Antimicrobial Testing

The purpose of this experiment is to see effects of ampicillin, tetracycline and ethyl alcohol to bacterial growth by disc diffusion method and dilution method.

At disc diffusion method, effects of ampicillin and tetracycline were observed effectiveness to E.coli and differences of these effects of them were observed and measured also. If these antibiotics have effects to E.coli, inhibition zones are formed by bacteria. Diameters of these zones can give information about sensitivity of bacteria to these antibiotics. Dilution method was done for a different purpose. Effects of different concentrated EtOH can be observed by O.D measurements at 600 nm and minimum concentrations of antibiotics can be measured.

An antibiotic, antimicrobial agents, is used for kill or inhibit bacterial growth. Antibiotics are generally produced from secondary metabolites of microorganisms and it is thought that sporulation is an effect in the formation of antibiotics. Antibiotics have basic two effects to decrease growth, static and cidal effect. Static effect is inhibitory for bacteria. It decreases rate of growth by interacting enzymes, proteins or pathways of bacteria. Cidal effect is lethal side of antibiotic. Vidal processes of bacteria can be stopped by this effect and bacteria can be killed. If antibiotics are classified according to spectrum, there are two types of antibiotics. Some antibiotics have a large spectrum; it means that it can affect all wide of bacterial range. This kind of them is named as board spectrum antibiotics. Narrow spectrum antibiotics have limited range as antibiotic; it means that these kinds of antibiotics can affect some specific kinds of bacteria. (1,4)
There are two basic methods to measure effect of antibiotic according to concentrations to observe sensitivity of bacteria. Disc diffusion and dilution methods are most known. Disc diffusion method is used with a piece of paper. Antibiotics dropped onto paper passed through paper and affect bacteria. Disc diffusion method forms inhibition zones on plates. These zones can give visible source about effect of antibiotic. Largeness of zones demonstrates effectivity of it. However this method is not sufficient to show a minimum concentration for effectivity. In despite of non-visible results, dilution method can give minimum inhibitory concentration (MIC). At samples having same amounts of bacteria, different concentrated antimicrobial agents show different decreasing rate of growth. O.D measurement can give a result of decreasing bacteria on numeral values. These values can reveal MIC. The lowest concentration gives no bacterial growth; it is named minimum bactericidal concentration. (2,3)
Material and methods:
Determination of Minimum Inhibitory Concentration,
Overnight E. coli culture
LB broth
Pipettes and tips
96-well plate
Bunsen burner
At this step, EtOH that have different concentration was prepared stock solution with LB broth. E.coli was transferred later to wells. At blank solutions, E.coli was not be used but LB was used. For control groups, E.coli and LB were prepared and only LB was added to empty wells to find E.coli value without EtOH.
Firstly, 5ml of bacteria samples were diluted to 10-4 to be used for 10-4 dilution part of this experiment. Then, %2 stock solution of EtOH was taken desired volume of EtOH according to M1*V1=M2*V2 to get %0.2 solution. 100µl of EtOH having different concentrations was transmitted first 5 horizontal lines on 96-well plate and 5 lines to Line B. Next, LB broth was added to complete solution to wells poured. 100µl of diluted E.coli samples was transmitted to first and second 5-horizontal lines (Line A and B). At Line D and E, instead of E.coli, LB broth was added as blank. For control, 100µl of E.coli and 100µl of LB broth were used at same volumes at first-wells of Line G and H. Other wells of G and H were used for only 200µl of LB to find control of O.D value of E.coli.
The 96-well plate was sent for measuring O.D value at 600 nm and then waited for 24 hour in incubator at 370C. The 96-well plate was measured again after 24 hour. After calculations from data, standard deviations and graph were formed.
Disc Diffusion Method
Overnight E. coli culture
LB agar plates
Pipettes and tips
Sterile filter paper discs
Bunsen burner
Firstly, labelled agar plate for ampicillin and tetracycline were prepared. The two discs were placed, each disc to one part. 25µl of ampicillin (50µg) was dropped to side speared for ampicillin and to the other part, 25 µl of tetracycline (25 µg) was dropped aseptically onto discs. After that, distilled water was dropped too onto discs. The plate was placed into incubator for a day at 370C. Next day, Diameter of zones were measured by using ruler and recorded.

Tetracycline (25µg) Ampicillin (50µg)
Diameters: 20mm 24mm
Table 1.1-The diameters of zones after tetracycline and ampicillin were applied with amount of antibiotics
Time (hour) Control 0.1% EtOH 0.3% EtOH 0.5% EtOH 0.7% EtOH 1% EtOH
0 -0,005718 -0,00359 -0,02137 0,038435 -0,031213 -0,03393
24 0,892181 0,824214 0,813324 0,792511 0,797294 0,738407
Table 2.1-The results of OD values (600 nm) of E.coli diluted to 10-3 after calculations
Control 0.1% EtOH 0.3% EtOH 0.5% EtOH 0.7% EtOH 1% EtOH
Standard Deviation: 0,0714277

Table 2.2-The values of standard deviation of results at 24th hour for 10-3 diluted bacteria samples

Graph 2.3- The graph that shows O.D (600 nm) at initial and 24th hour with standard deviations at different EtOH concentrations for 10-3 diluted bacteria samples

Time (hour) Control 0.1% EtOH 0.3% EtOH 0.5% EtOH 0.7% EtOH 1% EtOH
0 -0,0110466 -0,0069583 -0,03778333 -0,03321 -0,01102333 -0,01076667
24 0,948564 0,82718067 0,745006333 0,794412 0,688869333 0,486184333
Table 3.1-The results of OD values (600 nm) of E.coli diluted to 10-4 after calculations

Control 0.1% EtOH 0.3% EtOH 0.5% EtOH 0.7% EtOH 1% EtOH
Standard Deviation 0,200969

Determination of Minimum Inhibitory Concentration,
EtOH is known as antimicrobial agent and used. Alcohols, mostly isopropyl alcohols, are often used for inhibitory or killing bacteria. EtOH does that by denaturating proteins. The presence of EtOH shows an inhibitory effect and increasing concentration of EtOH decreases bacterial growth. After a point, 70%, at concentration, EtOH begins to demonstrate killing effect or biocide on bacteria. At low concentrations, isopropyl alcohols, EtOH is an isopropyl alcohol, function as inhibitor for microorganisms. The amount of concentration of alcohol is an important for this effect. In this experiment, effect of EtOH with different concentrations (0.1%, 0.3%, 0.5%, 0.7% and 1%) was indicated.
According to graphs (2.3 and 3.3) and tables (2.1 and 3.1), inhibitory effect of EtOH can be seen obviously at different concentrations. For each data, firstly an average values of test groups, blank groups and control groups according to time and amount of dilution of bacteria. Average values are used for getting O.D values of only bacteria by subtracting. Then each value was placed onto graphs. On these graphs, changes of bacterial growth can be seen easily. However, the information must be confirmed to see clear observations. Calculated O.D values being within 0.05 interval standard deviation can give information rate of growth. Standard deviations (Table 2.2 and 3.2) were calculated to show which data is consistent. According to standard deviation, values or calculated data must be within 0.05 range to confirm consistency of experiment.
When the results of 10-3-experiment with tables (2.1 and 2.2) and graphs (2.3) was observed, they don’t give so clear results is obvious. According to standard deviation table (2.2), the values of only 0.7% and 1% EtOH can give more accurate consequences. The main reason of that may occur because of a mistake of rapid and serial filling wells. On the other, if consistent results are observed, there is a decreasing at growth. General data also with Graph 2.3 reveals that. At 10-4, except control group, values have more consistent. Values are within 0.05 standard deviation range (Table 3.2). For 10-4, the results are more consistent than 10-3. On graph, decreasing and effect of EtOH concentration can be more distinguishable. A decreasing rate can be noticeable easily. For both of them, there is decreasing on growth, but there is no a point for no growing. Or the other word, a MIC (minimum inhibitory concentration) cannot be seen. The growth is slow, but still it continuous. MBC (minimum bacterial concentration) means the lowest concentration of an antibacterial agent that can kill a kind of bacteria. 0.1% of EtOH can kill E.coli bacteria for both bacteria dilutions. 0.1% of EtOH can be MBC for E.coli bacteria because at even this lowest concentration, it can form lethal effect for bacteria.
Disc Diffusion Method,
After observations, there is a difference between 2 antibiotics. According to Table 1.1, effect of ampicillin could be seen that his effect was larger in size. 4mm difference against amount of antibiotics reveals that ampicillin is more effective on E.coli bacterial culture than tetracycline. These values provide that E.coli is quite sensitive against ampicillin. With disc diffusion method, MIC or MBC cannot be determined. Only difference between antibiotics cannot be seen according to kind of bacteria.

[1] Antibiotics (3 May, 2017),
[2] Antibiotic Sensitivity Testing Methods (2 May, 2017)
[3] Types of antibiotics (2 May, 2017)
[4] Norrell A. Stephen, Messley E. Karen, 2003 ,Microbiology Laboratory Manual:Principles and Applications,2nd Edt. page 1667

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