soil, the microorganism observed has the morphology of bacillus  bacteria which can be classify in group of bacteria and in Vitagen that being cultured in the agar, the microorganisms has the mixed morphology which is coccibacilli.

RECOMMENDATIONS

For the recommendation, the experiment must be done followed the correct procedure in order to get the accurate result. First, students need to wear gloves during laboratory  procedure to prevent contamination and injuries. Second, the culturing method for the sample need to be done in the laminar flow cabinet to apply aseptic technique concept which prevent any foreign microorganism from invade the nutrient agar. For the procedure that using sample from pond, the sample that being observed must be taken from the  bottom of the universal bottle to make sure the presence of either protozoa or algae in sight of the microscope with various magnifications.

REFERENCES

Bruns, A., U. Nubel, H. Cypionka, and J. Overmann. 2003. Effect of signal compounds

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and incubation conditions on the culturability of freshwater bacterioplankton. Appl. Environ. Microbiol. 69; 1980–1989

Davis, K.E.R., S.J. Joseph, and P.H. Ja nssen. 2005. Effects of growth medium, inoculum size, and incubation time on the culturability and isolation of soil bacteria. Appl. Environ. Microbiol. 71; 826–834

Doyle, M.P., and J. Meng. 2006. Bacteria in food and beverage production. In M. Dworkin, S. Falkow, E. Rosenberg, K-H Schleifer, E. Stackebrandt (Eds.) The Prokaryotes; 1; 797–811

Guggenberger, G. 2005. Humification and mineralization. In F. Buscot, and A. Varma (ed.) Microorganisms in soils: roles in genesis and functions. Springer, New York; 85106

J.M. Willey, L.M. Sherwood and C.J. Woolverton, Prescott’s Microbiology, 8 th edition, Mc Graw Hill, 2011

R. H. Garret and C. M. Grisham, Biochemistry, 4 th edition, Brooks/Cole, 2010. Harley and Prescott, Laboratory Exercises in Microbiology, 5 th edition, McGraw-Hill Company, 2002. John, P. Harley. and Lansing, M. Prescott. (2002), Laboratory Exercises in Microbilogy, 5th Edition, McGraw-Hill

APPENDICES

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