Posted at 12.11.2018
Bacteria are located almost everywhere (Campbell & Reece, 2005). They be capable of colonize almost all habitats this means they come in huge varieties of forms. Understanding bacterial composition can therefore be able to comprehend their functions and survival. For example, simply by learning the capsule layer of bacteria, one can get a concept of the pathogenicity of the bacteria. But they expand among diverse and mixed populations, where it is impossible to study a single kinds of bacteria. Thus it's important that the pure culture of bacterial colonies be obtained for learning. Pure culture is defined as a mass or band of cells due to the same parent or guardian cell. Genuine culture techniques, such as streak dish, pour plate and spread dish, isolate bacterial colonies from mixtures so that colonies composed of of exactly the same organisms can be analyzed. Isolation of 100 % pure culture is vital for characterizing a single species of bacterias otherwise occurrence of contaminants can lead to inaccurate observations (Prescott et al. , 2005)
Then the isolated bacterial colonies undergo incubation at 37C for 24 hours for all the three plates, after which, a colony can be chosen and isolated again to acquire pure ethnicities. Another part of the experiment is to execute a differential staining method known as Gram staining, which mainly differentiates the bacterias into two categories: Gram positive and Gram negative based on their cell wall membrane constructions (Campbell and Reece, 2005).
The types of bacteria within the given broth culture were Escherichia Coli, Staphylococcus Aureus and Bacillus Cereus. The performances of these bacterias known theoretically declare that Escherichia Coli are Gram negative and rod-shaped; Bacillus Cereus are Gram positive and rod-shaped while Staphylococcus Aureus are Gram positive and cocci.
So the test was carried out to see if it was possible to acquire pure cultures from blended broth by applying streak, pass on and pour dish techniques.
The main goals of this test were to understand the purpose, guidelines and techniques of Gram staining as well as the isolation of natural cultures. The experiment further targeted to compare the different isolation techniques, specifically streak plate, get spread around plate and pour plate in terms of producing real cultures, and also compare the morphological top features of three types of bacteria.
The treatment was completed based on the instructions stated in the pages 32-36 of the Microbiology lab manual (MIC2011, Course Notes, 2011).
The entire test was carried out in three different consultations.
A broth culture, labelled as Culture 2, was provided and it was known to contain a combination of Staphylococcus aureus, Escherichia coli and Bacillus cereus. Test from the broth culture was heat-fixed and then gram stained for observation under the light microscope. Three different types of bacterias were seen and their identifiable characteristics were matched with the three varieties of bacteria regarded as present in the mixture.
After the occurrence of all three types of bacteria was proved, loopfuls from the broth culture were taken in order to prepare pure civilizations using three isolation techniques - streak plate, spread dish and pour plate. Once the three isolation plates were ready, they were incubated over night at 37C for the next session.
After overnight incubation at 37C, the colonies which produced on the streak, pass on and pour dish were observed under the microscope to see the distribution and the morphology of bacterial colonies. These isolation techniques helped determine which bacterias were accountable for the colony morphology.
Since different and well-isolated colonies were not found from the pass on plate and pour plate techniques, collection of distinct and clear colonies of most three types of bacteria were created from the streak plate and noticed under the microscope.
The isolated colonies were incubated overnight at 37C and inoculated onto new nutritional agar (NA) plates, and pure plates of each type of bacterias were ready and Gram stained accompanied by the observation of sole colonies of bacterias under the microscope.
(1) As seen in the table-4 of results, Bacillus cereus acquired the most significant colony with abnormal shape, raised elevation, tough and boring surface, undulating ends, opaque and white in shade. They are Gram positive and rod-shaped, that happen to be set up in chains.
The moderate-sized colony was the Escherichia coli with a circular shape, convex elevation, easy and glistening surface, even edges, transparent and yellowish in colour. These are Gram negative and are seen under the microscope as brief rod-shaped buildings existing in pairs or single cells.
The smallest of all colonies belonged to the Staphylococcus aureus, with a circular shape, smooth and glistening surface, undulating edges, opaque in pale yellowish in colour. They can be Gram positive cocci arranged in grape-like clusters plus they do not form spores (Mahon et al. , 2007). The cellular morphology of bacterias was seen by Gram staining as the colony morphology was seen using streak plate technique. The morphology of each bacterium observed in the pure cultures matched up with those in the original mixture, however the colony size in real culture was much bigger than those in mixed culture. The probable reason for this is that multiple bacterial colonies experience competition for nutrition while colonies in genuine culture possessed sufficient food and space. No contamination was found and specific bacterial colonies were separated with an agar surface.
(2) On the list of three techniques used, streak dish is the most efficient way to produce well-isolated colonies, or quite simply, 100 % pure colonies. In streak dish technique, the microorganisms comprising many viable cells, were straight plated. However, for the technique to work well, there should be large populations of the organism in the original mixture (Sumbali and Mehrotra, 2009). Otherwise, the ultimate streaks lead to less or no colonies because in the streak plate method, the bacterial suspension is diluted more and more as it arises from the primary inoculums for the last mentioned streaks. Streak dish is a cost-effective and rapid-active method for separating bacteria in mixed ethnicities of high density and can even be repeated for achieving the desired purity. Therefore, streak plate strategy is trusted in laboratories (Pommerville, 2010). One downside of streak plate is that the chance of contamination is higher since the plate is subjected to air for a number of times (Sumbali and Mehrotra, 2009). For additional diluted populations, pass on dish and pour plate are considered being that they are easy to locate colonies among low density of consistently distributed populations (Willey et al. , 2011). Serial dilutions reduce the microbial people (Willey et al. , 2011). For get spread around dish, 0. 1 ml of the bacterial suspension was taken from 10-2 dilution and disperse over the agar whereas for pour dish, 1 ml of the suspension was extracted from the 10-2 dilution. Although pass on plate can divide a bacterial colony and it is more aseptic than streak dish, it isn't simple for isolating colonies from a combination because the method is time-consuming and the colonies aren't easily differentiated. However streak dish may be used to rely microbial populations (Prescott et al. , 2005). Likewise, pour plates are also used for keeping track of microbial populations. Although it has the least threat of contamination, the procedure is time-consuming and the colonies are hard to tell apart or count because the colonies also grow inside the agar, that this technique is not used to isolated colonies from a mixture. Pour plate is employed for isolating and keeping track of anaerobic bacteria since these microbes cannot survive in atmospheric degrees of oxygen and are therefore only found within the agar rather than on the surface of the agar (Hogg, 2005).
(3) Gelatin is colourless, brittle and translucent medium (Willey et al. , 2011). In this test, agar was used as the culture medium. Agar is preferred over gelatin for making solid mass media because of several reasons. First of all, unlike gelatine, agar includes sophisticated polysaccharides that cannot be nutritionally digested or degraded by most microorganisms to form precipitates, since the incubation temperature of agar is approximately 20C, which is below the most effective temperature of all organisms and this permits the agar to stay sturdy when microorganisms are growing onto it (Hogg, 2005). Secondly, agar sets securely and firmly, providing a well balanced culture medium (Willey et al. , 2011). Furthermore, agar has an array of pH from 5 to 8 and also solidifies below 40C, for which liquid agar can be poured in the temperature sensitive nutrients without harming them. Furthermore, the melting point of solid agar is above 100C and therefore may be used to culture thermophiles (Bauman, 2006). Finally, agar, being porous, can also show motility of the bacterias (Prescott et al. , 2005).
(4) Unlike bacterial colonies with heavy growth, the well-separated colonies do not have shortage of food or space, which helps prevent over-crowding and competition for resources and thus the bacterial expansion rate is higher, that they appear much larger. Progress rate of bacterial cells in well-separated areas is further enhanced by the bigger diffusion rate of the air uptake by the cells and launching of harmful metabolites from the cell (Willey et al. , 2011). Alternatively, bacteria in areas of high development face competition for food and space for which their growth rate as well as survival rate is lower. This is probably the reason Staphylococcus aureus, grew primarily, but then ceased.
In this test, once the bacteria were isolated, these were Gram stained and checked to ensure that no contaminants had taken place and that each colony had sole species of bacterias. (5a) An individual colony might contain much more than one kind of bacterium credited to contamination. Common known reasons for contamination are incorrect sterilization of inoculating loop or lack of aseptic techniques or sometimes even under sterilized environments. Contaminants are also present in the extracellular slime part of bacteria and also in those bacterias which are joined in a network of chains. Another reason for devoid of the same kind of bacterium in a colony, although very exceptional, is mutation and this leads to the presence of multiple mutant strains of bacteria being present in a single colony (Pommerville, 2010). (5b) Bacterias reproduce asexually by an activity called binary fission in which a parent or guardian cell divides into and succeeding divisions happen. In this manner, a colony can arise from multiple skin cells of same or different types of bacteria which are deposited along on a good medium and this is how a colony might not exactly always have the same mother or father cell. Even when bacteria are present in clusters, definitely not are they from an individual parent or guardian cell (Hogg, 2005).
(6) Moving the dried smear through the Bunsen flame is recognized as heat fixing. In so doing, the microorganisms are wiped out and attached tightly to the glide because of the coagulation of the proteins; otherwise they might rinse off with the stains. Heat correcting also alters the framework of the microorganisms, protecting the overall morphology, in order that they become permeable to discolorations (Sumbali and Mehrotra, 2009).
(7) A complete colony would cause a heavy smear, which not only seem overlapping and packed under the microscope, but also avoid the diffusion of the dyes over the cell, that the alcohol would not have the ability to effectively decolorize the skin cells, causing majority of the cells to be stained crimson. Also different types of bacterial skin cells have different staining methods which might get difficult. Therefore, it could get very difficult to identify specific cells.
(8) There is no relationship between bacterial cell shape and reaction to Gram stain. Gram staining depends upon the thickness of the bacterial cell wall which comprises peptidoglycan. Peptidoglycan consists of numerous teichoic acid, a thin level of periplasmic space and a peptide interbridge (Prescott et al. , 2005). Both Gram positive and Gram negative bacteria come in various shapes. For instance, Staphylococcus aureus (coccus-shaped) and Bacillus cereus (rod-shaped) are both Gram positive.
(9) The bacterial cell wall structure decides its Gram stain reaction. Bacterial cell wall space are comprised of peptidoglycan which maintain rigidity and form of the bacterial cell and also give security from osmotic lyses (Prescott et al. , 2005). However, in Gram positive bacterias, 90% of the cell wall structure comprises of peptidoglycan, that the cell wall space in Gram positive bacteria is thicker than in Gram negative bacteria where the peptidoglycan content is merely 5 to 20%. The decolourisation depends on the peptidoglycan content in the cell wall and therefore can determine if the cell will be Gram stained or not. Upon addition of liquor, the skin pores of the heavy peptidoglycan layers reduce in the cell wall structure of Gram positive bacterias, which results in dehydration of the coating and retaining of the stain, that they appear crimson under the microscope. As for Gram negative bacteria, the alcohol wash opens the pores of the peptidoglycan coating and the stain is not retained in the peptidoglycan covering (Prescott et al. , 2005).
(10) The step which is very crucial in determining the results of Gram staining is the decolourisation with alcoholic beverages. The cell wall structure of Gram positive bacterias allows the crystal-violet iodine dye organic (CV-I organic) to be maintained within the heavy peptidoglycan level when alcohol is added. On the contrary, the cell wall structure composition of Gram negative bacteria allows the CV-I complex to be taken off the peptidoglycan covering upon addition of alcoholic beverages. It is for the decolourization step that the Gram negative bacteria appear pink when counterstained with Safranin and Gram positive ones appear purple, otherwise CV-I complex would be present in both types of bacteria and both would be seen purple under the microscope.
(11) Nonetheless every part of Gram staining is necessary to obtain the correct effect. If the steps is eradicated, the results can change and are misleading. The desk below shows the many final results when the steps in Gram staining are evolved.
When crystal violet rinse is taken out in step A, nothing of the skin cells are certain to get the purple shade and thus all skin cells are stained red when washed with Fuchsin. In step B, when the iodine wash is taken out, the CV-I complex will not form and the crystal violet substances are not large enough to be retained in the peptidoglycan. Thus they can be washed off with this inflatable water and alcohol, that even the Gram positive skin cells appear green. In step C, with the elimination of alcohol wash, decolourization is not done, for which all cells, like the Gram negative one where in fact the CV-I complex masks the red color, are stained purple (Willey et al. , 2011). When Fuchsin clean is eliminated in step D, no change is brought on the Gram positive skin cells, however the Gram negative skin cells appear colourless, this is because after washing off of the crystal violet with alcoholic beverages, no counterstain is added to the Gram negative cells (Willey et al. , 2011).
A limitation in this experiment is not undertaking the Gram stain when the species of bacteria were discovered on the streak dish and again streaked on the nutrient agar plates. The bacterial varieties were of taken based on their colony morphology. However a Gram stain could have proved if the colonies made up of the expected bacterias or not.
It was required that aseptic techniques be used when experimenting with microorganisms. Compared to spread plate and pour dish techniques, streak plate was found to be the most effective as well as the easiest way to isolate bacterial colonies. Pure civilizations of three types of bacterias which were obtained included Staphylococcus aureus (rod-shaped and Gram positive), Bacillus cereus (cocci-shaped and Gram positive) and Escherichia coli (rod-shaped and Gram negative). Besides that, importance of heating correcting and Gram staining was grasped. Gram staining proved to be a very good method for watching the bacterial mobile features and therefore, variations between Gram positive and Gram negative bacteria were clarified. It was also recognized that reaction of a bacterial cell to the Gram stain is determined by the decolourization step.