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

Environmental Factors Affecting Growth of Bacteria

Aim:
The purpose of this experiment is to determine optimal conditions for kinds of bacteria by using E.coli and G.vulcani according to temperature, pH and different NaCI concentrations.
Introduction:
Every living organism needs some optimal conditions to grow, reproduction etc. These conditions can be changed by chemical or physical events. In biological applications, bacterial growth can be controlled by these conditions. Rate of growth can be increased or decreased or made stabile. In microbiology, two basic aspects exist, characterization and control. (1)
Firstly optimal conditions of organisms are obtained. It means best physical or chemical conditions that give highest rate of growth. After obtaining optimal conditions, bacterium has been characterized. After that, lethal limits are determined by researchers. It means that conditions kill bacterial population. Each kind of bacteria has these limits. These environmental limits are used for aseptic techniques and also minimum lethal conditions can help to kill pathogenic bacteria. (1)
Temperature, oxygen and hydrogen ion concentrations (pH) are basic conditions for testing. Additionally nutrients may be important for bacterial growth.
Effects of temperature:
Every organism have enzyme that works at between a certain temperature ranges. Evolutionary conditions caused these ranges. Optimum temperature is a term to define best temperature for bacterial growth. Bacteria can be specially named according to optimal temperature range. (2)
Psychrophiles: between -10° C and 20° C
Psychrotolerants: between 5° C and 30° C
Mesophiles: between 10° C and 47° C
Thermophiles: between 40 ° C and 75° C Hyperthermophiles: at 65 ° C to 120° C
Effect of pH or Hydrogen ion concentration:
Like temperature, enzymes needs suit pH range to work well. Environmental conditions have different pH or Hydrogen ion concentration. Best pH value for a kind of bacteria is called optimum pH. Many of bacteria live in natural pH range, but some bacteria have evolved to acidic environment and they are called acidophilic ones or some of them have evolved for higher pH values and they are called as alkaliphilic microorganisms. Hydrogen ion concentration is also depended on other conditions like temperature, nutrients. These also can change concentration and affect pH value.
Osmatic pressure:
Concentration difference can form a pressure between environment and inside of cell. This is osmotic pressure. Environment that has higher or lower concentrations than inner side of cell caused that some bacteria improve their survival skill to live these types of places to not be plasmolyzed. Some of bacteria live in different concentrated environment. Halophiles are one of these kinds of organisms. They live in minimum concentration of salt. Obligate halophiles need %13 salt concentration. As to osmophiles, their living environment is high concentrated organic solute such as sugar.(4)

Material and Methods:
Effects of Temperature,
E.coli and G.vulcani cultures
Broth
Bunsen burner
Incubator, water bath, refrigerator
The tube was labelled according to temperature values and kind of bacteria before experiment. Prepared bacteria samples were transmitted into the tubes with aseptic techniques and then the tubes with bacteria was placed to incubators, water or refrigerator according to interested temperature value (40C, 20-250C , 370C, 420C, 550C, 620C, 700C). Next day, the samples was observed and recorded on the tables by comparing each other.
Effects of pH,
E.coli culture
LB broth with different pH values
Bunsen burner
The bacterial samples were transmitted into the LB tubes that are different pH. The samples in LB tubes were waited into incubator at 370C for overnight. Next day, the samples were observed and recorded to table.
Effects of Osmotic Pressure,
Spreader
E.coli culture
LB agar plates including several concentrations of salt (NaCl)Spreader
Bunsen burner
100 µl of bacterial samples were inoculated to agar plates that include NaCI concentration in certain ratios (0.5, 1, 5, 10 and 20). Next day, the plates were observed.
Result:
Bacteria Culture
Temperature Growth of E.coli (Broth) Growth of G.vulcani
(Broth)
40C – –
20-250C (room temperature) + –
370C + –
420C ++ +
550C – ++
620C – +++
700C – +++

pH Bacteria Growth of E.coli (LB Broth)
3 –
4 –
5 +
6 ++
7 ++
8 +
9 +
10 –

NaCI(%) Growth of E.coli (LB Broth)
0 ++
0,5 ++
5 +++
10 –
20 –
Discussion:
The aim of the experiment was to obtain optimal conditions and lethal limits of bacteria according to temperature, pH and salt concentration.
At first step, effect of temperature was examined on bacteria. With different degrees of temperature, bacterial (E.coli and G.vulcani) samples were tested. E.coli is a mesophilic bacterium. As optimal temperature, 370C is best for E.coli growth. Experimental data (Table 1.1 and Graph 1.2) confirm expectations and theoretical information. Observations show that E.coli bacteria grow easily between room temperature and 420C. On the other hand, G.vulcani likes hotter environment according to data (Table 1.1 and Graph 1.2). After 420C, there is an increasing at rate of growth and as maximum 62-700C can be optimum for G.vulcani. Higher temperatures can be added to optimum for G.vulcani.
At second step, pH was examined as range for only E.coli. After observing results, E.coli can live in more neural pH value (Table 2.1 and Graph 2.2). E.coli culture started to grow at between 3 and 9, but as optimum pH value the best pH values are 6 and 7. It shows that enzyme activity of E.coli is highest at 6 and 7
Lastly, salt concentration is used for E.coli. E.coli is known as a bacterium living at normal conditions for temperature, pH and osmatic environment. Table 3.1 and Graph 3.2 show that E.coli can live at high salt concentration and it also indicates that E.coli doesn’t have any evolutionary mechanism for osmatic pressure. Cells cannot resist and overcome from this pressure. For better transport from out and inside of cell membrane, a certain concentration difference is also good for cells. For E.coli cells, this range is until 5%. 5% slat concentration can give a good environment for nutrient transport for cells (Table 3.1 and Graph 3.2).
References:
[1] Koch AL (2002). “Control of the bacterial cell cycle by cytoplasmic growth”. Crit Rev Microbiol. 28 (1): 61–77.
[2] Daniel RM, Peterson ME, Danson MJ, et al. (January 2010). “The molecular basis of the effect of temperature on enzyme activity”. Biochem. J. 425 (2): 353–60. .
[3] (http://www.biologyreference.com/Dn-Ep/Enzymes.html)
[4] Madigan, Michael T., and Barry L. Narrs, “Extremophiles” Scientific American, April 1997: 82-88

Environmental Factors Affecting Growth of Bacteria II

Aim:
The aim of this experiment is to observe lethal effects of temperature, heavy metals and UV light on E.coli cells for obtaining how microbial growth can be controlled; and determine thermal death point and thermal death time.
Hypothesis in this experiment is to determine thermal death time by groups studied at different temperatures to figure out thermal death point and to expect to see increase at proper temperature range and also decrease out of optimum temperature range ; to determine increase or decrease according to each different heavy metals and lastly UV light decreases bacterial growth.
Introduction:
Lethal effects of temperature,
In organisms, organized structure and units are so important for optimum workplace. High temperature causes structures and organizations. Especially, enzymes firstly are affected. Their dimensional structures are broken and then they cannot work properly. By increasing temperature, membrane dispersed. Eventually, cells start to die. (1)
Thermal death point refers to a certain temperature to kill organism in 10 minutes. Thermal death time is a term for using to refer the time that how long it takes to kill an organism at a certain temperature. These terms are used often for these kinds of microbial growth researches. (2)
Oligodynamic action,
Oligodynamic action refers to effects of heavy metals to organisms in low concentrations. Heavy metals can attach enzyme structure and they can affect their activity easily. Many of them have dangerous impact for living organisms because of them. Some of them are also important with benefits. Some of them can join structure or take a place on enzymes as a cofactor like Mg+2 joining to photosynthesis. Each heavy metal has different effectiveness according to kind of organism. (3)
Effect of UV light on microbial growth,
UV light can pass and give damage cells by its own high energy-wavelength. Mostly, lethal effect is to DNA. DNA structure can change. Especially changes in genes on DNA are so essential for organism. Generally this change is a dimerization. Close thymine nucleotides can form dimer structure between each other. The dimerization is to prevent transcription and cells cannot produce proteins they need. Some essential limits of UV light are necessary to damage. Exposure time and intensity are some of them. Also sporulation is important. Bacteria that can produce spores can resist against UV light than the others. (4)
Material and Methods:
Materials:
E. coli culture
Heavy metal solutions (0,5M Cr3+ , Cr6+ , Mn2+ , Ni2+ , Co2+ , Cu2+ , Fe2+ , Fe3+ , Zn2+ , boric acid , Mg2+ and 0,05M Cd2+ )
Heater
Beaker
Bunsen Burner
Drigalski spatel
Ethanol
Agar media plates
UV source

Methods:
Lethal effects of temperature,
For determining thermal death point and thermal death time, each group chose a certain temperature (50, 60, 70, 80 and 900C) and each group measure by using same time interval (10 min) and using same steps. First of all, a portion of water was put into the beaker and it was put onto heater arranged to 800C, the degree of temperature our group used. After that, five agar plates were prepared. A thermometer was placed into beaker to check temperature. Before the time measurement, 100µl of E.coli was inoculated to agar plate for control group by spreading with Drigalski spatel and then bacteria were aseptically transferred to the tube immediately before being obtained in 80 degree warm water. The tube was put into 80 degree warm water and it was waited for 10 min. After 10 min, the tube was taken from beaker and 100µl of the sample in the tube was inoculated to agar plate by spreading with Drigalski spatel aseptically. This process was repeated for 40 minutes at 10 minute intervals and at each step, the plates were labelled with date, degree of temperature, name of group. The plates were incubated for one day at 370C. Next day, agar plates were observed and the results were recorded.

Oligodynamic action,
At this point of experiment, each group used two different heavy metals of 0,5M Cr3+ , Cr6+ , Mn2+ , Ni2+ , Co2+ , Cu2+ , Fe2+ , Fe3+ , boric acid , Mg2+ and 0,05M Cd2 for this step.
0,1 ml of E.coli culture was inoculated by spreading with Drigalski spatel for each two agar plates near flame. 0.2 ml from Fe3+, Cu2+ ; the heavy metals of our group. 20µl of Fe3+ was poured onto the middle of plate and then same process was done with 20µl of Cu2+ to the other plate. The two plates were labelled with date, name of heavy metals. The plates were left in incubator at 370C. Next day, diameters of circles heavy metals formed on plate by ruler was measured and recorded.
Effect of UV light on microbial growth,
Each group determined effects of UV light with different time intervals (30 sec, 1 min, 5 min, 10 min, and 15 min). Each group prepared one agar plate with E.coli sample by using aseptic techniques. Then, sample was waited according to time interval under UV light. Lastly, observation of effects of UV light was observed and recorded.
Results:
500C 600C 700C 800C 900C
Control +++ +++ +++ +++ +++
10 min +++ +++ +++ +++ –
20 min +++ – + + –
30 min ++ ++ – – –
40 min +++ ++ – – –
Table 1- the results of observation of lethal effect of temperature to determine thermal death point and thermal death time. (-: no growth, +++ : high growth, ++ : normal growth, + : low growth) after incubating at 370C.

Heavy metal solutions: Cr3+ Cr6+ Mn2+ Co2+ Cu2+
Diameter: 2.5 cm 1.5 cm 2.8 cm 3.3 cm 2.5 cm
Heavy metal solutions: Fe2+ Fe3+ boric acid Mg2+ Cd2+
Diameter: 0.4 cm 1.5 cm – – 3.5 cm
Table 2-The results of diameter of circles on plate after heavy metal effects (-: no effect, +++ : high growth, ++ : normal growth, + : low growth) after incubating at 370C.

UV exposure time Growth rate
30 sec +++
1 min +++
5 min +++
10 min +++
15 min +++
Table 3- The results of UV exposure effect by time according to comparison. (-: no effect, +++ : high growth, ++ : normal growth, + : low growth) after incubating at 370C.
Discussion:
The aim of this experiment is to determine effects and exposure of UV light, heavy metals and also thermal death point and thermal death time to know how the bacterial growth can be controlled.
Lethal effects of temperature,
At this step of the experiment, each group aimed to observe effects of a certain temperature to bacterial growth according to time. At 50, 60, 70, 80 and 900C, observations were done (Table1).
At 500C, bacterial growth is as expected. E.coli is a mesophilic bacterium (5). It means they can grow at not too hot or cold environment. 500C is so proper condition for E.coli bacteria. In Table 1, only at 30 min, a small reducing is seen. This small reducing may have occurred because of sudden heat change between beaker and spreading onto plate. At 600C, until 20 min, there is high growth as expected. Sudden heat change and out of optimum conditions may have caused a going down at growth rate. At 20 min, no growth may have formed because of experimental error while transferring or waiting for a long time or not keeping the temperature within a certain range or experimenter may have forgotten cooling spatula and bacteria may have died because of that. At 700C, within 20 min and after 20 min, decrease can be seen. Effect of high temperature on bacteria shows itself by time. At 800C, also same results are seen with 700C. Bacteria can tolerate high temperature for a while. After a while, enzymes cannot tolerate these conditions and bacterial growth is decreasing. 900C is so lethal for E.coli cells. Enzymes begin to be degraded at even first 10 min. Bacterial growth cannot be observed at that point.
Control group was used for only comparing.
Oligodynamic action,
Heavy metals can affect bacteria with various ways in even low concentration. Many of them interact with enzyme, protein or amino compounds and then their activity is affected (6). Each kind of bacteria shows different effect for heavy metals. Some genes on their plasmids can have resistance for these. In this experiment, 0,5M Cr3+ , Cr6+ , Mn2+ , Ni2+ , Co2+ , Cu2+ , Fe2+ , Fe3+ , boric acid , Mg2+ and 0,05M Cd2+ were used for observing effects on E.coli cells.
When Table 2 was observed; Cd2+, Co2+, Mn2+, Cr3+, Cr6+, and Fe3+ have high influence onto E.coli cells (order of metals according to effects by diameter relatively). Even so low concentration of Cd2+ and Co2+, they have huge Oligodynamic impact for cells. Especially, 0,05M Cd2+ is so low concentrated solution and owns a strong lethal impact on E.coli bacteria. Also some metals like Fe2+can have weak effects on cells. On the other hand, boric acid and Mg2+ do not seem to have an influence. E.coli bacteria may tolerate these with help of genes in their plasmids.
UV light is a type of light having high energy. UV light can damage cellular structure. Especially damages on DNA can kill bacteria easily. At some microbial applications, a technique as sterilization can be used. At this experiment, Table 3, it can be seen that bacterial growth is not affected by UV rays. Such an observation cannot be expected due to the endospores since this bacterium does not produce spores (7). The main reason can be E.coli bacteria have resistance genes for UV light or the machine producing UV light may not have given light with proper wavelength.
References:
[1] Russell AD. 2003. “Lethal effects of heat on bacterial physiology and structure”.  Science Progress.(86): 115-37. Science Reviews 2000 Ltd
[2] Jay, J.M. (1992). Modern Food Microbiology. 4th Edition. New York: Chapman & Hall. pp. 342–6.
[3] Cowan, Marjorie Kelly (2012). Microbiology: A Systems Approach. 3rd ed. pp. 320–321
[4] David S. Goodsell (2001). “The Molecular Perspective: Ultraviolet Light and Pyrimidine Dimers”. The Oncologist. 6 (3): 298–299.
[5] Fotadar U, Zaveloff P, Terracio L (2005). “Growth of Escherichia coli at elevated temperatures”. Journal of Basic Microbiology. 45 : 403–4.
[6] Harke, Hans-P. (2007), “Disinfectants”, Ullmann’s Encyclopedia of Industrial Chemistry. 7th ed. Wiley. pp. 1–17
[7] (http://www.redorbit.com/reference/escherichia_coli/)

Bacterial Growth Measurements II: Total Cell Count

Aim:
In this experiment, cell counting with direct (direct cell count) and indirect (spectrophotometer) methods were realized as the purpose.
Introduction:
Cell counting,
Cell counting is essential basic at microbiology. Cell counting has different methods.
As a classical method, direct microscopic count is known. A counting chamber or hemocytometer is used for this method. It is a kind of specialized slide including squares with certain lines in it. A drop of cells is poured onto chamber. After placing under microscope, cells are counted. If possible, all cells on the camber are counted to obtain the best results. It can be done by counting all of them instead of a certain part. These squares have a certain area and depth. The number of cells per milliliter can be calculated by this volume by using these certain area and depth at the end of the cell count calculated. According to non-written rules, this counting is done by including the cells in the upper left corner. (1)
As indirect method, turbidity measurement is used often. Cell suspensions have turbid structure. Cell suspensions have an absorbance value according to cell concentrations. In spectrophotometer, cells can absorb or scatter light. These amounts of absorbance can give information about biomass in sample. The light should have a certain wavelength for better results. 600nm-wavelength is used for this measurement.(2)
Cell growth,
When cells were placed onto fresh culture, they don’t grow immediately. For a while, cells can adapt to environment and then prepare for dividing. This phase is called lag phase. At lag phase, a growing cell culture is not observed. (3,4)
Main growing on culture starts with log phase or exponential phase. Cell population increases rapidly by using fresh media. This increasing depends on other conditions such as temperature, pH. This temperature is changeable according to kind of microorganism. E.coli prefers 370C mostly. (3,4)
After a while, population is found in stabile line. Equal amounts of dividing and dying cells form this balance. Limited conditions creates stationary phase. (3,4)
At a point, lacks of growth factors cause a reducing. Cells can’t survive and death rate increases by time. This phase is named death phase because of that. (3,4)

Material and Methods:
Direct microscopic counting,
The yeast culture prepared for this experiment after an overnight was placed onto chamber and then the cover slide was put onto the camber. The chamber was observed under microscope and cells on 3 squares were counted.
OD measurement,
Firstly, 1 ml LB was measured in spectrophotometer for blank value at 600 nm. After getting “0” value at blank, same process was applied with 1 ml of bacterial sample for 3 times for each sample at 600nm.
400 l of inoculum of E. coli was diluted with 19,6 ml LB broth to get 2% inoculum of E. col for examinations of Group 1, 2 and 3 by using micropipette and aseptic techniques. Shaking aeration; shaking and no aeration; no shaking with aeration was examined the samples at 2% respectively. For Group 4, 5 and 6, 4 ml of E. coli was transferred into 16 ml LB broth to be diluted to 20%. These tubes were prepared in same conditions. In order, shaking aeration; shaking and no aeration; no shaking with aeration were applied. A tube including only LB broth was used as a blank in spectrophotometer and then each sample was measured for 3 times. After first measurement, the samples were measured by time intervals. Each 3-measurement was used for getting average value. Finally, calculations were applied on a graph.

Result:
Counting 1 Counting 2 Counting 3
Number of cell 76 60 81
Table 1-Cell counting of 10µl of samples at 10-2 dilution under 100x microscope.

Calculation for direct cell counting,
(76+60+81)/3=72
72x104x100=72×106 = 7,2x 107 cells/ml
Calculations for values of OD measurements,
Average measurement= (measurement 1+ measurement 2+ measurement 3)
Measurement 1 Measurement 2 Measurement 3 Measurement 4
Group 1 0.032 0.164 1.149 0.742
Group 2 0.034 0.151 0.914 1.043
Group 3 0.021 0.084 0.825 1.182
Group 4 0.350 0.563 1.173 0.794
Group 5 0.254 0.480 0.917 0.960
Group 6 0.304 0.472 0.809 1.102
Table 2-The values of absorbance by time intervals; 0, 1.30, 18 and 25 hours by spectrophotometer, each value on table is average of 3 measurements.

Discussion:
The purpose of this experiment is to count cells in culture by using direct and indirect methods. At first step, direct cell counting, cell culture prepared before was used. For direct cell counting, yeast(S. cerevisiae) cells were used because of their size. Yeast cells are bigger than bacteria cells. They can be seen easily under microscope. Cell culture should be diluted. Diluted cell culture can prevent to incorrect counting. Naturally, some cells will be cluster in high concentrations and this will affect our results. Low concentration is a problem too. Distance between cells will give too low counting and this won’t be clean results. In our examination, at first attempt with 10-2 diluted solution didn’t give expected. Cells were so far from each other. Because of forgotten vortex process, observation was repeated with vortex. After that, cells could be distinguished. They weren’t so close or so far from each other. 76, 60 and 81cells could be counted at this part. After calculation, average of these counting was found 72. 100 mm and 10000 mm; of lengths and depth of chamber were used for calculated volume between chamber and cover slide. The dilution factor was calculated with 106 ml of volume of chamber calculated by multiplying 100 mm and 10000mm. The cell/ml was achieved as 72×106. As significant number, it is 7,2x 107 cells/ml.(Table 1)
Secondly, OD measurement was used for cell counting. The light passing through a turbid solution and having a certain wavelength can’t pass totally. Particles in solution can prevent to a total passage. If absorbance of particle is used, mass of particles can be known. Same principles can be used for cells. Cells can absorb 600 nm optimally. Spectrophotometer measures this absorbance. For our experiment, different dilutions and conditions were used. For 2% E.coli, shaking aeration; shaking without aeration; no shaking with aeration was applied. Same processes were prepared for 20% E.coli too. When only Tube 1 and 4 was investigated, population of bacteria increased and then decreased at last point was seen. E.coli is a facultative anaerobic bacterium. E.coli can live with oxygen or not, however it prefer anaerobic environment much more. In Tube 1 and 4, shaking causes contact with oxygen and that reduce number of bacteria in tubes. Shaking and aeration can decrease number of bacteria. In the other tubes (2, 3, 5 and 6), bacteria can reproduce, because less contact of bacteria with oxygen give a good environment to grow. Even in the tubes including aeration (3,6), bacteria can increase. If there is no shaking, bacteria can grow in middle sites of solution to not touching oxygen.

References:
[1] Priego, R.; Medina, L.M.; Jordano, R. (2011). “Bactometer system versus traditional methods for monitoring bacteria populations in salchichon during its ripening process”. Journal of Food Protection. 74 (1): 145–148.
[2] Grossi, M.; Lanzoni, M.; Pompei, A.; Lazzarini, R.; Matteuzzi, D.; Riccò, B. (2010). “An embedded portable biosensor system for bacterial concentration detection”. Biosensors & Bioelectronics. 26: 983–990.
[3] Fankhauser, David B. (17 July 2004). “Bacterial Growth Curve”. University of Cincinnati Clermont College. Archived from the original on 13 February 2016
[4] Zwietering MH, Jongenburger I, Rombouts FM, van ‘T Riet K (1990). “Modeling of the Bacterial Growth Curve”. Applied and Environmental Microbiology. 56 (6): 1875–1881

 Isolation of Pure Cultures and Culture Storage Techniques

Aim:
The objective of this experiment was to isolated a colony from mixed culture; and prepare long-term, mid-term and short-term culture storages.
Introduction:
Bacteria have some specific morphological features while growing. These specifity bacterial colonies have is observable by naked eye. Only interested bacterial colony can be taken from culture, a method called isolation. Scientists use this feature to achieve a culture that only a specific kind of bacteria live, it is called pure culture, and isolate it from environment.(1)
Mixed culture is the culture that at least two kinds of bacteria exist on plate. Mostly, pure culture is formed from a mixed culture. Scientists take a specific colony from mixed culture and then place onto pure culture by serving a media that only these interested bacteria can live there. Pure cultures are ideal to study on them.(1)
Colony is an apparent cluster of bacteria that grow from a single cell and living on solid media. Colony can include millions bacteria and this huge numbers cause that they are visible for naked eye. Also these bacteria are same genetically because they come from a single mother cell.(1)
Differences of colonies morphologically can be classified as size, shape, pigmentation, opacity, margin, texture, elevation. The most used morphological feature is texture. Colony can have circle borders between and media, it is called smooth colony, but some colonies have irregular border between and media, it is called rough colony.(1)
Storage of pure culture:
Bacterial colonies must be storage for a while to study on them. According to duration of studying, they can be storage in long-term, mid-term and short term. (2, 3 and 4)
Firstly, short-term is used for one-week researching. Bacterial solution is placed on solid media as streaks or spreading. After one-week incubation and generally 40C are used for short-term. (2, 3 and 4)
Secondly, mid-term is used for one-month researching. Semi-solid media is proper to mid-term storage. This semi-solid media is formed by slant agar, it is produced by solidifying. It can give a large surface area for bacteria can place. By time periods, bacteria can be placed into new media and time can be expended. (2, 3 and 4)
Thirdly, bacteria can be waited for years by long-term storage. Long-term storage is based on to get slow metabolism of cells. It is realized by taking away water from cells without hurting them. For long-term storage, some techniques are used and these are (2, 3 and 4):
⦁ Lyophilization( Freezing-drying): this technique is used that frozen water is heated rapidly and is vacuumed from cells. The technique can include differences on values or according to kind of microorganism. Basically, lyoportectant is added to help freezing by protecting cells. It can be skim milk, or a special glucose solution. After freezing, in a machine water is vapored directly and then cells are left without water immediately.
⦁ Liquid Nitrogen: Liquid nitrogen is used as freezer for cells. Liquid N is -1960C. This method needs a protective buffer like solutions including glycerol, it is called cyroprotectant. The temperature is reduced 10C by 10C. According to species, it is stopped at a point.
⦁ Glycerol: Frozen cells are kept at -800C in glycerol by protecting cyroprotectant. 40% glycerol can serve a protection against osmotic pressure.

Material and Method:
Needle
Loop
Mixed culture
Dishes with solid media
Tubes including semi-solid media
Bunsen burner
Micropipette
40% Glycerol
Water
Slant agars
Eppendorf tubes
Liquid media

First of all, the tubes that have been numbered (13, 14, 15) differently was used for this experiment. Each tube has one of; E.coli, B.subtilis or mixed culture of both of them.

For Preparation of Short-term Media:
The bacterial colonies that have been prepared before on agar plates were observed and written visual features of colonies with naked eye to detect whether mixed colonies which bacteria exist in these colonies. Unknown colonies were taken and then placed onto our agar plates by being careful about aseptic protocols. After placing, dishes were labeled with experiment’s name, date, name of the sample. Next day, bacterial colonies taken from unknown colonies were investigated according to morphology of colonies. Colonies were detected mixed colony or that colonies belong to kind of bacteria (E.coli, B.subtilis).

For Preparation of Mid-term Media:
At mid-term media, 2 techniques were used: slant and stab culture.
⦁ Slant Culture:
Firstly, needle was hold in flame until it has a red color to be sterilized. After opening the tube that includes bacteria, the tube was passed through flame. The needle was put into the tube and then a snake-like line was formed from beginning point to end point of slope in tube. After that, the needle was submerged into slant agar. Lastly, the tube was labeled with date, experimenter’s name and number of the tube that includes pure culture of one of bacteria or mixed culture. Next day, the tubes were observed and detected kind of culture and bacteria the numbered tubes have.

⦁ Stab Culture: The needle was waited in flame until red color. The sample was taken from the opened tube after sterilization with flame and was put in agar by stabbing upright. After transferring, used bacterial tube was passed through flame and then closed. The tube with agar labelled with same like the other steps. Next day, tubes were observed.

At 2 mid-term culturing methods. The tube with agar was not closed tightly to provide air exchange for bacteria.

For Preparation of Long-term Media:
1 ml of 40% glycerol and 1 ml liquid media was mixed in a tube, and then incubated E.coli was transferred into the tube before mixing. The solution was poured into fresh and clean Eppendorf tubes. At all steps, the tube was kept on ice. This culturing was prepared for just seeing how to prepare long-term culture. Last step, keeping at -800C wasn’t realized.

Result:

Figure 1- Bacterial colony that grow on slant agar after slant culturing(Left), colony can be seen as snake-like lines ;and bacterial colony that grow on stab line in agar after stab culturing (Right), colony can be seen as a line in figure.

Figure 2- The samples that taken from the numbered tubes. The dish where 2 differnet colonies grow, mixed culture including E.coli and B.subtilis colonies that was taken from Tube 13 (Left). B.subtilis can be said on the dish that was transferred form Tube 14 according to morphology of colonies (Middle). E.coli colonies that were transferred from Tube 15 are on the agar. (Right)

Shape Margin Elevation Size Texture Appearance Pigmentation Opacity
E.coli circular entire flat small smooth dull no pigment opal
B.subtilis irregular undulate convex moderate rough dull no pigment opal
Tube 13 circular and irregular entire
and undulate flat and convex small and moderate smooth and rough dull no pigment opal
Tube 14 irregular undulate convex moderate rough dull no pigment opal
Tube 15 circular entire flat small smooth dull no pigment opal
Table 1- The morphology of colonies, first two samples was used for detecting and differing kinds of bacteria after culturing on dishes. The others show that morphology of bacterial colonies according to “number” of tube transferred (Tube 13, 14 and 15).

E.coli and B.subtilis colony morphologies was recorded (Table 1) and observations according to table were done after overnight. Tube 13 was detected that it includes mixed culture because of 2 different morphologies of colonies (Table 1) by comparing with the previous observation. Tube 14 was B.subtilis. The observations (Figure 2 and Table 1) was confirmed this. Tube 15 was determined that it includes E.coli by Figure 2 and Table 1. Also any contamination couldn’t be seen.
Slant and stab methods have bacterial culture. Some bacteria could grow at slant method better and some at stab method better.
Long-term storage was just prepared to learn how to prepare.

Discussion:
What is intended in this experiment is to detect kinds of bacteria by using morphological features of their colonies and form storage for them.
At first step the dishes including unknown bacterial culture, E.coli, B.subtilis or mixed culture of both of them were observed for detected what kinds of culture are by using morphological features. Mixed culture could be distinguishable easily because mixed culture has 2 different colony types. Firstly, size can be shown for difference. Also shape, margin, elevation and texture include differences. However some morphological features were quite similar. These are appearance, pigmentation, opacity.
For observing differences better, the other cultures were investigated. The first observable colonies observed in petri dishes were small. Also they had regular colonies. Circular spread of them can be seen. The surface between outside of a colony and the colony was so clear and regular, so margin of this colony was entire. Bacteria tended to expend to surface, flat. As an entire colony, they were smooth colonies. Observations were showing that these bacteria are E.coli. Second group bacteria should have been B.subtilis and morphological critics was confirming. On the other hand, both different colonies have mutual features like being non-pigmentation, dull and opal at appearance, pigmentation, and opacity. (Figure 2 and Table 1)
At mid-term storage, bacterial cultures were observed that they relatively grow well for both methods. Some colonies have spread better slant method. B.subtilis, a kind of bacteria can do aerobic respiration, could be thought. (Figure 1)
Long-term storage was prepared for only seeing preparation of long-term storage.
References:
[1] Healthwise, Incorporated (2010-06-28). “Throat Culture”. WebMD
(http://www.webmd.com/oral-health/throat-culture)
[2] Old, D.C.; Duguid, J.P. (1970). “Selective Outgrowth of Fimbriate Bacteria in Static Liquid Medium”. Journal of Bacteriology. American Society for Microbiology. 103 (2): 447–456.
[3] Madigan, Michael T. (2012). Brock biology of microorganisms (13th ed.). San Francisco: Benjamin Cummings
[4] Uruburu, F. (2003). “History and services of culture collections”. International Microbiology. 6 (2): 101–103.

Bacterial Growth Culturing and Transfer Techniques

Aim:
The objective of this experiment is to transfer bacteria from liquid to liquid, from liquid to solid and to do these with different devices to grow them in these media; and secondly, also to observe sterilization techniques (autoclave and syringe filter) and to prepare media.
Introduction:
The growth media is a place that has been formed for growth microorganism to study on them. Media can be designed as solid, liquid or semi-solid. According to kind of growth of bacteria, it can vary.
The commonest growth media are nutrient broths or LB (lysogeny broth). These can form liquid media. For getting solid media, some materials are used. Agar can form solid media with liquids by solidifying. Liquid mixed with agar is poured into petri dish and cooling agar produces solid media.
Media can be prepared according to kind of bacteria. Some mammalian cells can be grown up in the blood serum. Media needs some nutrients also for specific bacteria or purpose. General nutrients are mutual in many of them: A carbon source like glucose is necessary for some microorganisms. Water is the most important nutrient. Vital processes works with water. Organisms need water to protect this balance. Energy is needed to run all activity in organism. Organisms solve this by different ways. Nitrogen is the simplest source of protein. The most known feature of nitrogen is found in amino acids and because of that, protein production or shortly living for organisms depends on it. Minerals join different parts of processes, but they are so necessary. Osmatic balance and enzymes as a cofactor are just some of them. Growth factor is used for spreading organism on artificial place. It is essential component and it can vary as vitamin, amino acid or anything else. Lastly pH or H ion concentration can regulate enzyme activity. pH limits are very important for a media.
There are many compositions of media. According to use of media, it can show differences. Enrich media have many nutrients to be required by mostly more kids of organism than one. Differential media give a difference for disguising organisms by time. Some nutrients cause changes in bacterial cultures and makes separations between organisms. Selective media is used for growing only specific kind or kind of organism.
For preventing any contamination, some sterilization techniques are used. Techniques can separate 2 main groups: physical and chemical methods. Heat, radiation and filtration can remove bacteria that can contaminate experimental area and samples from media physically. Autoclave is one of them used in this experiment. Hot steam can kill out of experimental organisms immediately and make sterilized materials. On the other hand, chemical sterilization can do with liquid and alcohol. As a filter; flame, alcohol and a special kits can be applied. By these ways, other bacteria can be stopped placing our samples.

Material and Method:
Yeast stock solution
Yeast extract
Petri dishes
Tryptone
Agar
NaCl
Inoculation loop
Spreader
Aluminum foil
Test tubes
Bunsen burner
Autoclave/ autoclave tapes
Syringe
Filter Kit
Before transferring, LB agar and LB broth were prepared. LB broth was made of 1 g tryptone, 0,5 g yeast extract, 0,5 g NaCl. These materials were mixed and transmitted into a flask. As last material, distilled water was added into flask until the volume of the solution will be 100ml. The brim of the flask was closed with foil and then the solution was covered with aluminum foil. The flask was labeled to not be confused. LB agar was formed from 1 g tryptone, 0,5 g yeast extract, 1,5 g agar and distilled water until it has a 100 ml volume. Prepared solution was closed and covered with aluminum foil. As last step for preparation of LB broth and LB agar, autoclave was applied for sterilizing at 1250C for 15 min.
From Liquid to Liquid:
First of all, micropipette was cleaned with tissue getting wet with alcohol. 80µl of liquid including E.coli bacteria was transmitted into a tube that contains LB broth with cleaned micropipette; after the tube with LB broth was opened and the mouth part of the tube passed through fire to prevent contamination. The tube containing bacteria was passed through fire and closed. The other tube including broth and bacteria was labelled with name of experimenters, date, name of bacteria (E.coli). Transfer from liquid to liquid was repeated with same values and processes.
From Liquid to Solid with Spatula:
Firstly LB agar was placed onto dishes including the steps with solid transfers
Before transferring, the beaker containing alcohol was prepared and drigalski spatula was put into the beaker that contains alcohol. Micropipette was cleaned like previous transfer and then 100µl of the bacteria was left as small drop from liquid onto middle of petri dish. After spatula was taken from beaker, it was put on the fire and gotten dry with the fire. Dried spatula was cooled by putting and waiting on an empty side of dish. The dried spread the sample onto surface of dish thoroughly. Petri dish was closed and labeled with name of experimenters, date, kind of bacteria (E.coli), and method according to transfer way and device. At opening and closing the tube including bacteria, it was passed through fire.
From Liquid to Solid with Loop:
Firstly, loop was waited in flame until banding part looks red. The loop was touched on agar surface to be cooled. The loop was inserted into liquid containing bacteria without touching the walls, and then streaks were formed by using the loop. 3 streaks were formed and at each step the loop was passed through flame and after the tube was opened and closed, the mouth of tube was passed through flame. The dishes were labeled with same way like others.
From Solid to Liquid with Loop:
The loop was sterilized with flame, and then a bacterial colony was taken from prepared culture with the loop. The sample was placed into tube with LB agar. The loop was sterilized again and also the mouth of tube was passed through flame. Lastly, the tubes were labeled.
Filtration:
The sample that will be filtered was drawn into syringe. A special apparatus (Kit) including filter was placed to syringe. Solution was filtered by syringe slowly into a beaker near flame to prevent any contamination.
Result:

Figure 1- Cultured liquid media by transferring from liquid to liquid (Left) with E.coli and liquid media, LB broth (Right)

Figure 2- Expended bacterial (E.coli) culture by transferring from liquid to solid with spatula.

Figure 3- Cultured bacterial (E.coli) colonies by transferring from liquid to solid with loop
The bacteria grow up in a media was purposed by transmitting from a different media to other media. After experiment, all colonies have been formed very well. At transfer from liquid to solid with loop, colonies can be selected with naked eyes easily (Figure 3). Any contamination was not observed on these samples. From liquid to solid with spreader, it is good also. Bacteria culture has been expended onto dish successfully (Figure 2). These were not contaminated too. Culture transfer from solid to liquid can be seen that bacteria grow healthily. Contamination was not observed on these cultures too.
Discussion:

The purpose of this experiment was to grow a bacteria culture in a media and transfer it from a media to growth media by different techniques and also to observe filtration techniques. Mediums have three main differences; solid liquid, semi solid. In this experiment, experimenters applied transfers from liquid to liquid, from liquid to solid, from solid to liquid. For culturing bacterial growth, LB broth, LB agar, minimal media was formed, but at transferring, prepared solutions were used for forming except LB agar. Autoclave and syringe filtration were applied. Autoclave was observed how it works by experimenters. After autoclave, solution was controlled by hands to check its suitability to pour dishes. Syringe filtration was observed also. After transferring bacteria, cultures were observed. After observation, cultures were seen clearly. In liquid samples, a blurry image shows that bacteria can grow up. In solid media, colonies could have kept on surface and developed. And at the other samples, bacteria can expend on all surface. Contamination couldn’t be observed at any samples.
At all steps, the samples near flame or any contamination by using alcohol was be watched out.
References:
[1] Madigan M, Martinko J, eds. (2005). Brock Biology of Microorganisms (11th ed.). Prentice Hall.
[2] Hans Günter Schlegel (1993). General Microbiology. Cambridge University. p. 459. Retrieved 6 August 2013.
[3] Parija, Shubhash Chandra (1 January 2009). Textbook of Microbiology & Immunology. Elsevier India. p. 45.
[4] Cooper GM (2000). “Tools of Cell Biology”. The cell: a molecular approach. Washington, D.C: ASM Press

Staining Techniques on Microbiology

Introduction:
The purpose of this experiment is to learn staining microorganism techniques, to apply them in experimental procedures, and observe them under light microscope.
Microorganisms or microbes are simple and single living cells that are found on every corner of the earth. They can be used for beneficial reasons or can be dangerous for human kind. They have a huge family such as bacteria, protozoans, archaea but most known group is bacteria. Bacteria are prokaryotic organisms that have circular DNA and also bacteria have membrane bound organelles like Golgi, or nucleus. (1)
Escherichia coli is one of the most famous kinds of bacteria. They are often used for scientific purposes such as vector, gene isolation etc. E.coli has some specific features to differ than other bacteria. These are that to give pink (gram-negative) colour with gram staining, to be facultative anaerobic respiration, to have rod-shape. They are known that they have pathogenic skills and also beneficial skills for living organisms. Rapid reproduction has made them one of successful kinds in microorganisms. Bacillus subtilis is one of the important bacterial species. Its shape is rod like E.coli. In contrast to E.coli, the B. subtilis gives gram-positive with gram staining. B. subtilis does not show any pathogenicity. B. subtilis has a feature to form endospore against extreme situations to survive. (2) (3)

Microorganisms commonly have transparent image under microscope because of property of their own plasma membrane. Scientist uses stains to have clear image of bacteria. Principal of stains are based on retaining plasma membrane according to its specificity. There are some techniques for different purpose and different cells. The most known methods are negative staining, simple staining, Gram staining, and spore staining. A good staining must include a good smear of organism for quality observation. Smear must be prepared according to medium bacteria grows; solid or liquid. Smear will affect binding of dye to bacteria. Negative staining spread background and size and morphology of transparent bacteria are visible by passing light. Stains like India ink or nigrosine will be effective for this. However simple staining is based on an attraction between membrane and stain. The stain (generally methyl blue, basic fuchsine, or crystal violet) have positively charged ions giving colour, or chromophores. Generally negatively charged membrane is attracted to stain and gives a colour by this way. Gram staining has some complex method. Some bacteria have thicker peptidoglycan (~90% of cell wall). Crystal violet and Gram iodine, a kind of mordant, form a complex and this complex attaches to thick peptidoglycan structure and gives violet or purple colour. These kinds of bacteria are named as gram-positive. This complex also binds to membrane of bacteria having thinner peptidoglycan. However, thin peptidoglycan can’t retain stain as thick as against washing with ethyl alcohol for several times. Counterstain, safranin stains all membranes, but its lighter colour can’t be disguisable on the dyed membrane with crystal violet complex, but thinner membrane can be. It gives a pink colour, and named as gram-negative. Endospore is an organic structure formed at hard or extreme conditions that protect life of bacterium. Very rough structure of endospore doesn’t allow to be stained easily. Boil water and stain open pores on surface and give stain retains. Malachite green enters into membrane and hold onto spore. Safranin added as counterstain again binds to vegetative surface. Endospore and vegetative portion of bacterium can be seen under the light microscope.(4)

Material and Methods:
Slides and coverslips,
Nigrosine dye,
Culture of B.subtilis and E.coli ,
Methylene blue,
Crystal violet,
Safranin,
Malachite green,
Heater
Gram’s Iodine,
95% Ethanol,
Flame,
Distilled water,
Drying paper,
Beaker,
Aluminium foil for prevent pouring of bacteria sample onto bench
For smear preparation,
From liquid bacteria culture, two loopfuls from 20µl bacteria culture were taken and spread onto slide by forming a circle for entire organism was provided being on all of area. The sample has been dried for a while. Other dyeing techniques were in progress while drying. After drying, the flame on burner was used for few times to fix organisms by passing through for 3 times with different positions and directions.
For negative staining,
The few drops of nigrosine (~300µl) was taken from the tube and spread on slide including 20µl organisms (B.subtilis) by using micropipette. The other slide was put on one side of the slide including the sample with 45o angle and the suspension was dispersed by moving the spreader slide on entire slide with stain. After this process, the sample was dried with air and sometimes it was driven by flame to speed up drying. Next, it was observed under microscope and then oil immersion was used for 100x observation. Finally it was observed under 40x and 100x with oil immersion.
For simple staining,

The sample with bacteria was stained with 300µl methyl blue on a beaker used for collecting experimental trash. While staining, slide has been moved gently for stain expedition. After staining, it was waited for 6 minutes to stain the sample. Then, excess stain has gone with pure water by washing. Water drops were cleaned by paper gently to not remove bacteria from surface. Lastly, it was observed under 40x and 100x with oil immersion.

For Gram staining,
First of all, a mixture including culture of B.subtilis and E.coli was placed onto slide by preparing as smear. After waiting for 1 min, 300µl crystal violet was dropped by micropipette onto the sample and it was waited for 20 sec. For removing excess stain, the smear was washed under the trash beaker for a few sec. 300µl from Gram’s iodine was dropped onto the smear and then it was waited for 1 min. As next step, 300µl of 95% ethyl alcohol was used onto smear to remove colorful dye from slide for almost 20 sec. The sample on slide was washed for 2 sec. As last dye, 300µl of safranin was dropped and waited for 20 sec. After last washing process, it was ready to observe microorganisms under 40x and 100x with oil immersion.

For Endospore staining,

A piece of paper was covered on the smear on the slide that was prepared before and then green malachite was dropped on paper. The smear was placed in such a way to not touch water on beaker containing the water which has previously been heated on heater. Under these conditions, the smear has been waited for 5 min over boiling water. After observing that dye didn’t pass through paper, additional dye was dropped onto the smear directly. After the slide lost its heat a little, the paper was removed and washed with water for 30 sec. As counterstain, 300µl safranin was added for almost 20 sec. Excess safranin was removed by water washing, and then the sample was dried with paper to take water drops and waited for a while. After observation under 40x light microscope, a good image and observation weren’t achieved. The experiment, method, was repeated for a clear image. After 100 x observations gave same problem with oil immersion, only 40x examination was clear.

Results:

B.subtilis culture was stained by negative staining method and observed under 40x by light microscope. At first observation was not clear because of it was repeated by lecturer. At the second observation, rod-shaped bacteria could be visible under microscope in figure 1.1. Because of oil immersion and cover slide was forming some technical issues by clinging, observations under 100x light microscope weren’t realized except Gram staining method. At Gram staining (figure 2.1 and 2.2), positive gram (purple) bacteria weren’t visible. Only negative gram (pink) bacteria were visible under microscope and shape and size of pink bacteria was be observed under especially 100x light microscope. The repeated gram staining served also positive bacteria to appear (figure 3.1). E.coli culture and their form were appeared by simple staining method with malachite green like in figure 4.1. Schaeffer-Fulton method was used for staining B.subtilis bacteria formed endospore form, but the image was not clear enough under 40x microscope (figure 5.1).

Discussion:
The aim of this experiment is to learn to apply these kinds of stains for observation, prepare smear and how work principle of these methods by using E. coli and B. subtilis.
Firstly negative staining technique was used for observations on B. subtilis. Negative staining includes so simple method. Dye, methyl blue or nigrosine, spreads onto all ground, but it doesn’t strain bacteria. This opposite visualization technique serves image of transparent bacteria by passing through the cells of light. At first attempt, bacteria weren’t selected from nigrosine. The thick and dried stain could have prevented observation. Stain should have spread onto all bacteria and light could have passed through bacteria. Because of that, staining was repeated. The second observation had clearer image. The rod –shaped cells could be noticeable (Figure 1.1).
Gram staining technique was applied to differ B.subtilis and E.coli from each other. Gram stain is formed by some simple processes. Gram iodine and crystal violet create a form as positive (purple) on positive kinds of bacteria. B.subtilis is one of these kinds of bacteria. Counterstain, safranin, stains bacteria that can’t keep stain after washing with alcohol. These are called gram- negative. In figure 2.1 and 2.2, gram-negative (pink) bacteria, or E.coli, can be disguisable easily; on the other hand gram-positive (purple) bacteria, or B.subtilis, can’t be seen on our mixture. Crystal structures can be seen, but it couldn’t have seen on bacteria. It can include some basic reasons like washing attentively or dropping alcohol too much. Alcohol could have taken desired cells too or wash. The most doubted reason might be to forget to place B.subtilis onto slide because crystal structures can be seen but bacteria cannot. It shows that this can be real reason for unsuccessful observation, but then the image of gam- negative bacteria was so clear under 40x and 100x with oil immersion light microscope (Figure 2.1 and 2.2). Gram staining was repeated like negative staining and gram-positive could be seen under microscope for this time together with not so clear under 40x light microscope (Figure 3.1)
Simple staining uses attraction of charged organisms and stains. Negatively charge bacteria have caused an attraction between bacterium and positively charged stain. This attraction stains bacteria and made them disguisable from background. After all preparation and staining, bacteria weren’t visible. This means that stain couldn’t create an attraction. This could be a mistake at washing, or timing. Washing could have caused that cells are removed from surface or timing was wrong and observation was done so early. Eventually, experiment was repeated and then rod-shaped blue bacteria could be observed under 40x light microscope. Oil immersion was used also but because of cover slip and oil formed a strong force by clinging, 100x lenses couldn’t be used. (Figure 4.1)
Some kinds of bacteria form spore-like structure to survive at extreme conditions. One of them is B.subtilis, used at the experiment. Before experiment, bacteria placed at artificial extreme conditions to form endospore were observed by Schaeffer-Fulton Method with malachite green and safranin as counterstain. Endospore staining technique has a different way to stain spore. It firstly goes inside and it should retain on surface of endospore. Because of that, the smear on the slide placed onto boiling water to not touch water. It caused those bigger pores on the membrane letting passage of dye. The aim of malachite green is to stain green, on the other hand endospore and counterstain, safranin, stain vegetative parts of cell to pink. After followed procedure, noticeable colour differences weren’t discernible. Also, already so small spore couldn’t be noticeable under 100x light microscope because of oil immersion sticky issue to the lens. After a second preparation, under 40x only cells could be visible clearly. (Figure 5.1)
References:
[1] Madigan M; Martinko J, eds. (2006). Brock Biology of Microorganisms (13th ed.). Pearson Education. p. 1096. ISBN 0-321-73551-X.
[2]  Singleton P (1999). Bacteria in Biology, Biotechnology and Medicine (5th ed.). Wiley. pp. 444–454. ISBN 0-471-98880-4.
[3]  Madigan M, Martinko J, eds. (2005). Brock Biology of Microorganisms (11th ed.). Prentice Hall. ISBN 0-13-144329-1.
[4] Clark G (1981) Staining Procedures, 4th ed., Baltimore: Williams & Wilkins, p. 412

Growth Measurements: Viable Counts

 

INTRODUCTION 

The aim of this laboratory hour was to provide both theoretical knowledge and practical skills about the process of counting different organisms present in a specific sample.  

Process of counting can be realized by using either direct or indirect method. There are some factors that affect the counting, such as: pipetting, optimized incubation time, maintenance of homogeneity and a proper amount of the mixture [1]. 

Direct method includes two different techniques: total cell count and viable count, where the only difference between them is that in the viable count, only the cells that have the potential to give rise to new colonies are counted, while the total cell count technique involves counting both living cells and dead cells. Viable count is known also as plate count or colony count method Viable count technique is unable to give every time accurately the total number of the viable cells, because of the possibility it exists that the groups of cells get disrupted before being plated or the cells to get clumped and form a colony together. In order to take in account the possibility that the colony forming unit can contain one or more cells, the number of the viable cells is calculated as colony forming unit (cfu). Viable count can be realized either by spread plate method or pour plate method [2]. 

Serial dilution of bacterial culture is a crucial process in the process of enumeration a sample with unknown number of bacteria and it is realized by diluting the sample either with broth, saline solution or phosphate buffer in the ratio 1:9. The range of the number of colonies is 30-300 and it is an optimized value, as a number less than 30 colonies does not fulfill the statistical requirements, while a number bigger than 300 would cause colonies to compete for nutrition and could also lead to the suppress of the colony growth [3]. 

MATERIALS & METHOD 

Dilution of Bacteria Cultures for Spread Plate and Pour Plate Techniques 

Materials 

  • Bunsen burner 
  • Pipettes & tips 
  • E. coli culture 
  • Test tubes consisting of LB broth 
  • Vortex 

Method 

1 ml from the Eppendorf that holds overnight cultured E. coli is taken by using the micropipette.  Tube where 9 ml of broth is positioned is opened and sterilized through the flame of the Bunsen burner. Then 1 ml of E. coli is poured inside the broth and the tube`s mouth is sterilized again through the flame. Tube is vortexed and labeled with the specific data about the experiment. The procedure of dilution is repeated 7 times by using each time 1 ml from the tube that was diluted and vortexed in the preceding step.  

Pour Plate Technique 

Materials: 

  • Water bath  
  • Diluted E. coli culture 
  • Pipettes and tips 
  • 15 ml LB agar in tubes 
  • Petri dishes 

Method  

Initially, 1 ml of the diluted sample of E. coli (10-4, 10-5, 10-6, 10-7) is positioned in the Petri dish.  Afterwards, 15 ml melted agar that was molten in the water bath at a temperature of 50OC, is added in the Petri dish. The plate is covered and moved over the bench in order that the samples inside get swirled. After that, the Petri dish is positioned there till the solidification of agar occurs and then placed in the incubator for 24 hours at a temperature of 37OC. 

Spread Plate Technique  

Materials 

  • Drigalski spatula 
  • Bunsen burner 
  • Pipettes and tips 
  • LB agar plates 
  • Diluted E. coli cultures 

Method 

From the diluted samples (10-4, 10-5, 10-6, 10-7) 0.1 ml is taken by using the micropipette and it is thrown into the agar plate. The Drigalski spatula is first sterilized inside the alcohol and later through the fire. After it cools down, this spatula is used to spread the inoculums throughout the area of Petri dish. The plate is covered and placed over the bench for a short period of time so that the inoculums get absorbed by the agar. Afterwards, the Petri plate is positioned upside down and incubated for 24 hours under the temperature of 370C. 

Standard Plate Count of Milk 

Materials 

  • Milk 
  • LB agar plates 
  • Pipette and pipette tips 
  • Drigalski spatula 
  • Tubes consisting of 9 ml saline solution  

Method  

1 ml from the milk sample is taken by micropipette and poured inside the tube that contains 9 ml of saline solution whose mouth was sterilized through the Bunsen burner. The sample is vortexed for a short period of time. This process is repeated by transferring 1 ml of sample of the preceding step`s culture to the next buffer till the dilution of 10-4. After the dilution procedure is over, the dilutions 10-1, 10-2, 10-3, 10-4 plates are spread by using Drigalski spatula. The plates are covered and positioned over the bench for a short period of time till the inoculums are absorbed by agar. Afterwards, the plates are placed in incubator for 24 hours under the temperature of 37oC. 

 RESULT 

Plate count about each dilution factor  Cigdem` s group 

(raw milk) 

Ayhan` s group 

(raw milk) 

Nergiz` s group 

(pasteurized milk) 

Oyku` s group 

(raw milk) 

Deniz` s group 

(pasteurized milk) 

Elvan`  s  

group 

(raw milk) 

10-2    314  0  2    43 
10-3    32  0  3  0  1 
10-4  11  2  0  4  0  0 
10-5  0  1  0  2  0  0 
10-6  0        0   
10-7  0        0   

Table 1: Serial dilutions of the samples of raw milk and pasteurized milk and series of plates inoculated with diluted cultures 

Dilution factor  10-2  10-3  10-4  10-5  10-6  10-7 
Plate count  314; 0; 43  32; 0; 0; 1  11; 2; 0; 2; 0; 0  0; 1; 0; 4; 0; 0  0; 3; 0  0; 2; 0 
Colony-forming units  (314+43)/2 x1/10-2= 

1.79 x 104 

32 x 1/10-3= 

3.2 x 104 

0 (as no one of the values are in the range [30;300]  0 (as no one of the values are in the range [30;300]  0 (as no one of the values are in the range [30;300]  0 (as no one of the values are in the range [30;300] 

Table 2: CFU calculations of the standard plate count of the milk by using the formula [CFU= Plate Count x Dilution Factor]. The values are represented either as duplicate or triplicate, because of the experiment performed by the different lab groups. 

Dilution factor  10-4  10-5  10-6  10-7 
Plate count  >300  >300  135  13 
Colony-forming units  >300 x 1/104= 

More than 3 x 106 colony forming units 

>300 x 1/10-5= 

More than 3 x 107 colony forming units 

135 x 1/10-6=1.35 x 108  As it is not in the range [30;300] it is not calculated 

Table 3:  CFU`s calculations of the serial dilutions of samples inoculated by the spread plate technique 

DISCUSSION  

By comparing the data from the standard plate count of the raw milk and pasteurized milk, it is seen that there is a relationship between the number of bacteria and whether the milk is raw or pasteurized. In the pasteurized milk, the plate count leaded to a number of 0 bacteria. Additionally, in the raw milk it is seen that as dilution factor`s value decreases, the number of bacteria decreases. In the calculations of the colony forming units, most of the calculations are not performed because the values of bacteria are outside the range [30; 300].  

In the pour plate method, it is observed that most of the bacteria have accumulated in the upper part of the medium and this occurs, because they have a high requirement of oxygen. The other part has accumulated in the down part of the medium, because they are micro-aerophiles and do not need lots of oxygen.  

In the spread plate method, it is seen that as the dilution factor decreases, number of bacteria decreases too. The reason for that is that as the dilution process is made the ratio of the sample to the medium continues to proceed in this form: 1/100, 1/1000, 1/10000.  

 REFERENCES 

  1. http://biolabs.tmcc.edu/Micro%20Web/BacterialCounts.pdf retrieved 31.03.2016 from 
  1. https://www.boundless.com/microbiology/textbooks/boundless-microbiology-textbook/culturing-microorganisms-6/counting-bacteria-63/viable-cell-counting-384-5695/ retrieved 31.03.2016 from  
  1. http://textbookofbacteriology.net/growth_2.html retrieved 31.03.2016 from 

 

 

ISOLATION OF PURE CULTURES AND CULTURE STORAGE TECHNIQUES

INTRODUCTION 

Aim of this laboratory hour was to provide theoretical knowledge and master experimental skills about differentiating different bacteria and store them properly.  

Isolation of a microorganism is the process of discerning different organisms from each other, in such a way that a pure culture of organisms is attained. This process`s initial part consists of the differentiation of microorganisms in a solid medium [1].  

Mixed culture is the term used to describe the cultures where more than a specific type of a microorganism is found, while in the pure culture only a specific type of a microorganism is found [2].  

Colony is the term used to describe a considerable number of microorganisms observable without the help of microscope, that developed from the same ancestor cell. Colony morphology is crucial about discerning microorganisms from each other due to their physical differences, such as shape, elevation, size, appearance, pigmentation, opacity, margin and texture, that is the most distinguishing characteristic and the mostly used in morphological analysis. The texture of microorganisms can be either rough, characterized by an irregular outer space or smooth, characterized by a circular space [3].  

Storage of the microorganisms is a crucial step about ensuring the survival of the microorganisms and eliminating a possible contamination of the microorganisms. Short-term maintenance is realized by inoculation of the pure colonies inside the Petri dishes that contain solid media and streak plating or spread plating, followed by over-night incubation. Those pure cultures can only sustain inside their medium for one week at 40C and after that period of time they need a new solid medium [4].  

Mid-term storage is realized by the solidification of the semi solid media inside the slant agar by providing an increase in the total area of the media. Those pure cultures can sustain for one month in 40C and then after that period of time, they need a new slant agar [5].  

Long-term storage enables the maintenance of pure cultures for a couple of years. This method is not appropriate for being used frequently. They need to be placed on a fresh medium before any experiment performed with them. This method can be realized in three different forms: lyophilization, usage of liquid nitrogen and usage of glycerol [6].  

Lyophilization is the method that enables the removal of water due to a decrease in water`s sublimation temperature. The time of storage ranges for any specific microorganism. Additionally, the liquid nitrogen enables the sample to freeze when in contact with liquid Nitrogen (-1960C) inside the nitrogen tanks. Before this technique is realized, overnight incubation and suspension of cells in cryoprotectant, a buffer that protects the cells from damage.  [7].  

Glycerol enables the freezing of pure cultures at a temperature of -800C and it serves as a cryoprotectant of the cells. Generally, the glycerol reacts more efficiently when it is 40% concentrated [8].  

 MATERIALS & METHODS 

Isolation & Observation of Pure Cultures by Examining Colony Morphologies 

Materials  

  • Bunsen Burner 
  • Inoculation loop 
  • Known cultures on agar plates 
  • LB broth 
  • E. coli 
  • Subtilis bacillus  

Each of the 4 members of the lab team performed the same procedure. Initially, the morphologies of the microorganisms on the agar plates were observed. After the inoculation loop was sterilized, a piece of sample was taken from the unknown liquid culture and streak plating was performed inside the Petri dish. Afterwards, the Petri dish was labeled with the information needed to identify each specific result. In the end of each of the processes, the Petri dishes and the tubes were incubated for 24 hours.  

 Mid-Term Storage & Long Term Storage 

Materials 

  • Slant agars 
  • Bunsen burner 
  • Eppendorf tubes 
  • Innoculation needles 
  • Glycerol (40 % in dH2O) 
  • Test tube 
  • Vortex 
  • Pippetes 
  • Tips 
  • Mid-term Storage 

Slant Culture 

Initially the inoculating needle is sterilized in the flame. Afterwards, the needle is put inside the Eppendorf tube containing E. coli and then moved through the upper surface of the sloping tube and then placed inside the slant for a short period of time. Needle is sterilized again after taking it out from the slant. Finally, each of the tubes is labeled specifically. 

Stab Culture 

Initially, the inoculating needle is sterilized in the flame. Then it is put inside the Eppendorf tube where E. coli culture was present. Afterwards, the needle was inside the center of the agar. After the needle was taken out from the tube, it was sterilized again on the flame.  

  • Long-Term Storage 

Initially, 1 ml 40% glycerol was taken by pipette and positioned inside a sample and afterwards 1 ml media was taken by pipette and added in the same sample. Then, 1 ml E.coli was taken from the Eppendorf tube by pipette and positioned inside the same sample. The sample is put in the vortex. Afterwards, the mixture was separated as 500 microliters in each of the 4 sterile Eppendorf tubes. Then the tubes were put in the laboratory refridgerator in a temperature of 

-800C. 

RESULTS 

 

  Denisa  Busra  Cagla  Roza 
Inoculation from the tube 1  Colonies of bacteria were grown, except in one side growth did not occur.  Colonies of bacteria were grown, except in one side growth did not occur.  Colonies of bacteria were grown, except in one side growth did not occur.  Colonies of bacteria were grown, except in one side growth did not occur. 
Inoculation from the tube 2  Colonies of bacteria were grown in every part of the Petri dish where they were inoculated.  Colonies of bacteria were grown in every part of the Petri dish where they were inoculated.  Colonies of bacteria were grown in every part of the Petri dish where they were inoculated.  Colonies of bacteria were grown in every part of the Petri dish where they were inoculated. 
Slant method  New colony of bacteria was grown very well near the upper part of the tube.  New colony of bacteria was not fully grown as in the first slant tube.   New colony of bacteria was not fully grown as in the first slant tube.  New colony of bacteria was grown very well near the upper part of the tube. 
Stab method  New colonies of bacteria grew very good near the upper surface, and not as much in the bottom part of the tube.  New colonies of bacteria grew very good near the upper surface, and not as much in the bottom part of the tube.  New colonies of bacteria grew very good near the upper surface, and not as much in the bottom part of the tube.  New colonies of bacteria grew very good near the upper surface, and not as much in the bottom part of the tube. 

Table1: Growth results of each lab member`s 4 performed experiments. 

 DISCUSSION 

Due to observation after the incubation of each of the samples inoculated either with the bacteria inside tube 1 or with the bacteria inside tube 2, a conclusion was reached, that the bacteria inside the 1.tube was E. coli, while the one inside the 2. tube was Subtilis bacillus. This conclusion was based in the general features that characterize E.coli, such as: shiny appearance under microscope, circular shape, lack of pigmentation, entire margin, being opaque, having a convex elevation and a smooth texture. Additionally, the Subtilis bacillus is characterized by those features, such as: lack of pigmentation, having an irregular shape, an undulate margin, shiny appearance under microscope, rough texture and having a convex elevation [9]. 

The results from the stab method, showed that the colony of the new grown bacteria had accumulated in the end of the tube apart from the lack of oxygen that characterizes the end of the tube. From this can be deduced that the bacteria that grew near the end of the tube were anaerobic. However, the results from the slant method, showed that the colony of new grown bacteria had grown very little in the end of the tube and in a high intensity in the side of the tube that was in contact of the oxygen, showing that these bacteria were aerobic. Another reason that can be about why the newly grown bacteria spread in the entire upper surface of the tube can be because these bacteria are slightly motile, compared to the ones that have accumulated in the end of the tube [10]. 

In the Petri dishes where the inoculation was made by using either the 1st tube or the 2nd tube, there were parts where the growth did not occur and this can be because of using a scarce quantity of bacteria, the death of bacteria due to the highly heated loop or because of technical mistakes during the culturing process.   

REFERENCES 

  1. http://vlab.amrita.edu/?sub=3&brch=76&sim=1109&cnt=1 retrieved 23.03.2016 from  
  1. http://education.seattlepi.com/mixed-culture-biology-6739.html retrieved 23.03.2016 from 
  1. http://delrio.dcccd.edu/jreynolds/microbiology/2421/lab_manual/colony_morph.pdf retrieved 23.03.2016 from 
  1. http://www.wakemed.org/body.cfm?id=237 retrieved 23.03.2016 from  
  1. http://www.thermoscientific.com/content/tfs/en/about-us/general-landing-page/storing-bacterial-samples-for-optimal-viability.html retrieved 24.03.2016 from  
  1. http://www.ruf.rice.edu/~bioslabs/bios318/318manual.htm retrieved 24.03.2016 from 
  1. http://www.rpi.edu/dept/chem-eng/Biotech-Environ/LYO/ retrieved 24.03.2016 from  
  1. http://www.ncbi.nlm.nih.gov/pubmed/6804123 retrieved 24.03.2016 from 
  1. http://dnaresearch.oxfordjournals.org/content/4/1/9.full.pdf retrieved 25.03.2016 from 
  1. http://academic.pgcc.edu/~kroberts/web/slant/slant.htm retrieved 25.03.2016 from