IMViC Tests and Catalase Test

Aim:
The aim of this experiment is to determine kind of Enterobacteriaceae bacteria that are found in the tubes labelled as α and β by using IMViC tests.
Introduction:
In microbiology, kind of bacteria in Enterobacteriaceae of determination is done by using some methods. The rapid and useful test is IMViC. For checking danger or safety of water or food, this test is so important. (1)
Enterobacteriaceae is a huge family in bacteria. They have Gram-Negative bacteria including harmless and pathogenic kinds. Coliforms are found in this family. Coliforms are separated according to fermenting lactose with gases and acid. Coliforms are rod-shaped, Gram-Negative, non-spore forming bacteria. They mostly live in aquatic environment and fecal contamination is considered with coliforms. Because of that, they are so important as indicator for these purpose. They are used for pollutions or determining pathogenic organisms. For example, Escherichia coli is one of the most known coliform like Klebsiella pneumonia and Enterobacter aerogenes. Coliforms are obtained by using IMViC test. IMViC include some serial test to determine species of coliform. Each letter except “i” refers to first letter of tests. (1, 2 and 3)
Indole test is first test and used for obtaining present of indole amino acid. Some coliforms use tryptophan as substrate and they produce indole after reaction. Kovac’s reagent, so dangerous with air, is used for that. The reagent reacts with presence of indole and gives pink/red colour on top layer of the tube. E.coli is one of bacteria giving positive result for indole test. Many Bacillus gives negative result like this experiment. (4, 5 and 6)
Methyl-red or shortly MR test is based on pH value in solution. It acts like an indicator giving red colour with acidic solution. Glycoses is first step for generating energy for living organisms. At end of glycoses, pyruvates or pyruvic acid are produced from glucose. Naturally pyruvic acid makes acidic. Bacteria convert them to more stable and safer acidic forms such as formic acid, acetic acid or lactic acid. Eventually acidic media causes change of colour with reagent. E.coli forms acidic media and it causes positive or red colour at MR test. B.subtilis demonstrates negative result or yellow-brown colour. (4, 5 and 6)
VP test or Voger-Proskauer test is the other step of IMViC. VP is for determined existence of acetone in bacterial culture. Some bacteria live by using glucose and turning into acetylmethylcarbinol, shortly acetone. Two solutions are added for that. First one is alpha-naphthol. It binds to acetone and the second solution, potassium hydroxide forms cherry red colour if it is positive. Negative is to see yellow-brown colour for this test. B.subtilis is in positive group; however same test gives negative with E.coli. (4, 5 and 6)
Citrate Utilization test is a test to control bacteria use whether citrate or not as energy-carbon source. When bacteria are added into a media that have sodium citrate and pH indicator like bromothymol blue as basic test materials, citrase enzyme in bacteria breaks down citrate to oxaloacetate and acetate. Oxaloacetate becomes to pyruvate and CO2. On the other hand, after some reaction with acetate, sodium citrate and ammonium salts cause changes in alkaline pH value. These changes forms colour media to blue from green if it is positive like in B.subtilis. Negative result doesn’t demonstrate any growth on media. E.coli can be given an example for negative. (4, 5 and 6)
Catalyst test is one of simple tests. Some bacteria can break down H2O2 to water for producing energy. In this test, H2O2 is dropped onto cells directly and if bubble is observed, it means test is positive. For E.coli, it is variable, but for B.subtilis, it is positive. (4, 5 and 6)
Material and Methods:
Indole Production Test,
0.3 ml of Kovac’s reagent was taken and transmitted into the sample tubes α and β with Tryptone in hood. After a while, colours of samples were observed.
Methyl-Red Test,
1 ml of methyl-red indicator was added into the samples with glucose phosphate broth and the samples were waited for colour change.
Voges-Proskauer Test,
0.9 ml of solution A and 0.3 ml of solution B of Barrit’s reagent were added into samples and they were observed.
Citrate Utilization Test,
Tubes with Simmon’s Citrate Agar were transmitted into test samples and they were waited for 48 hours at 37°C in incubator. After that, they could be observed.
Catalase Test,
3% H2O2 solution was dropped onto bacterial colonies of the test samples that were incubated at small amounts onto a plate before. The samples form bubble was observed.
Results:
Tests
Samples Indole Methyl-red Voger-Proskauer Citrate Utilization
α – + – +
β + – – –
Table 1- the results of IMViC tests for 2 strains (α and β)
Catalase Test
α +
β +
Table 2- Catalase Test for 2 strains (α and β)
α and β refer to E.coli and B.subtilis as unknown samples.
Discussion:
In this experiment, it is aimed to determine kinds of the samples in coliforms by using some serial tests according to metabolic activities.
According to the results of this experiment, it can be seen that α and β have different metabolic activity. For first test for IMViC, indole test was done for each sample. Indole test is bacteria use tryptophan and turns it into indole amino acid. At presence of indole in media, Kovac’s reagent gives a pink layer on top of tube by forming a complex with indole. The sample β gave positive for indole test and the bacteria in sample β uses tryptophan for its metabolism. E.coli is one of kinds of bacteria that are positive at indole test. (5,6)
At second test, methyl-red test, reagent is used for obtaining pH value in sample basically. Methyl red acts as indicator to shows colour change according to pH value. Some microorganism metabolizes glucose to pyruvic acid and it makes environment acidic, but they convert pyruvic acid to some forms such as lactic acid, acetic acid or formic acid for preventing accumulation of pyruvic acid. However environment is still acidic and methyl red can give a reaction. Positive test that bacteria produce low pH value about 4 forms red colour from yellow. Negative gives yellow with more basic environment. α and β indicate different and unexpected result for MR test. The sample α may be B.subtilis and the sample β may be E.coli by comparing with the other tests, however α and β show unexpected results. There may be experimental error. The tubes that have been labelled as α and β could have been confused before experimental process. α and β may have been confused again at observation part. These kind of experimental errors may be and also contaminated culture with the other bacterial cultures or wrong aseptic techniques may give these results. (5, 6)
VP test or Voger-Proskauer is used for identified presence of acetone in bacterial culture. At VP is applied with two solutions for reacting with acetone and giving colour. These are alpha-naphthol and potassium hydroxide. If bacteria turns glucose into acetylmethylcarbinol at digestion, alpha-naphthol reacts and potassium hydroxide gives cherry red colour for positive result. At negative result, yellow-brown or copper colour indicates. In this experiment, there is no positive result. Both cell cultures don’t give acetone after their metabolic activity as expected. E.coli and B.subtilis must have negative result after VP test. (5, 6)
Citrate utilization test is to detect use of citrate by bacteria. Some bacteria use citrate for their metabolic activity. Citrate utilization test uses reagent that can change their colour after reaction in pH value. If test is positive, a change in colour forms from green to blue. B.subtilis can cause this kind of change on media. The sample α can be seen a B.subtilis culture. E.coli culture doesn’t look like that like at β culture. (5, 6)
Catalyse test is the easiest test than the other tests. Drops of H2O2 were poured on two cultures that were found on different places of same slide. Bubbles were observed at both samples. Already forming bubbles were expected at B.subtilis. The sample α that is thought it is B.subtilis shows same results with catalyse test. On the other hand, E.coli bacteria were variable about forming bubbles. In this experiment, β sample is E.coli that can form bubble with H2O2. (5, 6)
Except MR results, samples can be identified which kind of bacteria exist in tubes. According to Table 1 and Table 2, α sample is B.subtilis and β sample is E.coli. (5,6)

Reference:
[1] Received on 24 May,
MacFaddin J.F. 2000. Biochemical Tests for the Identification of Medical Bacteria, 3rd ed. Lippincott Williams & Wilkins, Philadelphia, PA, USA.
[2] Received on 24 May,
Don J. Brenner; Noel R. Krieg; James T. Staley (July 26, 2005) [1984 (Williams & Wilkins)]. George M. Garrity, ed. The Gammaproteobacteria. Bergey’s Manual of Systematic Bacteriology.  2nd ed. New York: Springer.
[3] Received on 24 May,
(http://www.moldbacteriafacts.com/what-are-bacteria/what-is-coliform/)
[4] Received on 24 May,
MacFaddin, Jean F. “Biochemical Tests for Identification of Medical Bacteria.” Williams & Wilkins, 1980, pp 173 – 183.
[5] Received on 24 May,
(http://www.microbiologyinfo.com/biochemical-test-and-identification-of-e-coli/)
[6] Received on 24 May,
(http://www.microbiologyinfo.com/biochemical-test-and-identification-of-bacillus-subtilis/)

Antimicrobial Testing

Aim:
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.

Hypothesis:
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.

Introduction:
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
Ethanol
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
Forceps
Sterile filter paper discs
Bunsen burner
Ampicillin
Tetracycline
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.

Result:
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
0,056639
0,088438
0,218017
0,0084146
0,010738

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
0,023451
0,065697
0,042138
0,015642
0,021695

Discussion:
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.

References:
[1] Antibiotics (3 May, 2017),
(https://www.boundless.com/microbiology/textbooks/boundless-microbiology-textbook/antimicrobial-drugs-13/overview-of-antimicrobial-therapy-153/antibiotics-and-selective-toxicity-773-727/)
[2] Antibiotic Sensitivity Testing Methods (2 May, 2017)
(http://amrls.cvm.msu.edu/microbiology/detecting-antimicrobial-resistance/test-methods/examples-of-antibiotic-sensitivity-tesing-methods)
[3] Types of antibiotics (2 May, 2017)
(http://www.biotopics.co.uk/g11/antibiotic_types.html#tetras)
[4] Norrell A. Stephen, Messley E. Karen, 2003 ,Microbiology Laboratory Manual:Principles and Applications,2nd Edt. page 1667