Journal of Xi'an University of Architecture & Technology ISSN No : 1006-7930

Growth Characteristics of and aureus on Alternative Medium Leaves of Lamtoro (Leucaena leucocephala)

Meidawati Suswandari*, Department of Primary School, Faculty of Teacher Training and Education, Universitas Veteran Bangun Nusantara, Sukoharjo, Indonesia

Lamtoro leaf has a high content. The protein content is very suitable for bacterial growth. Because of the high cost of bacterial growth media for educational and research institutions, lamtoro leaves can be used as an alternative medium for bacterial growth in general. The purpose of this study was to determine the potential of lamtoro leaf as an alternative medium for bacterial growth in general. This research is descriptive. Alternative mediums of lamtoro leaf were tested for the growth of Escherichia coli and . Escherichia coli bacteria grow on three alternative medium plates. After final identification, there are Escherichia coli bacteria. Whereas the Staphylococcus aureus bacterium did not grow on seven plates of alternative medium despite being incubated for 48 hours. Lamtoro leaf has less potential as an alternative medium for bacterial growth in general. The lamtoro leaf medium can only be used as a for gram-negative bacteria. While the growth of gram-positive bacteria there is no growth due to the presence of active substances in lamtoro leaves.

Key words: Leaves of Lamtoro, Alternative Media, Escherichia coli, Staphylococcus aureus

Introduction Bacteria are single-celled creatures that are very small or microscopic. Hans Christian Gram divides bacteria based on the characteristics of walls through the Gram system, namely Gram Positive and Gram Negative bacteria (Elferia, et al, 1996; Elliot, 2013; Harvey, 2001; Clausen, Gildberg, and Raa, 1985). Gram-positive bacteria have a thick layer of peptidoglycan and a thin lipid layer on the cell wall, so after Gram staining, Gram-positive

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bacteria will be colored purple by violet crystal dye. Examples of Gram-positive bacteria include , Staphylococcus sp., Staphylococcus sp., Etc. While Gram-negative bacteria have a thin layer of peptidoglycan and a thick lipid layer on the cell wall, then after Gram staining, Gram-negative bacteria will turn red by safranin coloring. Examples of Gram- negative bacteria include cereus, Clostridium botulinum, Escherichia coli, and so on (Elferia, et al, 1996).

Bacteria can live in nature, are grown and developed on a substrate, called a medium. Most bacteria will grow on an artificial medium. Under the right conditions (nutrition, temperature, and atmosphere), a bacterium will grow in size and then divide through the process of binary fission into two identical cells. Both of these cells are able to grow and divide at the same speed as stem cells provided that their environmental conditions remain stable (Elliot, 2013; Harvey, 2001). Besides requiring suitable conditions, bacteria also need nutrients for growth in the medium. Nutrition in growth must contain all the elements needed to make a new organism biologically (Clausen, Gildberg, and Raa, 1985). The required elements include sources, sources, sources, and mineral sources. Carbon sources can be organic carbon, carbon dioxide or carbohydrate groups because carbohydrates are composed of carbon atoms, hydrogen and oxygen. The carbon source is used to support the growth of fermentation or respiratory growth in many organisms. For the growth process, bacteria also need nitrogen because nitrogen is a major component of protein and nucleic acids, generally about 10% of the bacterial dry weight (Thomas, Keat, Walker, and Mackinnon, 2013). Like nitrogen, sulfur is a component of many organic materials in cells. While the mineral source is needed by bacteria for enzyme function (Elferia, et al, 1996).

From the classification of types of medium based on their form and function, the type of medium that is often used in educational, research and health service institutions is a general growth medium, for example, medium Nutrient , and Nutrien Broth. Nutrient Medium Agar is a type of general medium with a solid form that is widely used because many bacteria can grow on the medium and the growth of the colony can be seen macroscopically. In educational institutions in general or for research activities, the need for a general growth medium must be fulfilled. However, in order to provide this common growth medium, it sometimes encounters cost constraints. Another obstacle is the place that provides the growth medium, most of the distributors are located in big cities, so that the general growth medium such as Nutrients is difficult to obtain even more so if the inspection site is located in rural areas or places with difficult to access locations while the availability of the medium very needed. Then an alternative medium is needed to replace the medium as a medium for bacterial growth.

According to Leveau and Lindow (2005) and Willey, Sherwood, and Woolverton (2014), the best food base for bacterial growth is a medium that contains organic substances such as meat stew, vegetables, food scraps or herbs made by humans. Indonesia as an agricultural country has many sources of raw materials, including Lamtoro (Leucaena leucocephala) (Jones, 1979; Bairagi, Ghosh, Sen, and Ray, 2004). So far, lamtoro is often used by the community as a food supplement and as medicine. However, compared to lamtoro seeds, people often use lamtoro trees as reforestation, shade trees, prevent erosion or use wood sources of lamtoro trees as furniture.

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In terms of its function, many people use lamtoro seeds and trees for household or furniture industry needs. Whereas lamtoro leaf is only used by the community for animal feed. Although it can be used as animal feed, according to research conducted by Askar (1997) on ruminant animals and by Yurmiaty (2007) on rabbits, the results show that animal feed from fresh lamtoro leaves causes health problems in animals because there are anti-nutrient substances that are affect the work of enzymes in farm animals. So to overcome this problem, lamtoro leaves are used in other fields.

According to Poedjiadi dan Supriyanti (2005), in 100 g lamtaro leaves contain 12.4 g carbohydrates, 6.5 g fat, 12.0 g protein, 128 g calories, 1500 mg calcium ions, 100 mg ions and 2.5 mg iron ions . Seeing the presence of protein and carbohydrate content in lamtoro leaves, the lamtoro leaves have the potential as a medium for bacterial growth, because to support the growth of bacteria needed growth factors in the form of carbon and nitrogen sources found in lamtoro leaves in carbohydrate and protein content. In addition, the use of lamtoro leaves as an alternative medium for bacterial growth can also be a solution and a substitute for synthetic medium in the form of Nutrient Agar medium, which now has a quite expensive price for educational institutions and researchers.

Research Method This type of research is a descriptive study to describe the growth of Escherichia coli and Staphylococcus aureus bacteria on an alternative medium of lamtoro leaf. This research was conducted data collection to obtain information by presenting and analyzing data that has been collected or deliberately collected (Luknis, 2014).

Identification of Escherichia coli and Staphylococcus aureus Bacteria on Agar Nutrient Medium According to Waluyo (2008), identification of Escherichia coli bacteria can be done by carrying out a biochemical, confectionery and growth test on the Mc Conkey medium. The ose needle is fixed by spreading it to the flame. Pure bacterial inoculants in the were fixed by passing the test tube mouth to the fire. Escherichia coli bacterial inoculants were taken using ose needles and dipped or wiggled ose needles on indole, MR, VP medium and aseptic test sugars. Whereas in Simon Citrat and Mc Conkey medium, bacterial inoculants that have been taken are stripped on the surface of the medium aseptically. The test medium is labeled with the name of the bacterial species that has been cultured. The new culture was incubated in an at 37 °C for one night

Catalase, and culture tests on MSA medium The ose needle is fixed by spreading it to the flame. Pure bacterial inoculant medium is fixed by passing the test tube mouth to the fire. Staphylococcus aureus bacterial inoculants were taken using an ose needle and cultured or scratched on MSA medium on the surface of the medium. The medium was labeled and incubated 24 hours. In the test, inoculant bacteria Staphylococcus aureus was etched on the slide and then added 1-2 drops of H2O2. On the slide the object is noticed that there are small bubbles generated from the preparation. Whereas in the coagulase test, plasma citrate was dropped on a slide as much as 1 drop, then inoculant bacteria Staphylococcus aureus were taken one ose on the different ose and glass

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objects that had been labeled. Plasma citrate and bacterial inoculants were homogenized and the presence of fine lumps in the preparations was noted. Making Medium Nutrient Agar According to the quality control procedure contained in the Nutrient Agar packaging with the Oxoid brand, the making of the Nutrient Agar medium can be done by dissolving as much as 2.8 g of Nutrient Agar into 100 ml of distilled water in the Erlenmeyer flask while being heated on fire. During the heating process, the solution continues to stir until it boils. After boiling, the medium awaits the temperature down so it is not too hot. Then the tube mouth is closed using a tube stopper and coated with aluminum foil. The medium is sterilized in an autoclave at 121 ° C for 15 minutes. Sterile medium was transferred into sterile petri dishes with a volume of ± 20 ml in each aseptically in laminar air flow. The medium is waited for to cool and labeled the medium.

Making of Lamtoro Agar Leaf Medium According to research conducted by Nugroho (2016), the manufacture of lamtoro leaf decoction can be done by separating fresh young lamtoro leaves from the leaf stems. Lamtoro leaves are weighed using an analytical balance with a weight of 1 kg to 1 L distilled and refined. Then boiling is carried out at a temperature of 90 ° C for 20 minutes. Lamtoro leaf boiled water is filtered and squeezed using clean gauze and accommodated in a beaker. According to Deivanayaki and Iruthayaraj (2012), making alternative media can be done by weighing so that it weighs 15 grams for 1 L of lamtoro leaf cooking water. 1 L of lamtoro leaf boiled water is heated in an Erlenmeyer flask. Then added with 5 grams of NaCl and 15 grams so that the stem and stir until well blended. After the medium is homogeneous and boils, the medium is waited until it is not too hot. The mouth of the tube is closed using a tube stopper and coated with aluminum foil. The medium is sterilized in an autoclave for 15 minutes at 121 ° C. Sterile medium was transferred into sterile petri dishes with a volume of ± 20 ml in each petri dish aseptically in laminar air flow. The medium is waited for to cool and labeled the medium

Escherichia coli and Staphylococcus aureus Bacteria on Alternative Medium Leaves of Lamtoro Agar According to Samidjo (2014), bacterial culture can be done by ose needle fixation by spreading it to fire. Pure bacterial inoculants in the test tube were fixed by passing the test tube mouth to the fire. Bacterial inoculants were taken using ose needles and strips of the bacteria were made on an aseptic alternative lamtoro leaf medium. Alternative mediums of lamtoro leaf are labeled based on different bacterial species that have been cultured. The new culture was incubated in an incubator at 37 ° C for one night. The growth of Escherichia coli and Staphylococcus aureus colonies on the alternative medium of lamtor leaf was observed.

Results and Discussion Identification of Escherichia coli bacteria Identification of Escherichia coli bacteria is to ensure that the bacteria that will be inoculated are really E. coli bacteria. To identify E. coli, a series of Gram staining tests were carried out, culture on Mac Conkey medium, Indol test, MR test, VP test, Simmon , motion test on semi-solid medium and carbohydrate fermentation test. At the Gram staining stage, bacterial inoculants that have been stained, the staining results are observed under a

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microscope with a magnification of 10x100. The results of the staining showed red bacterial cells in the form of cocco bacill. The results of microscopic observations of Gram staining can be seen in Figure 1.

(a) (b) Figure 1 Gram staining (a) Escherichia coli and (b) Staphylococcus aureus under a microscope at 10x100 magnification Gram staining is used to separate groups of bacteria. According to Hans Cristian Gram, bakeries are divided into two groups based on the characteristics of the cell wall, namely Gram positive and Gram negative. In bacteria that have been done Gram staining, it was found that E. coli bacteria were Gram negative bacteria with the results of red Gram staining, while S. aureus bacteria were Gram positive bacteria. When adding the first basic dye, violet crystal, transparent colored bacteria will be colored purple. To maintain this color, a lugol solution is added, then an allohol alcohol solution is added. The addition of 96% alcohol is used to dissolve the lipid layer on the bacterial cell wall. Gram-positive bacteria have a thin lipid layer, so after adding 96% alcohol, only a few lipid layers are dissolved so that even a few pores of the cell wall are open. So the coloring agent that has colored the bacteria only dissolves in small amounts. While Gram negative bacteria have a thick lipid layer, then after adding 96% alcohol, many lipid layers dissolve so that many pores of the cell wall are open. Then the coloring agent that has colored the bacteria can come out through the pores of bacteria that have been opened. This causes Gram negative bacteria to be transparent. To color the bacterial cell, a red safranin base dye was added. In bacterial culture in Mac Conkey medium, the results showed that the growth of the bacterial colony was pink, shiny and convex elevation. Macroscopic observations can be seen in Figure 2. In the Mac Conkey medium, there is a colony of pink E. coli bacteria. The presence of pink is because the E. coli bacteria can ferment . This medium is equipped with carbohydrates (lactose), bile salts and as a pH indicator that is able to distinguish enteric bacteria as the basis for its ability to ferment lactose. E. coli bacteria produce acid from lactose fermentation and precipitation of bile salts followed by absorption of neutral red.

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Figure 2 Colonies of Escherichia coli bacteria on Mac Conkey Medium which have been incubated for 24 hours at 37 ° C At the stage of culture in the IMViC medium, it was found that the indole medium obtained positive results because the indicator formed a pinkish ring-like layer on the medium surface. In the MR medium, a positive test result is obtained because the color changes in the medium to red. Whereas the VP and Simmon Citrate does not change color. In the urea medium, negative results showed no color change in the test medium. As for the semi-solid medium there is a white culture on the puncture marks during inoculation, meaning that the inoculated bacteria can move or have motility. In the TSIA medium there is a color change in the medium. The culture results in the medium are yellow in color on the whole media and positive gas with the agar being lifted from the bottom of the medium.

1 2 3 4 5 6 7

Figure 3 Biochemical Test Results (IMViC) on Escherichia coli isolates used in the study

Information : 1. Test results on the Indol medium 2. Test results on MR 3. Test results on VP medium 4. Test results in Urea medium 5. Test results on the Simmon Citrate medium 6. Test results in Semi-solid medium 7. Test results on the TSIA medium

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IMViC test uses indole, MR, VP and Simmon Citrate medium. The IMViC test is used to distinguish between bacteria, based on their ability to ferment and lactose, the breakdown of tryptophan which produces indole and the presence of permease citrate enzymes capable of breaking down sodium citrate from the specific medium used. In indole medium, E. coli bacteria show color changes with an indicator of the presence of a red ring on the top surface of the medium. This is because the E. coli bacteria can produce indole using the enzyme tryptophanase (Hallman, et al., 1997; Backman and Sikora, 2008). E. coli bacteria can utilize amino acid tryptophan. The amino acid tryptophan when hydrolyzed by the enzyme tryptophanase will produce indole, pyruvic acid and para-benzaldehyde compounds which are not soluble in water, thus forming red compounds on the surface of the medium. In the MR medium, there is a change in the color of the medium to red, because the E. coli bacteria can produce and maintain a stable acid final product from glucose fermentation, so that in an acidic state the MR medium will turn red (Backman and Sikora, 2008). In the VP medium, there was no discoloration in the medium after inoculation of E. coli bacteria. This is because the E. coli bacteria cannot produce 2.3 butanadiol. In the medium Simmon Citrate bacteria E. coli also there is no change in color in the medium. Because, E. coli bacteria cannot use citrate as the only source of carbon and energy. The urea test showed negative results, because E. coli bacteria cannot hydrolyze urea and do not have the enzyme. The results of observations on the E. coli bacterial TSIA test showed yellow, positive gas and negative H2S results. This means that E. coli bacteria can ferment glucose, lactose and sucrose. Fermentation results are indicated by the presence of gas at the bottom of the medium. Whereas in the motility test, E. coli bacteria can move with the presence of colony growth in the former stabbing semi-solid medium (Hallman, et al., 1997). In the carbohydrate fermentation test stage, 5 types of carbohydrates are used, namely glucose, sucrose, lactose, maltose, mannitol. Before inoculation, the medium is blue because an indicator solution in the form of BTB (Bromtimol Blue) was added. But after inoculation and incubation, the color of the medium changes to yellow, so that positive results are obtained in the carbohydrate fermentation test. The observations can be seen in Figure 4. In the carbohydrate fermentation test, five types of carbohydrates are used, namely glucose, lactose, sucrose, maltose and mannitol. Before inoculation, the carbohydrate medium was blue due to the presence of the Brom Timol Blue (BTB) indicator in the medium. The results obtained after inoculation of E. coli bacteria is that there is a change in the color of the medium to yellow. The yellow color is due to E. coli bacteria fermenting carbohydrates, the atmosphere will become acidic and the BTB indicator will turn yellow (Toelle, 2014).

1 2 3 4 5 Figure 4 Biochemical Test Results (Carbohydrate Fermentation Test) on Eschericia coli Isolates used in research

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Information : 1. Results of glucose fermentation test culture 2. Sucrose fermentation test culture results 3. Lactose fermentation test culture results 4. Maltose fermentation test culture results 5. Results of mannitol fermentation test culture After the Gram staining test, culture on the Mac Conkey medium and biochemical tests on the isolates to be used in the study, suitable results were obtained according to Hallman, et al. (1997). Escherichia coli is a Gram negative bacterium in the form of cocco bacill which can ferment lactose, produce acids and gases, indole and positive, negative oxidase, do not use citrate as an energy and motile source, while other tests give varied results.

Identification of Staphylococcus aureus bacteria Identification of Staphylococcus aureus is to ensure that the bacteria that will be inoculated are really Staphylococcus aures. To identify S. aureus bacteria, it was carried out through Gram staining, culture on Mannitol Agar medium, catalase test and coagulase test. In inoculants which have been tested for Gram staining, microscopic results were obtained with a magnification of 10x100, namely the presence of purple bacteria with coccus shape and clustered like grapes (see Figure 1). Then the growth test in medium, showed the results of the growth of yellow and shiny colonies. Macroscopic examination results can be seen in Figure 5. Medium Mannitol Salt Agar is a medium with a high salt concentration and can inhibit bacterial growth, except Staphylococcus bacteria. This media contains mannnitol carbohydrates and phenol red (pH indicator) as indicators to detect the presence of mannitol fermented acids. Staphylococcus bacteria can show a yellow colony zone around its growth, while Staphylococcus bacteria that cannot ferment so that there are no yellow zono colonies (Lugtenberg, Kravchenko, and Simons, 1999).

Figure 5 Staphylococcus aureus Bacteria Colony on Mannitol Salt Agar Medium

The next identification test is the catalase test. Bacterial inoculants placed on the slide are given a hydrogen peroxide solution and homogenized. The catalase test results show the presence of bubbles in the preparation, meaning that the results are positive because the S aureus bacteria can convert H2O2 to H2O and O2. Then proceed with the coagulase test using plasma citrate. Inoculant bacteria were added with plasma citrate and homogenized. Obtained

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positive results, namely the formation of coagulation or lumps on the slide. The observations can be seen in Figure 6. The catalase test is used to detect the presence of the cytochrome oxidase enzyme. After 3% H2O2 solution is dripped on the slide, and a small amount of bacterial growth is placed in the solution. Bubble formation (release of oxygen) shows positive results. Staphylococcus bacteria can produce catalase that converts H2O2 into water and oxygen. The catalase test can be a test to distinguish Staphylococcus bacteria which are catalase positive and Staphylococcus bacteria that are negative in the catalase test (Elferia, et al, 2014).

Figure 6 Results of Staphylococcus aureus Bacteria Catalase Test that will be used in the study

After the Gram staining test stage, culture on Mannitol Salt Agar medium, catalase test and coagulase test on the isolates to be used in the study, obtained the appropriate results according to Toelle (2014). The isolates which had been done were Gram staining in the form of coccus and colored purple, followed by the catalase test. Staphylococcus aureus will form air bubbles and in the coagulase test, the bacteria can coagulate plasma citrate. Coagulase test is used to detect protein A and clotting factors. S.aureus produces extracellular coagulase, an enzyme-like protein that freezes plasma oxalates or crates. Coagulase is bound to prothrombin, which together become enzymatically active and start fibrin polymeration. Coagulase may precipitate fibrin on the surface of Staphylococcus, it may alter its digestive process by phagocytic cells or its destruction in these cells (Elferia, et al, 2014).

Figure 8 Results of Staphylococcus aureus Bacterial Coagulase Test that will be used in research

Test results for E. coli and S. aureus bacterial growth on Lamtoro agar leaf medium After an alternative medium was made from lamtoro leaves, each bacterial species was cultured in the medium and observed after 24 hours incubation in the incubator at 37 ° C. After being incubated for 24 hours, a macroscopic observation of the growth of Escherichia coli and Staphylococcus aureus bacteria on an alternative medium was carried out. Observational data are presented in Table 1 and Figure 10.

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Table 1 Bacterial Growth in Lamtoro Leaf Medium

The observed Escherichia coli Staphylococcus aureus aspect Nutrient Lamtoro Nutrient Lamtoro Agar agar leaves Agar agar leaves Shape Round Round Round - Color Milky white Chocolate Milky white - Elevation Convex Convex Convex - Edge Align Align Align -

In the medium of lamtoro leaf control, there was no bacterial growth after incubation at 37 ° C for 24 hours. The observations can be seen in Figure 9.

Gambar 9 Medium Kontrol Daun Lamtoro

Pertumbuhan E. coli pada Pertumbuhan E. coli Pertumbuhan E. coli medium daun lamtoro 1 pada medium daun pada medium daun lamtoro 2 lamtoro 3

Figure 10 Escherichia coli Bacteria Growth on Alternative Lamtoro Leaf Medium

In the alternative medium of lamtoro leaf, inoculated E. coli bacteria by means of the quadrant streak and repeated three times. In all three mediums, there is a growth of bacterial colonies with a brownish color, round in shape with flat edges and convex elevation. The colony grows in two quadrant parts and a single colony is obtained. The results of macroscopic examination can be seen in Table 1.

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To find out the species of bacteria that grows is E. coli bacteria, the final identification is in the form of Gram staining. After the Gram staining test, microscopic results resembling those of 1. The bacteria that grow on the alternative medium are red and in the form of cocco bacill (see Figure 11).

Gram staining of Gram staining of Gram staining of E. coli Escherichia coli bacteria Escherichia coli bacteria on bacteria on lamtoro leaf on lamtoro leaf medium lamtoro leaf medium medium (3) (1) lamtoro leaf medium (2)

Figure 11 Results of Gram Staining of Escherichia coli Bacteria on Alternative Medium of Lamtoro Leaves

On the alternative medium, lamtoro leaf inoculated with Staphylococcus aureus was also repeated three times. Bacterial inoculation is done by quadrant streak. In the three alternative media, there was no bacterial growth after inoculation and incubation at 37 ° C for 24 hours. In order to obtain accurate results regarding the growth results of S. aureus bacteria, all three bacterial inoculants in the medium were re-incubated at 37 ° C for 24 hours. But there is still no bacterial growth in the alternative medium. Then repeated with inoculating S. aureus bacteria on three alternative media and incubating at 37 ° C for 48 hours. The same results were obtained for repetition of the inoculation. The observations can be seen in Figure 12.

Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus growth on lamtoro leaf growth on lamtoro leaf growth on lamtoro leaf medium (1) medium (2) medium (3)

Figure 11 Growth of Staphylococcus aureus in Alternative Medium of Lamtoro Leaves

After 24 hours incubation time was achieved, macroscopic observations were made in the form of colors, shapes, elevations and edges of the colony. In the control medium and bacterial inoculant medium Staphylococcus aureus there was no bacterial growth. The shape

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and color of the alternative medium did not change as before bacterial inoculation. Whereas in the inoculant medium Escherichia coli there is a very large growth of the colony. The colony is brownish, round in shape, with a convex elevation with flat edges. Each Escherichia coli inoculant on the alternative medium was carried out the final identification, namely the Gram staining differentiation test. The staining of the three bacteria showed the same results after microscopic observation. The three preparations contained red Gram-negative bacteria in the form of cocco bacill. This shows that the species that grow on these alternative mediums are Escherichia coli species. In this study, the use of lamtoro leaf as an alternative medium was considered less optimal because this alternative medium can only be used to see the growth of Gram negative bacteria only. In terms of nutrition, lamtoro leaves are suitable for the growth of E. coli and S. aureus bacteria, but there are differences in the results of growth in the two species of bacteria due to the active substance content in lamtoro leaves and the different types of Gram bacteria. Lamtoro leaf contains active substances in the form of alkaloids, saponins, flavonoids, tannins, mimosin and leukins. Where these substances are able to inhibit and suppress the rate of microbial growth (Andika, 2018). The secondary metabolites of flavonoids are polar, so they easily penetrate the peptidoglycan layer of Gram-positive bacteria that are also polar, so S. aureus bacteria are more sensitive to flavonoids (Karlina, Ibrahim, and Trimulyono, 2013). The Gram positive bacterial layer has a peptidoglycan structure, less lipids and theatricic acid. Theatricic acid is a water-soluble that is polar in nature. So the flavonoid compound is easier to penetrate the polar peptidoglycan layer than the nonpolar lipid layer as in the Escherichia coli bacteria. The outer wall of E. coli has high permeability, the active substance contained in the extract cannot enter the bacterial cell, and the bacteria is not damaged or stunted, so E. coli can utilize protein in the form of amino acids to its growth (Karlina, Ibrahim, and Trimulyono, 2013). In addition to flavonoid compounds, other compounds such as tannins can also inhibit the growth of Gram-positive bacteria. This is because the active ingredient of tannins has hydroxyl groups (-OH) which are polar as flavonoid compounds (Sari, Rita, and Puspawati, 2015). So that Gram positive bacterial cells can be damaged because of the two active compounds.

Conclusion Lamtoro leaf has less potential as an alternative medium for bacterial growth in general. The lamtoro leaf medium can only be used as a growth medium for Gram negative bacteria. While the growth of Gram-positive bacteria there is no growth due to the presence of active substances in lamtoro leaves.

Acknowledgements

The researcher would like to thank Lembaga Pengelola Dana Pendidikan (LPDP) Indonesian endowment fund for education, Finance ministry of Indonesia for supporting this research.

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Thomas, L., Keat, S., Walker, A., and Mackinnon, R. (2013). Microbiology And Infectious Disease On The Move. Jakarta: Indeks. Toelle, N. N. (2014). Identifikasi dan karakteristik Staphylococcus dan dari infeksi ovarium pada ayam petelur komersial. Journal of Animal Science Padjajaran University. 1(7): 32 – 37. Waluyo, L. (2008). Teknik Dan Metode Dasar Dalam Mikrobiologi. Malang: UMM Press. Willey, J. A., Sherwood, L. M., and Woolverton, C. J. (2014). Prescott’s Microbiology. 9th Ed. New York: Mc Graw Hill. Yurmiaty, H. (2007). Penggunaan daun lamtoro dalam ransum terhadap produksi pelt dan kerontokan bulu kelinci, Journal of Animal Science Padjajaran University. 7(1): 73-77.

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