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CHAPTER 4 RESEARCH METHOD

4.1 Research Design The study design used is the in vitro experimental study (true experiment-post test only control group design) using the tube dilution test. The purpose is to observe the antibacterial effect of caffeine on the growth of Escherichia coli and to compare the potential concentrations of caffeine towards the colonial growths of Escherichia coli. The concentrations of caffeine used are 100 %( negative controlled), 1.8%, 1.6%, 1.4%, 1.2%, 1.0 % and 0 %( positive controlled). The 0% concentration is the control group of this experiment.

4.2 Venue/Time of Research 4.2.1 Venue The research conducted in Laboratory of Microbiology, Faculty of Medicine Brawijaya University. 4.2.2 Time From June until October 2011

4.3 Repetition Estimate This research use Escherichia coli isolate collection from Laboratory of Microbiology, Faculty of Medicine Brawijaya University. The total sample was calculated by using this formula:

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p (n - 1) 15
Description: p = number of concentration tube (caffeine + control) n = number of repetition needed

In this experiment, the total intervention (p) = 7, so the total repeat supposed to be done was calculated as below: P (n - 1) 15 7 (n 1) 15 7n 7 15 7n 15 + 7 7n 22 n 3.1 n=4 The total repetition done in this experiment is same as or minimal four times. 4.4 Definition of Key Term Pure caffeine is a powder form bought at Pharmacology Laboratory Faculty of Medicine Brawijaya University. Caffeine solution is a solution obtained by diluting pure caffeine that had been heated up by a microwave with the distilled water. Isolates Escherichia coli is obtain from readily available cultures in the Laboratory of Microbiology, Faculty of Medicine Brawijaya University . The Escherichia coli inoculum is the inoculums with a concentration of 106 CFU/ml.

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The degree or concentration of caffeine solution which is used in this experiment obtains from (tube dilution test) method using in vitro technique of antimicrobe substance (caffeine) mix with bacteria of concentration 106 CFU/ml. Minimum Inhibitory Concentration is the degree or concentration at which the caffeine solution is able to inhibit the growth of the bacterial sample (Escherichia coli), marked by cloudiness solution in tube test. Minimum Bactericidal Concentration is the degree or concentration at which the caffeine solution is able to inhibit the growth of the bacterial sample (Escherichia coli), marked by without bacterial colonization growth (MBC<0.1% from total bacteria colony of original inoculums). In positive control (PC) is only fill with suspension bacteria concentration without mix with caffeine solution and distilled water (Caffeine concentration 0%). In negative control (NC), is only containing of caffeine solution in tube without bacteria suspension or distilled water (Caffeine concentration 100%). Control bacteria were growth on Nutrient Agar Plate with total 1 ose as original inoculums (106 CFU/ml) to determined MBC. Qualitative observation is used to observe the growth of Escherichia coli by using three black fix line which put at behind of test tube. It is to observe: a. Clear solution ( can see all line) b. Not very cloudy solution (only can see line one and two) c. Cloudy solution (only can see line one) d. Most cloudy solution (cannot see all line)

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Quantitative observation is to determine the growth of Escherichia coli bacteria colony by calculate total of bacteria colony with colony counter.

4.5 Research Variable 4.4.1 Independent Variable The independent variable used in this research is concentration of caffeine. It means that caffeine concentration uses in this experiment are 100 % ( negative controlled), 1.8%, 1.6%, 1.4%, 1.2%, 1.0 % and 0% (positive controlled). 4.4.2 Dependent Variable The dependent variable is the number of colonies of Escherichia coli colonizing in Nutrient Agar Plate. 4.4.3 Confounding Variable The confounding variables in the study are the working process between repetitions

. 4.6 Research Instrument

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4.6.1 Apparatus Inoculating loop Analytic weighing apparatus Reaction tube Filter paper Beaker Film container Incubation equipment Colony counter Microscope Object glass Immersion oil Cotton bud Vortex Calibrated pipette Forceps Tray with rack

4.6.2 Materials Pure caffeine Caffeine solution Pure Escherichia coli culture Nutrient Agar Plate media

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Gram stain dyes: crystal violet, Lugols iodine, 96% alcohol, safranin Distilled water

4.7 Research Procedure 4.7.1 Bacterial (Escherichia coli) preparation 4.7.1.1 Bacterial (Escherichia coli) Identification The colony that grown in Nutrient Agar Plate medium was identified by using Microbact System. E. coli was identifying as below. A single colony of the E. coli grown on a Nutrient Agar Plate medium was emulsified in a given amount of sterile water. The emulsified colony was inoculated in the wells of Microbact System kit and incubated overnight at 37oC. Reaction was scored with a reference of color code system and produced a unique four digit code number 4.7.1.2 Identification with Gram Stain Clean object glass with sterile cotton then pass it briefly over the flame to get rid of the fat. Allow it to cool. One drop of distilled water or saline solution is dropped on the object glass. With a sterile inoculating loop, a small amount of Escherichia coli colony growing on a Nutrient Agar Plate media is taken

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and suspended into the drop of distilled water or saline solution on the object glass. The smear should be done thinly. The smear is allowed to air-dry. Once dried, fix the smear by passing it briefly over the flame 3 times. The preparation is ready for staining. Flood the preparation with crystal violet for 1 minute, then rinse off with tap water. Flood the preparation with Lugols iodine for 1 minute, then rinse off with tap water. Flood the preparation with 96% alcohol for 5-10 seconds or until the stain fades, and then rinse off with tap water. Flood the preparation with safranin for 30 seconds, and then rinse off with tap water. Dry the preparation with a blotting paper. Observe under the microscope using 100x objective lens magnification. Positive result: rod shaped Escherichia coli stained red (Gram negative).

4.7.1.3 Escherichia coli Testing Suspension The bacterium was transferred into tube that contains NA broth and incubated in incubator using optimum temperature 35-37 0C for 18-24 hours.

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After that, dilution of liquid medium culture of bacteria was done until the 108 CFU/ml concentrations of bacteria were achieved. 1ml of culture (from the 108 CFU/ml concentrations of bacteria) was added to 9ml of NaCl to get dilution 107 CFU/ml concentration of bacteria. Then, the dilution process repeated again by adding 1ml of culture (from the 107 CFU/ml concentrations of bacteria) to 9ml of NaCl to get the final dilution which is 106 CFU/ml concentrations of bacteria. 106 CFU/ml concentrations of bacteria were used in this experiment.

4.7.1.4 Inoculation at Nutrient agar plate Escherichia coli bacteria are inoculated with streaking method at Nutrient Agar Plate at 37C for 1 day.

4.7.2 Antibacterial Testing of Caffeine (Tube Dilution Test) Procedure: 5 reaction tubes prepared and labeled as A (1.0%), B (1.2%), C (1.4%), D (1.6%), E (1.8%). 2 reaction tubes were used as positive control (PC) and negative control (NC). In each tube (A, B, C, D, E) there will be 1ml of caffeine concentration and 1ml Escherichia coli bacteria.

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Stock solution containing 1.8% of caffeine was prepared by weighting out accurately 36mg of caffeine and dissolving in 20 ml of distilled water.

Reactions tube A which contain 1.0% caffeine solution obtain by mixing with 0.56 ml caffeine stock solution with 0.44 ml of distilled water.

Reaction tube B which contain 1.2% caffeine solution obtain by mixing with 0.67 ml caffeine stock solution with 0.33 ml of distilled water.

Reaction tube C which contain 1.4% caffeine solution obtain by mixing with 0.78 ml caffeine stock solution with 0.22 ml of distilled water.

Reaction tube D which contain 1.6% caffeine solution obtain by mixing with 0.89 ml caffeine stock solution with 0.11 ml of distilled water.

Reaction tube E which contain 1.8% caffeine solution obtain from stock solution.

Negative control solution tubes were prepared by weighting out accurately 200mg of caffeine dissolved in 2ml distilled water.

Positive control solution tubes were obtain from 2ml pure solution of Escherichia coli bacteria.

1ml Escherichia coli bacteria added to each tubes (A, B, C, D, E)

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Control bacteria were growth on Nutrient Agar plate with total 1 ose as original inoculums.

All reaction tubes and original inoculums were incubated at 37C for 1 days

After the tubes were incubated, observe and evaluate the degree of cloudiness by compare the reaction tubes with positive control tube and negative control tube. The MIC (Minimal Inhibition

Concentration) was determined after the observation. The mixed solution from each reaction tubes will be streak on Nutrient Agar plate by using ose. All the Nutrient Agar plates are incubated at 37C for 1 days. After incubated, colony growths those appear on the agar were observed and calculate by using colony counter. The MBC (Minimum Bactericidal Concentration) from the caffeine solution determined after the observation and calculation. Analysis and conclusion prevailed from the data observed and calculated in the experiment.

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Summary

7 reaction tubes (concentration) Reaction tube A (1.0%) = 0.56 ml caffeine solution + 0.44 ml distilled +1 ml bacteria . Reaction tube B (1.2%) = 0.67 ml caffeine solution + 0.44 ml distilled water + 1 ml bacteria. Reaction tube C (1.4%) = 0.78 ml caffeine solution + 0.22 ml distilled water + 1ml bacteria. Reaction tube D (1.6%) = 0.89 ml caffeine solution+ 0.11 ml distilled water + 1 ml bacteria. Reaction tube E (1.8%) Positive Control (0%) = 1 ml caffeine stock solution (1.8%) + 1 ml bacteria. = 2 ml of bacteria

Negative Control (100%) =2 ml of caffeine stock solution (100%).

4.7.3 Schematic Procedure


Identification Escherichia coli by Gram staining and growth in EMB agar media Put in Escherichia coli in Nutrient Broth

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Preparation of bacteria Escherichia coli Preparation of Escherichia coli culture using Testing Suspension at 106 bacteria/ml concentration

Test for antimicrobial test with Tube Dilution Test (add caffeine)

Streaking at Nutrient agar to get the Original inoculums (OI)

0% PC

1.2 %

1.4 %

1.6 %

1.8 %

2.0% E

100% NC

Incubation at a temperature of 37C and observed after 18-24 hours Observe for cloudy broth (To identify MIC) Streaking at Nutrient agar Incubate agar plates at 37C for 1 day Calculate the colonial growth (to identify MBC) Analyze using One Way ANOVA statistic test

Figure 4.1Schematic procedure Instruction: PC = Positive Control (tube that only contain suspension bacteria of Escherichia coli without concentration of caffeine) NC = Negative Control (tube that only contain caffeine 100% concentration without suspension bacteria of Escherichia coli) Nutrient Agar = medium growth culture 4.8 Data Analysis

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The data collected was a quantitative data. This data obtained from the colony counting of Escherichia coli after intervene with different concentration of caffeine solution (100%, 1.8%, 1.6%, 1.4%, 1.2%, 1.0% and 0 %). Numeric variable was used to determine the differences in total colony of Escherichia coli according to the intervene factor in different concentration of caffeine solution (100%, 1.8%, 1.6%, 1.4%, 1.2%, 1.0% and 0 %). The statistics test used in this experiment was One Way ANOVA with SPSS 15.0 facility from Window with = 0.05. Hypothesis defined as Ho. Ho accepted when significance value p< 0.05 and rejected when p>0.05. One way ANOVA Analysis test used to determine significant differences in total colony of Lactobacillus acidophilus after intervene with different concentration of caffeine solution (100%, 1.8%, 1.6%, 1.4%, 1.2%, 1.0% and 0 %). In ANOVA statistic test, criteria for more than two groups were: Normal distribution data Same variant data Post Hoct Test (Tukey Test) was continued after ANOVA Analysis test showed significant differences. The purpose of Tukey Test was to determine which group of intervened caffeine solution (100%, 1.8%, 1.6%, 1.4%, 1.2%, 1.0% and 0 %) that causes significantly differences towards total colony of Escherichia coli. Regression Test was done to determine the influences of concentration after intervene with caffeine towards the total colony of Escherichia coli. Besides that, it also showed the relationship of concentration after intervene with caffeine towards total colony of Escherichia coli.

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