517

Antibacterial Activity of Extractof Non-Soybean Tempe

Widaningrum1, Endang Yuli Purwani1, Veni Issani2, Brata Abdalla2

1Indonesian Center for Agricultural Postharvest Research & Development Jl. Tentara Pelajar 12, Bogor 16114, Indonesia 2Food and Technology Department, Bogor Agricultural University Jl. Raya Dramaga, Bogor 16680, Indonesia

Keywords: Non-soybean tempe, hyacinth, jakbean, antibacterial activity

Abstract Soybean is one type of legume that is used in Indonesia mostly for processing it

into tempe. There was imbalance between demand and production of soybean in Indonesia. In 2011, national soybean demand was about 2.4 million tonnes, while soybean production was reduced from 974.51 thousand tons of dry beans in 2009 to 907.03 thousand in 2010 (BPS, 2011). This condition has forced Indonesia to import large quantity of soybean to meet local demand. In the current study, hyacinth (Lablab (L.) purpureus) and jackbean (Canavalia ensiformis L.) were selected as indigenous sources as substrate for tempe production. The objective of the study was to explore antibacterial activity of hyacinth and jackbean tempe. Research include the manufacture of soybean tempe and hyacinth and jackbean tempe, hyacinth and jackbean extraction, preparation of bacterial culture test, testing the antibacterial activity of soybean extract (well diffusion method), and the Minimum Inhibitory Concentration test (MIC) of those extract. Research results showed that hyacinth and jackbean tempe had visual appearance looks like soybean tempe, which has whte mycelium built a compact structure of tempe. Unlike extract of soybean tempe, hyacinth and jackbean tempe had not been proven to have inhibitory power neither against the bacteria S. aureus nor to E. coli. Insufficient evidence suggest that the antibacterial components in soybean is a protein degradation product of the raw material bean. It is suspected that soy has certain components that are not owned by hyacinth and jackbean that can be metabolized by the tempe molds to produce compounds that has antibacterial activity.

INTRODUCTION Legume play a vital role as protein sources in developing countries such as

Indonesia. Soybean is one type of legume that is used in Indonesia mostly for processing it into tempe. Tempe is a traditional fermented food in Indonesia and it is mainly derived from soybeans. In Indonesia, more than 80% of soybean is processed into tempe and tofu. However, there was the imbalance between demand of soybean for tempe production and low production of soybean in the country. In 2011, national soybean demand was about 2.4 million tonnes, while soybean production was reduced from 974.51 thousand tons of dry beans in 2009 to 907.03 thousand in 2010 (BPS, 2011). This condition has forced Indonesia to import large quantity of soybean to meet local demand. Use of indigenous sources to completely substitute for soybean in high quality tempe production would help Indonesia to reduce dependence on expensive soybean import.

Researchers have tried to incorporate non oil-seed legume such as pigeon pea, cowpea and tribal bean in the production of tempe (Damardjati and Widowati, 1995; Indrasari et al., 1992, Richana and Damardjati, 1999, Purwani et al. 2006, Haliza et al. 2007,

518

Djaafar et al., 2010). In the current study, hyacinth (Lablab (L.) purpureus) and jackbean (Canavalia ensiformis L.) were selected as substrate for tempe production due to its great potential to be developed. Hyacinth and jackbean are considered to be dispersed legume crop. They grow under many different ecological conditions, and show no marked seasonality compared to soybean. Both legumes were cultivated on marginal land especially in Central Java, East Java and Aceh. The objective of the study was to explore antibacterial activity of hyacinth and jackbean tempe.

MATERIALS AND METHODS

Materials The materials used in this study were hyacinth beans, jackbeans, soybean varieties

Grobogan, tempe yeast produced by PT. RAPRIMA Bandung, methanol, distilled water, Escherichia coli, Staphylococcus aureus ATCC 25923, Nutrient Broth (NB), Nutrient Agar (NA), Tryptone Soy Broth (TSB), Kovacs reagent, Methyl Red Voges Proskauer Broth (MRVP), a solution of alpha-naphthol, potassium hydroxide (KOH), Methyl red indicator, Koser Citrate Broth, Baird-Parker Agar (BPA), crystal violet, safranin, Lugol, 90% ethanol, dimethyl sulfoxide (DMSO), and amoxicillin.

The tools used in this study include boiling pot, steamer pot, fermentation rack, fermentation packaging (plastic PP), 35°C fermentors, blenders, shaking incubator, vacuum filters, rotary evaporator, drying oven, Chromameter (CR 300 Minolta), penetrometer (Precision), pH-meters, spectrophotometer (UV Kross 6500), test tubes, petri dishes, hemasitometer, micropipette, microscopy, and 37°C incubator.

Research Method Research include the manufacture of soybean tempe and hyacinth and jackbean

tempe, moisture content analysis, soybean extraction, hyacinth and jackbean extraction, preparation of bacterial culture test, testing the antibacterial activity of soybean extract (well diffusion method), and the Minimum Inhibitory Concentration test (MIC) of the extract of hyacinth and jackbean tempe.

Production of Hyacinth and Jackbean Tempe (modified method of van den Hil, Nout 2011)

The process begins with immersion of hyacinth and jackbean in water for 12 hours, drying in the drying oven with temperature of 70°C for 24 hours. Hyacinth and jackbean were dried and peeled with a mechanical peeler to separate the seeds and skin. Dried hyacinth and jackbean without skin then were boiled in boiling water for 15 minutes and soaked for 50 hours in water. Hyacinth and jackbean then subsequently washed, steamed for 10 minutes, drained, and cooled at room temperature (29 to 31°C). Then innoculated with 0.1% of yeast (0.1 grams to 100 grams of steamed yeast). Hyacinth packed in a PP plastic that has been perforated with a distance of 2 cm and incubated in a fermenter at temperature of 35°C ± 1°C for 24 to 36 hours.

Tempe Extraction (modified method of Pawiroharsono 1995) A total of 20 grams of tempe that had been crushed in a blender then extracted with

100 ml of absolute methanol, and the extract obtained tempe stored in a freezer to coagulate fat. Fat then was separated by vacuum filtration. Filtrate then evaporated with rotary evaporators at 40° C to obtain the methanol extract. Extracts stored in the cold room (4°C)

519

until the time of testing. Soybean extract obtained is used for further research. The yield of extraction expressed in ratio of weight of tempe’s extract to weight of tempe extrated (%w/w).

Preparation of Testing Culture Bacteria This stage aimed to ensure an uniformity of testing culture bacteria which will be

used on the research.

Preparation of Bacteria S. aureus and E. coli Testing culture bacteria were accepted in the form of bacteria growing on agar slant

culture Nutrient Agar (NA). Before use, a loop culture from agar slant culture NA was innoculated in 10 ml of liquid NB medium and incubated at 37° C for 24 hours. This culture was used as the working culture in each test. Escherichia coli purity test done by Gram Staining test, morphology and biochemical tests such as indole test, Voges Proskauer test, methyl red test, and test citrate (IMViC) (SNI 01.2332.1:2006). Staphylococcus aureus purity test performed by growing cultures in BPA media that had been added egg yolk and Gram Staining test. Calculation of total bacterial count done by total plate count method.

Preparation of Standard Curve by Turbidimetry Method (Waluyo, 2008) The number of bacteria was calculated by spectrophotometric method i.e. the

measurement of turbidity (Optical Disturbance (OD)) cultures with a spectrophotometer. Optical density of the suspension does not directly indicate the number of cells in the population but indicates the amount of light transmitted by the population. To obtain the number of bacteria, the OD values should be compared with the number of microbes (CFU/ml) (Waluyo 2008), by making a standard curve with the x-axis as the number of bacteria (CFU/ml) and the y-ordinat as OD. Once the standard curve is obtained, a large number of similar organisms can be measured quickly by measuring their turbidity and concentration. Serial dilutions of 1:1, 1:2, 1:4, 1:8, and 1:16 were made from 20-24 hours bacterial age, and the OD was measured at a wavelength of 620 nm.

Preparation of Testing Culture Bacterial Growth Curve (Khodijah et al. 2006) Preparation of testing culture bacterial growth curve was performed to determine the

optimum incubation time for each testing culture bacteria to reach the end of the log phase. A loop of pure culture of agar slant NA was innoculated in 10 ml NB and incubated at 37˚ C. Cell mass culture in NB calculated using the standard curve and then diluted to obtain cultures with a dilution rate of 104. Respectively 1 ml of culture were included in 10 ml of NB and then incubated in an shaking incubator at 37ºC (120 rpm). The growth of the culture was observed by measuring OD using a spectrophotometer at wavelength of 620 nm, with an interval of 60 minutes for 24 hours to enter stationary phase.

Samples Negative Testing Negative test of samples conducted by innoculating extract of samples (100%

concentration) in 1 ml of medium into the NA with pour method, then incubated for 24 hours at 37oC.

Testing of Antibacterial Activity (modified method by Zuhud et al. 2001) Testing of antibacterial activity of hyacinth and jackbean tempe were done using well

diffusion test. Tempe extracts were tested its effectiveness against two bacteria;

520

Staphylococcus aureus and Escherichia coli with total microbes in the plate about 105 colonies/ml. Total microbial confirmed with a standard curve. Dimethyl sulfoxide (DMSO) was used as negative control, since this chemical substance was able to dissolve non-polar and polar compounds. Amoxicillin was used as positive control. This antibiotic substance is an antimicrobial classified on penicillin class that has antimicrobial mechanism by inhibiting synthesize of cell wall of bacteria (Black 2005). Penicillin is a broad spectrum antibiotic and is usually effective against Gram-positive and Gram- negative strains, especially S. aureus and Enterococci are usually cause gastrointestinal infections (Parhusip 2006).

Antibiotics were dissolved in DMSO at a concentration of 0,01% for the bacteria S. aureus and at 0.05% for the bacteria E. coli. The concentration of extract used was 100% (without dilution), 40 µL respectively. Testing culture bacterial that had been refreshed (reached late log phase) diluted to a concentration of about 105 colonies/ml then pipetted about 1 ml onto a sterile petri dish and inasmuch as 20 ml of sterile liquid NA (in test tubes) then poured to a petri dish containing bacterial suspension, homogenized, and allowed to freeze. After the agar freezed, 5 wells then made and the sample then poured into each well, i.e. 40 µL soybean tempe on the 1st hole, 40 µL of market soybean tempe on the 2nd hole, 40 µL of hyacinth tempe on the 3rd hole, 40 µL of DMSO on the 4th hole, and 0.01% µL of amoxicillin 0:01% on the 5th hole. The similar method was conducted to the jackbean tempe. The plate was then incubated at 30°C for 24 hours with the plate position not reversed to allow the extract to diffuse into the agar, followed by incubation at 37°C for 24 hours, and measurement of the diameter of the inhibitory area.

RESULTS AND DISCUSSION

Production of Tempe To produce expecting seeds that are easy to peel, hyacinth needs 15 minutes boiling

time, jackbean 20 minutes, and soybean needs 40 minutes. Those texture will facilitate the process of immersion. Immersion process aimed to hydrate seeds and let the lactic acid fermentation occurs naturally to achieve the degree of acidity that is suitable for tempe mold to grow, i.e. pH 3.5 until 5.2, i.e. pH 3.5 to 5.2 (Hermana and Karmini 2001). In the immersion process, lactic acid bacteria (LAB) would be easy to use nutrition obtained from legumes (Syarif et al.1999).

For soaking time, hyacinth and jackbean need 50 hours and soybean needs 30 hours. According to Hermana and Karmini (2001), a long time soaking of soybeans traditionally is 20-30 hours. Soaking of soybeans was faster than hyacinth and jackbean because soybean was soaked with their skin so that the number of LAB were abundant that played a role in soaking process. Seeds will be hydrated during the process of boiling, soaking and steaming.

The measurement results indicated that the average of water absorption of hyacinth tempe was 68,98% and jackbean tempe was 72,54% meanwhile soybean tempe was 63,92%. The results showed that the more water absorbed by the bean will produced even greater yield of water absorption. Water absorption by seeds mainly occurs during the immersion. Hyacinth and jackbean immersion time were longer than the time of soaking so that the amount of water absorbed by hyacinth and jackbean would many more. Hyacinth and jackbean tempe had visual appearance looks like soy tempe, white mycelium grains of beans with a compact built and had normal scent. Visualization of hyacinyh, jackbean and soybean tempe were presented in Fig. 2 and 3.

521

Extraction Process of Tempe During tempe fermentation, the beans macromolecules such as carbohydrates,

proteins, and fats were broken down by tempe molds into smaller components. Antibacterial compounds in soybean probably derived from the breakdown of proteins by tempe molds (van den Hil, 2011). Results of bean protein breakdown by tempe can be peptides and amino acids. Pawiroharsono (2001) suggested that further studies to determine the chemical structure of antibacterial compounds of tempe had done with the polar solvent methanol. Methanol and water had polarity of 0.73 and 0.9, respectively (Moyler 1995), the higher the polarity of the compound, the polar the substance will. Methanol can extract groups of compounds, especially sugars, amino acids, and glycosides (Houghton and Raman 1998).

Preparation of Testing Bacteria Cultures Staphylococcus aureus and Escherichia coli Bacteria S. aureus and E. coli which will be used was first tested for its purity.

Visualization of S. aureus on BPA media and Gram stain of S. aureus is presented in Fig. 4 and Fig. 5.

Colonies that grew on media BPA was round, black, and there was a clear zone (halo) around the colony. Gram staining results showed cell purple (Gram positive) and round shape with a clustered arrangement. Both of these results showed that the test bacteria was S. aureus (SNI 2011, Fardiaz 1989). Bacteria E. coli to be used in testing has to follow the purity tested first. Visualization of E. coli on media EMBA and Gram stain E. coli is presented in Fig. 6 and Fig. 7.

Colonies that grew on the medium metallic green (EMBA) showed bacteria belonged to Enterobacteria. To ensure that the correct test bacteria was Entrobacteria, the IMViC test was conducted. Gram staining results showed red cells (Gram negative) and short rod-shaped with a single arrangement. These three results indicate that the test bacteria was E. coli (SNI 2006, Fardiaz 1989).

The number of bacterial cells was calculated by the method of turbidimetry using a spectrophotometer at a wavelength of 620 nm. Standard curves for the bacteria S. aureus is presented in Fig. 8 and for the bacteria E. coli is presented in Fig. 9.

Bacteria used on antibacterial activity was at the end of the log phase. According to Parhusip (2006), in log phase, cells in a state of high metabolic activity (labile) so it is more sensitive to tempe’s extract tempe and more easily to be damaged. At the end of the log phase, cells usually adapt to unfavorable environmental conditions. The rate of cell metabolism decreases in order to avoid the end-up of limited nutrient quickly. In unfavorable conditions the cells will have the highest resistance to antibacterial compounds (Purwoko 2009). End of log phase bacteria S. aureus occurred at 19 hours that marked by peak marked by the growth of bacteria. The number of bacteria was estimated to approximately 1010 CFU/ml. The growth curve of S. aureus is presented in Fig. 10, meanwhile end of log phase of E. coli occurs at 14 hours that marked by peak marked by the growth of bacteria. The number of bacteria was estimated to approximately 1010 CFU/ml. Growth curve E. coli is presented in Fig. 11.

Negative Test of Sample Negative test results showed that the extract of hyacinth tempe, jackbean tempe,

soybean tempe, and DMSO did not contain microbes that can be directly used in the next test.

522

Antibacterial Activity Testing Measurement of antibacterial activity was carried out by well diffusion method.

When the compound tested was able to inhibit the growth of bacteria, it will show a clear zone around the well (inhibition zone). The total area of this lymph is correlated with the work force of antibacterial size (Waluyo 2008). The ability of the extract to inhibit the growth of bacteria in the sample test is presented in Table 1.

Top of Form Soybean extract and market soybean extract had inhibitory activity against S. aureus

(Gram positive) but has no inhibitory activity against E. coli (Gram negative), these results together with the results of previous studies by Kobayashi et al. (1992) which stated that the soybean extract has inhibitory activity against S. aureus but did not inhibit Gram-negative bacteria such as E. coli. Differences in the ability of antibacterial compounds inhibit Gram positive and Gram negative due to differences in the structure of the cell wall of bacteria, Gram- positive bacterial cell wall more easily penetrated by antimicrobial agents. Purwoko (2009) stated that Gram-positive bacterial cell wall is composed mostly of peptidoglycan layers and acid teicoic so it is easy to be passed by a hydrophilic component. Gram negative bacteria has cell walls that having more complex outer membrane which protects the peptidoglycan layer, namely lipopolysaccharide (LPS). Suliantari (2009) explained that in Gram-positive bacteria, antimicrobial materials can be directly entered and will fill the peptidoglycan layer and then binds to the protein, further causing bacterial cell lysis while in Gram-negative bacteria. Such materials (antibacterial material) enter through the porin found in the outer layer (LPS), then entering the peptidoglycan layer and make bonds with proteins.

Extract of market soybean had higher inhibitory activity against S. aureus compared to soybean extract and hyacinth tempe and jackbean tempe extract. Nothing found by the inhibitory activity of soybean extract against S. aureus and E. coli.

Extract of hyacinth and jackbean tempe had no antibacterial activity againts both testing bacterial culture. It also explained that the antibacterial component found in soybean are not solely the result of protein degradation by tempe molds during fermentation but also influenced by the type of beans which are used as raw materials. Components in soybeans which were instrumental in the formation of antibacterial compounds is presumably not present in hyacinth and jackbean. In other words, the differences of inhibiting ability of each extract is suspected due to different chemical compounds of each of the extracts. These results are in accordance with the opinion of Naidu (2000), which stated that the antimicrobial activity is influenced by several factors including the chemical composition of the substances.

At higher incubation temperatures i.e. 37°C, the inhibitory activity of extracts of soybean tempe and market soybean tempe were not detected. This is presumably because the antibacterial component in the soybean extract was heat labile so it did not have antibacterial activity when the incubation temperature was raised.

Another suggestion which emerged that antibacterial component in the soybean extract has bacteriostatic effect, which inhibits the growth of bacteria but did not kill the bacteria, so antibacterial (inhibitory) activity was found on 30°C for 24 hours incubation but not found while the incubation was continued for 48 hours. It is also suspected that the extract used still contains a high water content, so it needs to be concentrated to obtain the ability to inhibit bacterial extract greater. This is consistent with the statement of Waluyo (2008) which reported that antimicrobial substances can be bacteriostatic at low concentrations but shall be bactericidal in high concentrations.

523

CONCLUSION AND RECOMMENDATION Hyacinth and jackbean tempe had visual appearance looks line soybean tempe, which

white mycelium grains of beans built a compact structure of tempe. Unlike extract of soybean tempe, hyacinth and jackbean tempe had not been proven to have inhibitory power neither against the bacteria S. aureus nor to E. coli. Insufficient evidence suggest that the antibacterial components in soybean was a protein degradation product of the raw material bean. It is suspected that soy has certain components that are not owned by hyacinth and jackbean that can be metabolized by the tempe molds to produce compounds that has antibacterial activity.

Hyacinth and jackbean tempe has similar appearances with soybean tempe, so it deserves to be developed further. Further research on the characterization of antibacterial components in soybean tempe needs to be done in order to know which compounds actually act as an antibacterial in tempe.

Literature cited Syarief R, Hermanianto J, Hariyadi P, Wiraatmadja S, Suliantari, Dahrulsyah, Suyatna NE,

Saragih YP, Arisasmita JH, Kuswardani I, Astuti M. 1999. Wacana Tempe Indonesia, Yee YB, Basry AA, Puruhita A, Supriyono, editor. Surabaya: Universitas Katolik Widya Mandala.

[FAO] Food and Agriculture Organization. 2012. Monthly Price and Policy Update: Oilseeds, Oils, & Meals no. 32. [terhubung berkala]

http://www.fao.org/fileadmin/templates/est/COMM_MARKETS_MONITORING/Oilcrops/Documents/MPPU_Feb_12.pdf [30 Desember 2012].

Kay DE.1979. Food Legumes: Crop and Product Digest No 3. London: Tropical Products Institute.

Pawiroharsono S dan Siregar E. 1993. Influence of incubation time on the bacterial activity of tempe produced by single strain R. oligosporus. Tempe Workshop, Bpp Teknologi. Februari 15-16 1993, Jakarta.

Pawiroharsono S. 2001. Microbiological Aspects of Tempe. Di dalam: Sapuan dan Soetrisno N (eds), Agranoff J (penerjemah). The Complete Handbook of Tempe: The Unique Fermented Soyfood of Indonesia. Jakarta: Indonesian Tempe Foundation. Terjemahan dari: Bunga Rampai Tempe Indonesia.

Kiers JL, Nout MJR, Rombouts FM, Nabuurs MJA, van der Meulen J, 2002. Inhibition of adhesion of enterotoxigenic Escherichia coli K88 by soya bean Tempe. Letters in Applied Microbiology 35: 311-315.

Kobayasi SY, Okazaki N, Koseki T. 1992. Purification and characterization of an antibiotic substance produced from Rhizopus oligosporus IFO 8631. Bioscience, Biotechnology and Biochemistry 56: 94-98.

van den Hil PJR, Nout MJR. 2011. Anti-Diarrhoeal Aspects of Fermented Soya Beans, Soybean and Health, Prof. Hany El-Shemy (Ed.), ISBN: 978-953-307-535-8, InTech, DOI: 10.5772/17997. [Internet]. [diunduh 2013 Mar 6]. Tersedia pada: http://www.intechopen.com/books/soybean-and-health/anti-diarrhoeal-aspects-of-fermented-soya-beans.

[SNI] Standar Nasional Indonesia 2332.9:2011Tentang Cara Uji Mikrobiologi - Bagian 9: Penentuan Staphylococcus aureus pada Produk Perikanan.

[SNI] Standar Nasional Indonesia 01-2332.1-2006 Tentang Cara Uji Mikrobiologi - Bagian 1: Penentuan Coliform dan Escherichia Coli pada Produk Perikanan.

[SNI] Standar Nasional Indonesia 3144:2009 Tentang Tempe Kedelai. Waluyo L. 2008. Teknik dan Metode Dasar dalam Mikrobiologi. Malang: UMM Press.

524

Pawiroharsono S. 1995. Metabolisme Isoflavon dan Faktor II (6,74’ Trihidroksi Isoflavon) pada proses pembuatan tempe. Di dalam Prosiding Simposium Nasional Pengembangan Tempe Dalam Industri Pangan Modern. 15-16 April 1995. Universitas Gajah Mada. Pp. 165-174.

Khodijah S, Tuasikal BJ, Sugoro I, dan Yusneti. 2006. Pertumbuhan Streptococcus Agalactiae sebagai Bakteri Penyebab Mastitis Subklinis pada Sapi Perah. Seminar Nasional Teknologi Peternakan dan Veteriner.

Zuhud EAM, Rahayu WP, Wijaya CH, Sari PP. 2001. Aktivitas Antimikroba Ekstrak Kedawung (Parkia roxburghii G.Don) Terhadap Bakteri Patogen. Jurnal teknologi dan Industri Pangan 12(1): 6-12.

Kaur SP, Rao R, Nanda S. 2011. Amoxicillin: A Broad Spectrum Antibiotic. Int J Pharm Pharm Sci, Vol 3: 30-37.

Hermana dan Karmini M. 2001. The Development of Tempe Technology. Di dalam: Sapuan dan Soetrisno N (eds), Agranoff J (penerjemah). The Complete Handbook of Tempe: The Unique Fermented Soyfood of Indonesia. Jakarta: Indonesian Tempe Foundation. Terjemahan dari: Bunga Rampai Tempe Indonesia.

Moyler DA. 1995. Oleoresin, tinctures and extracts. Di dalam: Ashurts, PR (ed.) Food Flavorings. New York: Blackie Academic and Profesional.

Naidu AS. 2000. Natural Food Antimicrobial Systems. London: CRC Press.

525

Tables:

Table 1. The ability of the sample extract in inhibiting the growth of testing bacteria (at incubation temperature 30°C, 24 hours).

Sample Pathogen Average of clear zone diameter (mm)

(+) Control (Amoxicillin) E. coli 20.5 S. aureus 17.2 (-) Control (DMSO) E. coli 0.0 S. aureus 0.0 Hyacinth tempe extract E. coli 0.0 S. aureus 0.0 Jackbean tempe extract E. coli 0.0 S. aureus 0.0 Soybean tempe extract (made in laboratory)

E. coli 0.0

S. aureus 7.0 Soybean tempe extract (bought in traditional market)

E. coli 0.0

S. aureus 8.9 Description: 0.0: there is no ability to inhibit (inhibition diameter 0); Sa: S. Aureus; Ec: E. Coli; Control +: Amoxicillin 0:01% (for Sa) and at 0.05% (for Ec).

526

Figures:

Figure 1a. Hyacinth beans

Figure 2a. Hyacinth tempe

Figure 2b. Jackbean

Figure 2c. Jackbean tempe

Figure 4. S. aureus on media BPA

Figure 5. Gram staining of S. aureus

527

Figure 6. E. coli on media EMBA

Figure 7. Gram staining of E. coli

Figure 8. Standar curve of S. aureus

Figure 9. Standar curve of E. coli

Remarks: OD = Optical Density, showing turbidimetry value, means the higher OD, the higher number of bacteria

Figure 10. Standar curve of S. aureus

Figure 11. Standar curve of E. coli

Time (hour) Time (hour)

Bacterial count (CFU/ml) Bacterial count (CFU/ml)

528