Abstract
Identification of three unknown bacterial strains was performed using simple agar plate pouring and differential staining analyses. The growth of the bacterial cultures was evaluated using colony counting methods. In addition, the morphology of the bacterial colonies was evaluated, as well as their reactions to lactose utilization assays. Bacterial plating methods involved streaking the unknown bacterial samples onto TSA and MacConkey agar plate for differentiation based on a growth in selective culture media. Further differentiation of the bacterial unknowns was performed through additional colorimetric tests, resulting in the validation of the identity of each bacterial species. The techniques employed in this experiment have resulted in the determination of the 3 bacterial species, namely Escherichia coli, Streptococcus pyogene and Yersinia enterocolitica.
Introduction
Identification of the precise species of bacteria is important because this information will provide information on the correct diagnosis of disease. More importantly, the identification of a bacterial pathogen will provide hints on the proper treatment that should be given to a patient. Currently, there are a number of laboratory tests that can be employed to identify the specific type of bacteria that is being tested.
There are currently several microbiological assays that can help in the identification of bacterial species. The principle behind these assays is that each species has a unique physiological feature that can be tested in the laboratory using in vitro conditions. For example, there are particular bacterial species that have the capacity of degrade red blood cells. This process of hemolysis is caused by exotoxins that are produced by a specific type of bacteria. Upon the destruction of a red blood cell, the contents are thus released to the red blood cell’s immediate environment. One of the major proteins that appear after such process is hemoglobin, which is a transport protein that carries oxygen to the rest of the body. In this experiment, three bacterial strains of unknown identities will be tested in order to determine the exact microbiological species that is present in the test tubes.
Materials and methods
The experiment involved the identification of three unknown bacterial strains which were provided as broth cultures. Bacterial agar cultures were initiated by plate streaking onto TSA plates which are considered as basic non-differential growth media. In addition, another culture was initiated by streaking the broth onto a MacConkey agar plate which selectively allows the growth of Gram-negative bacteria and selects for lactose fermentation through the decrease in the pH level of the culture medium. The MacConkey media also contains pH indicators which generate dark purple bacterial colonies. After 24 hrs incubation at 37oC atmosphere, the colonies that emerged on each plate were counted. In addition, the morphology of the bacterial colonies were analyzed.
In the first series of the analysis, Gram staining was conducted in order to classify each of the three unknown bacterial strains are either Gram-positive or Gram-negative. The three unknown bacterial samples were also inoculated on microbiological differential culture media following the aseptic transfer technique. In order to determine the Gram staining profile of the bacterial unknowns, the catalase test was performed. In this test, hydrogen peroxide was added to the bacterial sample and the reaction of the bacterial sample to the presence of hydrogen peroxide, in the form of bubbles, was taken note of.
Another assay that was conducted in our experiment involved the mannitol salt agar (MSA) plate, wherein the test sample was inoculated onto an agar plate that was supplemented with blood. It has been established that the MSA plate can only allow the survival of a specific bacterial strain which is Staphylococcus. After allowing the bacterial unknown to growth for 24 hours, the color of the agar culture media was checked. The initial color of the agar plate was red, due to the presence of the pH indicator, phenol red.
For those bacterial samples that tested negative in Gram staining, these were inoculated in citrate, triple sugar iron (TSI) and eosin methylene blue (EMB).
Results
The bacterial unknowns were cultured onto TSA and MacConkey plates and tested after 24 hours of incubation at 37oC atmosphere. The TSA plate was visually observed to carry two bacterial species because they could be distinguished according to the morphology of the colonies. One type of colony was composed of pin-shaped Gram positive bacteria, while the other type of colony was composed on pin-shaped Gram negative bacteria. Further culturing of the colonies in MacConkey agar showed that one of the two colony resulted in a color reaction, showing a purple colony on the plate. On the other hand, the other type of colony remained colorless on the MacConkey agar plate.
The other bacterial unknown that was grown in blood agar plate did not show any change in color. Furthermore, addition of hydrogen peroxide to the blood agar plate resulted in the generation of bubbles. The MSA plate containing another bacterial unknown did not show any change in the color of the media. Another bacterial unknown that was inoculated into EMB plates resulted in dark purple colonies. The bacterial unknown that was inoculated in the citrate culture slant resulted in a blue culture medium. The TSI culture tube with another bacterial unknown was found to generate bubble in the culture medium yet there was no precipitate that was observed. The other bacterial unknown that was grown in the EMB plate showed colorless bacterial colonies. The citrate slant was also found to be of blue color as well as the absence of bubble or precipitate. The techniques employed in this experiment have resulted in the determination of the 3 bacterial species, namely Escherichia coli, Streptococcus pyogene and Yersinia enterocolitica.
Discussion
In order to perform bacterial identification, one needs to collect a sample material that is suspected to carry the bacteria of interest. Upon collection of the biological sample that carries the bacteria, these microorganisms may be cultured in the laboratory for further analysis and possibly, manipulation. The growth of bacteria pertains to a process wherein a single bacterial cell generates two identical daughter cells. This simple doubling of bacteria is observed in cultures that are classically conducted in microbiological laboratories. The quantification of bacterial growth is generally performed through the use of either direct or indirect cell counting methods. Colony counting is an example of a direct counting technique while the measurement of turbidity is an illustration of an indirect counting procedure.
The progress of a bacterial curve is generally described through the use of a growth curve (Novick, 1955). Four different phases comprise a bacterial growth curve. The lag phase involves the adaptation of inoculated bacteria to the conditions of the culture medium. This phase denotes that time that the bacteria are undergoing maturation. The logarithmic or exponential phase involves the doubling of bacteria in culture. The rate of division is observed to logarithmically increase through time. The growth conditions and the chances of survival of the resulting daughter cells influence bacterial growth rate. The logarithmic growth of the bacterial culture is dependent on the availability of nutrients in the culture medium. The stationary phase pertains to the decrease in growth rate due to the exhaustion of nutrients in the culture medium and in turn, wastes have accumulated in the culture medium. During the death phase, the cultured bacteria lose nutrient resources and die.
In the first series of the analysis, Gram staining was conducted in order to classify each of the three unknown bacterial strains are either Gram-positive or Gram-negative. In this test, the addition of hydrogen peroxide determines that a bacterial species is Gram positive. On the other hand, when no bubbles are observed in the reaction of hydrogen peroxide to the bacterial sample, the test sample contains Gram negative bacteria.
Another assay that was conducted in our experiment involved the mannitol salt agar (MSA) plate, wherein the test sample was inoculated onto an agar plate that was supplemented with blood. It has been established that the MSA plate can only allow the survival of a specific bacterial strain which is Staphylococcus. The growth of this particular bacterial species is due to the presence of 7.5% NaCl which is also present in the MSA agar culture media. Another feature that allows the MSA agar plate to selectively allow growth of Staphylococcus in the presence of mannitol is the change in the pH level of the culture media. The fermentation of mannitol results in the lowering of the pH level and this can be monitored by incorporating a pH indicator in the culture media. In our case, phenol red was the pH indicator that was employed for determining any changes in the pH of the culture media. When the pH of the media decreases, the color of phenol red changes to yellow and indicates that the pH had changed to a lower level.
For those bacterial samples that tested negative in Gram staining, these were inoculated in citrate, triple sugar iron (TSI) and eosin methylene blue (EMB). The citrate culture media also contained a pH indicator of bromthymol blue which indicates when the bacteria utilize citrate for nourishment. When this occurs, carbon dioxide is produced as a by-product of the reaction, which in turn increases the pH of the culture media and changes the color from green to blue. The change in the culture media is due to the reaction of carbon dioxide with the sodium ions and the alkaline products that are present in the culture media.
The observations in the TSI culture media are mainly based on the bacteria’s capacity to use lactose and sucrose for nourishment. Phenol red is also present in the TSI media and thus indicates any changes in the pH that are associated with lactose and sucrose utilization. Alkaline production is determined when the red color of phenol red changes to yellow. When a bacterial species ferments the sugars present in the culture media, the media will then turn acidic and thus the bacteria will consequently not be able to ferment any sugar formats any further. In case that the bacteria produce sulfur gas while being cultured in the media, a black precipitate will result on the culture plate. In addition, when a bacterial species generates carbon dioxide or possibly hydrogen gas, one will observed bubbles that will appear in the media.
The EMB plate can differentiate bacterial strains through the principle of fermentation. When a bacterial strain can ferment lactose, acid will be produced and this will accumulate in the culture media, which in turn reacts with the dye that was incorporated in the EMB plate. This reaction can be observed through visual inspection of the plates after 24 hours. Should the bacterial sample be incapable of fermentation of lactose, the colonies will be of pink color and this is simply due to the media uptake.
Bacterial cells should be grown to its exponential growth stage in order to have a sufficient amount of DNA. Once the appropriate amount of bacterial cells is present, the bacterial cells can now be called competent cells because these are ready for DNA manipulation. Plasmid DNA from bacterial cells will be extracted with minipreparation techniques that involve lysing the bacterial cells and centrifuging the cellular solution in order to remove other organelles of the bacterial cell. The bacterial plasmid will then be exposed to the same restriction enzyme that was used in the human cytokine DNA segment, also generating sticky ends that are ready to reassociate with other sticky DNA ends (Zhu 3089). The cleaved human cytokine DNA fragments can then be introduced into the bacterial plasmid because both DNA molecules are sticky. The principle of reassociating foreign and host DNA molecules is to employ the same restriction enzyme so that the sticky ends have the same recognition sequences that are complementary to each other. Once the human cytokine DNA fragment is inserted into the plasmid, it is now possible to let the plasmid make more copies of itself inside the bacterial cell.
Bacterial cells multiply very fast and also, the transcription and translation rates of these cells are very short as compared to human cells (Duarte 107). Molecular biology techniques allow the manipulation of DNA segments of interest. After incubation of the bacterial cultures that contain plasmids that carry the human cytokine genes, it is then possible to allow the bacterial cells to perform the process of translation, which is the production of protein products based on the transcription results. Translation of the specific human cytokine genes in the plasmid allows that production of human cytokine which can then be collected using isolation techniques (Grunstein 3961). The human cytokine is then further purified using mass chromatographic techniques in order to remove any other unnecessary proteins and other smaller cellular material. The human cytokine protein is then resuspended in a stable buffer such as sterile double distilled water or a buffer such as phosphate buffer in saline solution so that the human cytokine protein remains in its native state. The bottled human cytokine products that are now sold in pharmacies are thus produced through the abovementioned techniques.
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