CHAPTER I INTRODUCTION 1.

1 Background

These days amount of water required and clean food very costly. So that many society choosing to consume water which improper to consumption. Like water of got residing in Ketintang Wiyata VII which consist of many bacterium. Bacterium disseminate considerably easy and expand by leaps and by bounds in body, water and air, hence needing to do examination of quality irrigate to know the quality of water. bacterium type which exist in the water by isolating bacterium irrigate got. (Pelczar, 1986 Microbial Population naturally is not apart by it self, only containing an mixture from different kinds . Microbial Population naturally around us is very complex and big. In microbiological laboratory, result of bacterium population earn insulation into pure culture consist of one type able to study form, size measure and type, and can be counted colony which grow ( Frobisher, 1974)

1.2 1. 2. 3. 4.

Problem formulation

How is water quality sample in the alley got of Ketintang Wiyata VII? What are the characteristics to different colonies on agar medium in a petri dish? How many colonies were formed on dilution 10-5-10-7? Why is the number of bacteria in each samples is different?

1.3

Objective

1. Knowing the quality of water samples in the alley got of Ketintang Wiyata VII 2. Knowing the characteristics of colonies on agar medium in petri dish 3. Knowing the number of colonies formed on dilution10-5-10-7 4. Knowing the cause of the difference in the number of bacteria in each sample

1.4

Beneficial

This research is very useful in recognition kinds of microbe (include form, size and amount of bacterium), preventive of microbe contamination, and also can know the quality of water of got of Ketintang Wiyata VII.

CHAPTER II BASIC THEORY 2.1. Isolation of Microorganisms Microorganisms are an important component of the ecosystem. In its natural habitat, they live in a community made up of different types of microorganisms, along with other biological species. Within this community, the microbial species can affect other species in many ways. Some have beneficial properties, but some other harm (Pelezer, 1988). In study of bacteria, the bacteria must be taken from the nature and isolated in a pure culture. Pure cultures are cultures that contain only one type of bacteria (Pelezer et al., 1988). Isolation is a process of taking the microorganisms present in nature and grow in an artificial medium (Sutedjo, 1996). Several factors need to be considered in isolation of microorganisms are: 1. The nature and types of microorganisms 2. Habitat microorganisms 3. Growth medium 4. How to inoculation and incubation 5. How to identify 6. How maintenance (Dwidjoseputro, 1998). There are various methods of insulation that can be used. Various methods of isolation include: 1. Single isolation is a method of isolation with the dropping of material that contains microorganism on the cover glass using micropipette shed by a microorganism containing a cover glass using a micropipette, then is observed under microscope. 2. Scratching isolation is isolation method by sliding or scratching the needle loop containing microorganisms carefully over the surface of agar in a zig zag order starting from base of the tube to the top of the tube. 3. Spreading isolation is isolation method by spreading materials containing

microorganisms on the surface of the tube.

4. Pouring isolation is a method of isolation by taking a small sample of the mixture of bacteria that had been diluted then the sample is spreaded in a medium from liquid of broth and gelatin. (Dwidjoseputro, 2003). Characteristic of bacterial colonies that is result of inoculation is one imprtant part in the identification of bacteria. Some specific forms colonies of bacterial colonies on flat agar medium are (Sutedjo, 1996): 1. Size Point, small, medium, and large. 2. Colony colour There are pigmented and non-pigmented 3. Forms colonies Mother, irregular and rhizoid (spread like roots) 4. The shape of the edge of the colony (Margin) Average (entire), uneven, bumpy uniformly (lobate), wavy (undulate), bergerig (serrate), and like filaments (filamentous)

2.2. Enumeration of Microorganisms Enumeration is the technique of counting the number of microbes in a medium without identifying the types of microbes (bacteria, fungi, yeast), which aims to determine the number of cells of a bacterial culture quantitatively (Cappucino and Sherman, 1983). Determination of the number of bacteria is done by counting the number of bacterial cells capable of forming colonies in culture medium in a solution or forming a suspension on the culture solution (Schlegel & Schmidt, 1994). Analysis of Quantitative microbiology of food is important to know the quality of food and calculate the curing process that will be applied to the food material (Fardiaz, 1992). There are several ways to measure the number of cells, among others: 1. Dish counting (plate count) Calculation using counting dish method is the minimum number of microorganisms. When grown on appropriate media and environment groups these bacteria would only produce

one colony of bacteria. Should only plates containing 30-300 colonies are used in the calculation. Slab with a high number of colonies (> 300 colonies) are difficult to be calculated so that the possibility of a huge miscalculation. Sample dilution helps to obtain a correct calculation of the amount, but the dilution is too high will result in the slab so that the number of colonies is low (<30 colonies) (Lay, 1994).

2. Direct microscopic count (direct microscopic count) or MPN (Most Probable Number) (Fardiaz, 1992). Determination of the number of bacteria can be done by counting the number of bacterial cells that can form colonies in culture medium or forming suspension in a culture solution (Schlegel and Schmidt, 1994). Enumeration can be done using the three techniques are: 1. Direct platting It is a technique of counting the number of microorganisms directly with the help of colony counter or other device. 2. Dillution platting There are two ways that can be done is to calculate the chemical components in the cell such as DNA or by counting oxygen or carbon dioxide is absorbed or produced during respiration or fermentation. The second way is by using a calculation of the number of microorganisms indirectly because it cannot determine the number of microorganisms but can only know the OD or Optical Density with using a spectrophotometer. 3. Calculation of number of living cells Unlike the previous method for counting cells that die and live cells, in this way specifically wanted to know life cell number only.

2.3. Bacteria - Bacteria Cause Rotten of Vegetables Some of bacteria that causes the rotten of vegetable are; (Fajriyati, 2012) 1. Soft Rot Bacteria (Bacterial Soft Rot) Commodity who attacked: BWG. red / white, carrot

Type: spp.

Erwinia

carotovora,

Pseudomonas

marginalis,

Clostridium,

Bacillus

2. Gray mold rot (Gray Mold Rot) Commodity who attacked: wine, spinach Type: Botrytis cinerea, Botrytis spp. 3. Downey Mildew Commodities were attacked: mustard / rapeseed Type: Phytophthora, Bremia, etc. Pickling Vegetables ( Fajriyati, 2012 ) With aseptic Eliminates microorganism contaminants The use of high temperature: blanching Use of low temperature: chilling, freezing Drying Use of Preservatives: Salt (2.25 to 2.5%) Irradiation: gamma rays (g)

2.4. The Limitation of Bacteria In Water that Can be Consumption Water is a vital necessity of life, especially human beings. Because water is making up almost 90% of the human body and other living creatures. According to the Health Department, the terms of drinking water is tasteless, odorless, colorless, contains no harmful organisms, and does not contain heavy metals. Drinking water is water that through processing or without processing that qualified health and can directly drink (KepMenKes No. 907 of 2002). Although water from natural sources can be drunk by humans, there is a risk that the water has been contaminated by bacteria (eg Escherichia coli) or harmful substances. Good drinking water must meet drinking water standards specified in the regulations of Ministry Health of the Republic of Indonesia No.416/MENKES/PER/IX/2002

which about drinking water standard. In test water quality, the parameter of physic, chemistry and biology is required. Biological parameter that is used in the test water quality is level of fecal coliform levels or to be more specific is the presence of E.coli bacteria. If in the ground water contained the E. Coli, so it could have been present the virus, bacteria, parasites and other amoeba in the water. But if there is no of E. Coli, so possibility there is no virus, bacteria or parasite and in there just present the non-pathogenic germs or isnt harmful. This is why E. coli can be used as a biological parameter water quality testing. Additionally the methods is used to detect the presence of E. coli is relatively simpler and more representative than other aquatic microbial detection. Based Permenkes No. 416 of 1990, the limitation of bacteria in the water so it capable to consuming as follows: Fecal Coliform number per 100 ml = 0 Total coliform (MPN) for piped water is not a maximum number per 100 ml = 50, and for the maximum amount of piped water per 100 ml = 10. Meanwhile, Water Requirements In bacteriological Figures TPC value in drinking water <100 / ml (POM DG, 1989). TPC is a quantitative examination in the sample of the bacteria. TPC is Total Plate Count, which is a count of the number of bacteria contained in 1 ml of sample. (Rahayu, 2011).

CHAPTER 3 RESEARCH METHOD 3.1 Date and Place

The research have done in Microbiology Laboratory, Surabaya State University on Tuesday until Thursday 25th 27th September 2012 3.2 Tools and Materials

Tools and materials will be described in the following table: Table 1. tools and materials for the experiment No 1 2 3 4 5 Tools Spirtus Lamp Ose Needle Incubator Petri dish with medium TA inoculation needle (injection) 1 1 1 5 (in group) 7 Amount Materials water of Gutter medium TA alcohol 70% Amount sufficienly sufficienly sufficienly

3.3

Procedure

a. Cultivate Sample of Microorganism Procedure

b. Enumeration the amount of Mikroorganism Procedure

First thing to do is to bring the water got in opaque containers and preparing tools and materials needed to capture and dilution in test tubes containing 9 mL dilution up to 10-7. Then the bacteria are grown on media TA obtained aseptically. After that, the media containing the bacteria were incubated with a temperature of 27-28 C for 24 hours. After 24 hours, bacterial colonies were observed growing and differentiated characteristics.

CHAPTER IV RESULTS AND ANALYSIS DATA 4.1. Analysis Data From the observation we have done, we got some data of it, such as : 1. Table of the amount of dilution colony from class data.

Sample 1. Gutter behind

The amount of colony 1,7 x 107 5,1 x 106 2,4 x 106 2,0 x 106 5,5 x 106

Unesas mosque 2. Gutter beside Unesas mosque 3. Gutter of Ketintang Wiyata III 4. Gutter of Ketintang Wiyata VII 5. Gutter of Ketintang Baru

We used 10-5, 10-6, 10-7 of dilution. From the table above, in first sample in dilution 10 -5 and 10-6 produce the number of colony between 30 until 300 and the comparing between a higher dilution and lower dilution is smaller or equal to 2, and we get 1,7 x 107. From the fourth dilution, the result in table have less than 30, so we report the result as less than 300 and multiple with amount of dilution, but the real result have been included in parenthesis. 2. Table of colony amount in 10-5, 10-6, 10-7 dilution. Sample 10-5 10-6 10-7 Amount of colony 6 5 4

From the table above, we can see that in each dilution, there many shape of microorganism inside. They have many differences such as optical, shape, elevation, and margin. It showed that they are different.

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4.2. Discussion Based on the class data, in first sample in dilution 10-5 and 10-6 produce the number of colony between 30 until 300 and the comparing between a higher dilution and lower dilution is smaller or equal to 2, and we get 1,7 x 10 7. In second sample the amount of colony is 5,1 x 106. In third sample in dilution, in higher dilution (10-7) in second petri dish have no bacteria amount (TNTC), it can caused the number of colony is too much so that the media in petri dish looks so full and almost cover up by bacteria colony, the result is 2,4 x 10 6. In fourth sample in dilution, the result in table have less than 30, so we report the result as less than 300 and multiple with amount of dilution, but the real result have been included in parenthesis, and the result is 2,0 x 106. In the last sample, in higher dilution (10-7) in first petri dish have no bacteria, it can caused the number of colony is too much, the result is 5,5 x 106. If there is amount of bacteria colony near with 300, it can caused the number of bacteria in media has been reduced because the number of dilution is greater. But if there is amount of bacteria colony is near with 30, it can caused the number of bacteria in media has been reduced because the repeated dilution. The other factors are how to take sample and put it in Petri dish and homogeneous. In here, homogeneous using centrifuge. In that data, the most bacteria colony is occurred in fifth sample, 5,5 x 10 6 . its mean the number of bacteria is higher. Beside that, the least bacteria colony is occurred in fourth samples, 2,0 x 106 . It means the number of bacteria is lower. The different bacteria in both sample is the number of bacteria inside of sample. The threshold of a sample to determining it feasible or not feasible is <100 cfu/ml. From the data which have been we got, there are some sample that exceed from the provision of threshold, it can be caused there are too many bacteria which live in the sample, so it means that the water can not be consumed. The other data show there are some sample that less than the provision of threshold, but it does not mean that water can be consumed, because over there are still there are bacteria. Before the bacteria are characterized, the water will not be saved. The pathogenic bacteria, even it amount are small, but it has big effect to water quality. Standart plate count method, in this experiment using 10-5, 10-6, and 10-7 and it using petri dish to grow the microba or microorganism. From the observation result in 10-51, there are 3 bacteria colony which growing with different shape, elevation, margin, etc. In 10-52 , there are 3 bacteria colony which growing in different shape. In 10-61 , there are 3 bacteria colony which growing. In 10-62 , there are 2 kinds of bacteria colony which growing and have different color and shape. In 10-71, there are 2 kinds of bacteria colony which growing and have different size and optical view. In 10-72, there are 2 kinds of bacteria colony which

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growing and have different optical view, surface color, and size. It can be concluded that higher concentration of dilution can caused the bacteria growing only slightly or nothing at all.

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CHAPTER V CONCLUSSION 5.1. Conclusion Based on the experiment, we can conclude that the water quality in Ketintang Wiyata VII gutter is still there are bacteria, even it is in small amount but that small amount is still not able to be characterized. It is worried if that small amount is pathogenic bacteria such as E. Coli. To characterized the bacteria, there are some characteristics that are used. They are optical, shape, elevation, pigmentation, surface and margin. From that characteristics are gotten 15 kinds of colonies for all dilution. That is divided as 6 colonies in dilution 10-5, 5 colonies in dilution 10-6, and the last is 4 colonies in dilution 10-7. The colony kinds are only 15, but the amount of the bacteria is so many. There are 5 water samples and they have different amount of bacteria. It is caused of many factors. It can be caused of the environment surround the water sample is placed, the turbidity of water, and the activity surround the water sample is placed.

5.2. Suggestion It will be better if every 6 months is conducted this experiment to know the bacteria development and doing experiment to make the water in each gutter is free or less in bacteria. It will make the gutter in surround UNESA campus is cleaned and saved to be consumed or used.

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BIBLIOGRAPHY Cappuccino, J.G and N.Sherman. 1983. Microbiology: a Laboratory Manual. Adison-Wesley. Publishing company: California DEPKES RI. 1990. Peraturan Menteri Kesehatan No.416/MENKES/SK/IX/1990 tentang standar air minum. DEPKES RI. 2002. Keputusan Menteri Kesehatan Republik Indonesia

No.907/MENKES/SK/VII/2002 tentang syarat-syarat air minum. Dwidjoseputro, D. 1998. Dasar-Dasar Mikrobiologi. Djambatan : Malang. Fardiaz, S. 1992. Mikrobiologi Pangan 1. PT. Gramedia Pustaka Utama. Jakarta. Frobisher, Hinsdill, etc. 1974. Fundamentals of Microbiology: Saunders Company.USA. Lay, B. M. 1994. Analisis Mikroba di Laboratorium. PT. Raja Grafindo Persada: Jakarta Masud, Fajriyati. 2012. Kerusakan Bahan Pangan Oleh Mikroorganisme accessed. http://pharzone.com/materi%20kuliah/mikrob/12.ppt September 2012 at 5 pm. Pelczar, Jr etal. 1986. Dasar-Dasar Mikrobiologi: Universitas Indonesia (U I Press), Jakarta Pelczar, M.J.Jr, and E. Chan.1988. Dasar-dasar Mikrobiologi. Penerbit UI Press. Jakarta. p:23-24. Rahayu, A. (2011). DETEKSI ADANYA BAKTERI PADA AIR MINUM DALAM KEMASAN GALON. Article , 8. Schlegel H. G. & K. Schmidt. 1994. Mikrobiologi Umum. Gajahmada University Press. Yogyakarta. Sutedjo, M. 1996. Mikrobiologi Tanah. Rineka Cipta : Jakarta. Waluyo, L. 2007. Mikrobiologi Umum. UMM Press. Malang, p: 61-67. accesed on Saturday 26th

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Attachment

Figure 1. Making quadrant area

Figure 2. Pouring Agar media to Petri Dish

Figure 3. Enumeration step (counting bacteria on colony counter)

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