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If more than one organism is growing in the same environment, one might prevent the growth of another. This research deals with the co-culturing of Escherichia coli and Brochothrix thermosphacta and Escherichia coli and Pseudomonas putida. Primarily, E.coli, P.putida, Br.thermosphacta and P.vulgaris were sub cultured separately and streaked out onto agar plates. The broth containing the primary culture was stored at 10°. The plates were stored in their optimum temperature and after a certain sub culturing; the dilution series was carried out. This process was carried on until the growth was too numerous to count and the colonies were counted and recorded. The growth curve for each organism was established in a graph. The second phase of the project involved culturing E.coli and Br.thermosphacta together in a broth solution and E.coli and P.putida in another solution. The same process was carried on and the former batch was streaked on VRBG agar and STAA agar and the latter was streaked on VRBG agar and PAB agar respectively. After the colonies were too numerous to count, they were recorded and the growth curve was made.
In the primary test where the organisms were grown separately, E.coli and Br.thermosphacta achieved a slow growth rate but P.putida and P.vulgaris were faster in growth. The organism's optimum temperature played a critical role as E.coli and P.vulgaris have an optimum temperature is 37°c while for Br.thermosphacta and P.putida, its 23°c.
The second test was in two phases, the culturing of E.coli and Br.thermosphacta and E.coli and P.putida. In the first phase, the growth count showed that Br.thermosphacta grew faster than E.coli. Even though for the first two days E.coli showed more growth, Br.thermosphacta started growing in a faster rate after that. When it came to the second phase, P.putida was growing in a faster rate than E.coli from day one. Hence the research, with reference to the discussion of Mellefont et al. (2008) showed that co-culturing of these organisms causes the exhaustion of essential nutrients by the dominant organism.
Growth, Temperature, E.coli, Br.thermosphacta, P.putida, Pr.vulgaris, Co-culturing.
When two organisms grow in the same environment, one can prevent the growth of another for many reasons. Production of inhibiting substances like antibiotics, change in the basic gene structure of the recessive organism can be some of the results that can be attained. In the other hand, some interactions can cause beneficial effects like the addition of lactic acid bacteria cultures to cheese. In some cases, microbial interactions are carried on for vaccination purposes. The use of native Lactococci as vehicles for delivery of DNA into mammalian epithelial cells can be a good example. When it comes to the food industry, salami can be a good example. The reduction in pH by the dominant species will make the recessive one to lose its ability to grow. It doesn't mean that the recessive organism is dead; it just means that the available environment isn't viable to grow on. Jameson effect deals with the limitations of growth when two organisms grow in the same environment. Mellefont et al. (2008) suggested that the consumption of nutrients by the dominant organism causes a decrease in the maximum population density of the suppressed organism.
This research deals with the investigation of growth of E.coli when co-cultured with Br.thermosphacta and P.putida. E. coli O157:H7 is the strain of E.coli that was used because it's a foodborne pathogen. The primary organisms which were cultured separately were E.coli, Pr.vulgaris, P.putida and Br.thermosphacta.
Proteus vulgaris is a rod shaped gram negative, oxidase negative bacterium species. It is under the group Enterobacteriaceae. They are known to cause food spoilage in fresh meat.
Pseudomonas putida is a saprotrophic soil bacterium and are rod shaped. They are also gram negative. It is also widely seen in the food processing industry because of its cross contaminating properties.
Brochothrix thermosphacta is a gram positive, catalase positive rod which is mainly associated with pork.
The four organisms were primarily mono cultured under selected conditions to determine the growth rate. When once they are recorded, sub culturing was carried out.
MATERIALS AND METHODS
The required materials for the experiments are Nutrient agar plates, VRBG agar plates, PAB agar plates, STAA agar plates, universal bottles, bijou bottles, septate lids, Duran bottles, Bunsen burner, wire loop, Nutrient broth, 0-20 µl pipette, 20-200 µl pipette, micro-titre plate, Maximum Recovery Diluent (MRD), sterile 2ml syringe with needle, glass spreader and alcohol.
This research can be divided into two experiments. The first experiment is the monoculture of all four organisms while the second one deals with the co-culture of E.coli and Br.thermosphacta and E.coli and P.putida.
Experiment I :
The four organisms were grown in monoculture to find the growth rate. The primary cultures were obtained from the culture collection of the Microbiology Research Unit. The cultures were E.coli, Pr.vulgaris, P.putida and Br.thermosphacta. Before the experiment started, the basic materials were prepared. This includes the Nutrient agar plates and universal bottles containing 10ml of Nutrient broth and 9ml of MRD. Using a wire loop, a loopful is taken from one culture and transferred to a 10ml bottle containing Nutrient broth aseptically. After sterilising the loop, another loopful from the same culture is taken and streaked into a Nutrient agar plate. Nutrient agar is the most preferred agar as it's not selective hence it can be a medium where any organisms can grow. This is useful to find if the culture is contaminated by any other organisms. This is done to examine the presence of growth from the primary culture. This process is carried out to all four organisms individually. Then they are later stored at their respective incubators which runs with specific optimum temperatures. For E.coli and Pr. Vulgaris, the optimum temperature is 37°C while for Br.thermosphacta and P.putida it's 23°c. The next day, the plates were checked for growth and once confirming the growth, one can be assured that the organism's growth in the Nutrient broth is positive. Then, from the first universal bottle containing the organisms in the nutrient broth, a loopful is taken aseptically and transferred to another bottle of fresh nutrient broth. After sterilising the loop, another loopful was obtained and streaked into an agar plate. This process is usually called as Subculturing. It's a method where a culture is transferred from a used medium to a fresh growth medium, in this case, Nutrient broth. This is done to increase the life span of the organism. When introduced into a new growth medium, which isn't contaminated by older cultures, they tend to grow in full capacity. This new culture is stored at their respective incubators. This process was done for the next four days and the fifth subculture can be claimed as a culture where organisms are still in the exponential phase. This is when the dilution series was carried out.
The concentration in all the four organism's subcultured bottle is 10^8. This concentration is high to find the growth curve, so it has to be reduced. This is done by the use of MRD. 1ml is taken from each organism's subculture using a sterile pipette tip and transferred to a universal bottle containing 9ml of MRD and mixed well. This bottle now has a concentration of 10^7. Then from this bottle another ml is taken and transferred to a fresh bottle of MRD and mixed well. This is done for another time to achieve a concentration of 10^5. So at this stage, all four organisms were diluted to 10^5 concentrations. Four 250ml Duran bottles containing nutrient broth was prepared and the bottle cap is replaced with septate lids. 1ml of solution from each of the four 10^5 concentrated bottles were transferred to each Duran bottle and they are named according to the organism present in it. These four bottles containing four organism cultures are the growing medium for the organisms. These bottles were stored at 10°c according to the protocol.
A sterile microtitre plate with lid was taken and the first three rows of wells for four rows were filled with 180µl of MRD using a sterile pipette tip. Using a syringe and a needle, one ml of solution is dispensed from each Nutrient broth bottle to a sterile bijou bottle. This process is carried out for all four organisms. Using a 0-20µl pipette, 20 µl is taken and dropped in the first well of the microtitre plate. Then using a sterile pipette tip, the first well was well mixed and one ml was transferred to the second well. Then another sterile pipette top was used to do the same procedure. This was done till the third well was reached. These steps are done to reduce the dilution from 10^5 (in the Nutrient broth bottle) to 10^2 which is the concentration in the third well. Hence the bijou bottle contains 10^5 concentration of solution and the first well contains 10^4 concentration and so on. This is individually done for all four solutions.
Four Nutrient agar plates were taken and each was marked into four sections. They are named as 0, 1, 2 and 3 with 0 being 10^5 and 3 being 10^2. For each organism, 20µl is taken from the microtitre plate and dispensed into the respective section in the agar plate starting with the lowest concentration. Using a sterile glass spreader, the droplet is spread starting from the lowest concentration. This process is carried out to all four organisms. The date and time is noted and the plates are stored at their respective incubators. The Nutrient broth bottles were stored at 10°. The next day, the colonies were counted and were recorded into the logbook. The same dilution series process was done every day from the solution from the Nutrient broth. When the 0 dilution became too numerous to count (TNC) the dilution was extended from 0-3 to 1-4 i.e.: to 10^1. This experiment was carried out until each organism reached a stage where the colonies were too numerous to count in the 5th dilution. All the colony counts were recorded in the log book. The log values were calculated and the growth curves were plotted. Hence, the optimum growth rate and maximum population density for each organism was discovered through mono culture.
Experiment II :
The second part of the research was almost same as the first process. But this involved the co-culturing. E.coli and Br.thermosphacta were co-cultured in this experiment. The subculturing process was the same as experiment 1. The same primary culture was used. After the fifth sub culture was obtained, the dilution series was carried on. The only difference is that both the solutions were transferred to the same Nutrient broth bottle. This bottle is where both the organisms grew the whole length of this experiment. This medium was also stored at the 10°c incubator. Instead of Nutrient agar plates, Violet Red Bile Agar (VRBG) and Streptomycinthallous Acetateactidione Agar (STAA) with supplement were used to retrieve E.coli and Br.thermosphacta respectively. The rest of the process was same as experiment 1. The STAA plates were stored at 23°c while VRBG plates were stored at 37°c. VRBG agar and STAA agar are selective medium and only E.coli and Br.thermosphacta can be grown in them. The experiment stopped when the colony count were too numerous to count in the 5th dilution. The readings were recorded in the log book and a graph was plotted with both the organism's growth curve.
Experiment III :
The co-culturing of E.coli and P.putida forms the third phase of the research. From the same primary cultures, the subculturing was done. The dilution series was carried out in the same way and the solutions from both the organisms were transferred to the same Nutrient broth. The rest of the process is same as experiment II. Since P.putida is used instead of Br.thermosphacta, the selective medium is changed to Pseudomonas Agar Base (PAB) with supplement. The PAB plates were stored at 23°c and the STAA plates were stored at 37°c. The readings were recorded and the growth rate was plotted.