Isolation And Observation Of Bacteria Using Pure Culture Methods Biology Essay

Isolation And Observation Of bacterium Using uncontaminated Culture Methods Biology EssayINTRODUCTIONBacteria argon put everywhere (Campbell Reece, 2005). They piss the ability to colonize almost alone habitats which gist they come in big varieties of forms. Understanding bacteriuml structure displace therefore make it possible to understand their functions and survival. For example, just by lavatoryvass the capsule story of bacterium, one nates get an idea of the pathogenicity of the bacteria. to a greater extentover they grow among diverse and fuse populations, where it is impossible to study a champion species of bacteria. Thus it is necessary that the pure conclusion of bacterial colonies be obtained for studying. unadulte vagabondd culture is defined as a mass or base of prison cellphones arising from the same recruit cell. Pure culture techniques, such as chevron scale leaf, pour rest home and stagger place, seize bacterial colonies from mixtures so that colonies comprising of the analogous organisms can be studied. Isolation of pure culture is vital for characterizing a single species of bacteria otherwise front end of contaminants can lead to faulty observations (Prescott et al., 2005)Then the dislocated bacterial colonies undergo pensiveness at 370C for 24 hours for all the terce cases, afterward which, a addiction can be selected and isolated once more to obtain pure cultures. Another step in the look into is to perform a contrastingial steeling mode cognize as guanine staining, which mainly differentiates the bacteria into two categories g substantiative and yard minus establish on their cell fence in structures (Campbell and Reece, 2005).The species of bacteria vex in the given broth culture were Escherichia Coli, staphylococcus Aureus and barn Cereus. The waitances of these bacteria known in theory state that Escherichia Coli atomic number 18 one thousand ostracise and rod-shaped group B Ce reus are yard confident(p) and rod-shaped while Staphylococcus Aureus are gravitational constant autocratic and coccus.So the experiment was carried turn out to hit if it was possible to obtain pure cultures from mixed broth by applying streamlet, spread and pour dwelling house techniques.AIMSThe main objectives of this experiment were to understand the purpose, principles and techniques of chiliad staining as well as the closing off of pure cultures. The experiment further aimed to compare the different isolation techniques, videlicet streak ordered series, spread plate and pour plate in call of producing pure cultures, and alike compare the morphological features of leash pillow slips of bacteria.MATERIALS AND METHODSThe procedure was carried out according to the instructions stated in the pages 32-36 of the Microbiology lab manual (MIC2011, menage Notes, 2011).RESULTS (combined with Morning Lab, Bench 1, Group A3)The entire experiment was carried out in three dif ferent sessions. academic session 1A broth culture, denominate as Culture 2, was provided and it was known to contain a mixture of Staphylococcus aureus, Escherichia coli and Bacillus genus Cereus. Sample from the broth culture was warmheartedness-fixed and then gram stain for observation under the light microscope. Three different types of bacteria were seen and their recognisable characteristics were matched with the three kinds of bacteria known to be set out in the mixture. hedge 1 Observation of Culture 2 (the broth culture) and characterisation of the types of bacteria seen under the microscopeSpeciesCellular MorphologyType of Bacteria universal gravitational constant brand skeletal systemArrangementType 1(Gram +ve) PurpleCoccus bunch togetherStaphylococcus aureusType 2(Gram -ve) Pink rod (shorter)Single/ pas de deuxEscherichia coliType 3(Gram +ve) PurpleRod (longer)ChainBacillus genus CereusAfter the presence of all three types of bacteria was confirmed, loopfuls fro m the broth culture were taken in order to prepare pure cultures using three isolation techniques streak plate, spread plate and pour plate. Once the three isolation plates were ready, they were incubated overnight at 37C for the next session.Session 2After overnight incubation at 37C, the colonies which organise on the streak, spread and pour plate were observe under the microscope to see the distri entirelyion and the sound structure of bacterial colonies. These isolation techniques helped determine which bacteria were obligated for the small town morphology. control panel 2 Distribution and observation of bacterial colonies present in streak plate, spread plate and pour plate, after one-day incubation at 37CAppearance of Bacterial ColoniesStreak musical scaleSpread PlatePour PlateDistributionPrimary inoculum and initial streaks were overlapping while the final streaks were distinct and separate imbrication and could not be severaliseOverlapping and could not be differe ntiatedDensityHighHigherHighestLocationFew ample colonies present on the fall out of the agar-agar-agar-agar-agarOverlapping colonies present on the surface of the agarDistributed on the agar surface as well as within the agar mediumOther FeaturesVarious features were noted among different colonies swarthy, guerrilla, dispassionate and flatWhite and opaqueSince distinct and well-isolated colonies were not base from the spread plate and pour plate techniques, selection of distinct and release colonies of all three types of bacteria were do from the streak plate and observed under the microscope.Table 3 Morphology of the bacterial coloniesAppearance resolution 1Colony 2Colony 3Shape nebCircularIrregularSizeSmallMediumLarge draw closeSmooth and glisteningSmooth and glisteningRough and dull heightFlatConvexRaisedColorPale yel beginning-bellied jaundicedWhiteEdgeUndulatingSmoothUndulatingOpacityOpaqueTransparentOpaqueIdentityStaphylococcus aureusEscherichia coliBacillus cer eusSession 3The isolated colonies were incubated overnight at 37C and inoculated onto new nutrient agar (NA) plates, after which pure plates of each type of bacteria were prepared and Gram stained followed by the observation of single colonies of bacteria under the microscope.Table 4 Observation of cellular and colonial morphologyType of BacteriaColony MorphologyCellular MorphologyGram StainShapeArrange-mentOther FeaturesStaphylococcus aureusSmallest in size gamin flat smooth surface and undulating edges pale yellow and opaque(Gram +ve) PurpleCoccus gatherPure culture was extracted. No contamination.Escherichia coliMedium in size circular convex smooth surface and edges yellow and transparent(Gram -ve) PinkRod (shorter)Single/PairPure culture was extracted. No contamination.Bacillus cereusLargest in size irregular raised rough surface and undulating edges snowy and opaque(Gram +ve) PurpleRod (longer)ChainPure culture was extracted. No contamination.DISCUSSION(1) As seen in the ta ble-4 of results, Bacillus cereus had the largest colony with irregular shape, raised elevation, rough and dull surface, undulating edges, opaque and white in colour. They are Gram positive and rod-shaped, which are arranged in chains.The moderate-sized colony was the Escherichia coli with a circular shape, convex elevation, smooth and glistening surface, smooth edges, transparent and yellow in colour. They are Gram negative and are seen under the microscope as short rod-shaped structures pull rounding 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 yellow in colour. They are Gram positive cocci arranged in grape-like clusters and they do not form spores (Mahon et al., 2007). The cellular morphology of bacteria was observed by Gram staining while the colony morphology was seen using streak plate technique. The morphology of each bacterium observed in the pure cultures matched with those in the trustworthy mixture, but the colony size in pure culture was much bigger than those in mixed culture. The probable actor for this is that fivefold bacterial colonies experience contestation for nutrients while colonies in pure culture had sufficient food and property. No contamination was found and individual bacterial colonies were separated on an agar surface.(2) Among the three techniques employ, streak plate is the most efficient way to enhance well-isolated colonies, or in other words, pure colonies. In streak plate technique, the microorganisms containing many viable cells, were directly plated. However, in order for the technique to reckon well, there must be large populations of the organism in the original mixture (Sumbali and Mehrotra, 2009). Otherwise, the final streaks result in less or no colonies because in the streak plate method, the bacterial suspension is diluted more and more as it proceeds from the primary inocu lums towards the latter streaks. Streak plate is a cost-effective and rapid-active method for separating bacteria in mixed cultures of lavishly density and can alike be repeated for achieving the desired purity. For these reasons, streak plate technique is widely used in laboratories (Pommerville, 2010). One damage of streak plate is that the risk of contamination is racyer(prenominal) since the plate is expose to air for several times (Sumbali and Mehrotra, 2009). For more diluted populations, spread plate and pour plate are considered since they are easy to locate colonies among low density of evenly distributed populations (Willey et al., 2011). Serial dilutions reduce the microbial population (Willey et al., 2011). For spread plate, 0.1 ml of the bacterial suspension was taken from 10-2 dilution and spread over the agar whereas for pour plate, 1 ml of the suspension was taken from the 10-2 dilution. Although spread plate can separate a bacterial colony and it is more uninsp ired than streak plate, it is not feasible for isolating colonies from a mixture because the method is time-consuming and the colonies are not easily differentiated. However streak plate can be used to count microbial populations (Prescott et al., 2005). Similarly, pour plates are also used for counting microbial populations. Although it has the least risk of contamination, the figure out is time-consuming and the colonies are hard to distinguish or count since the colonies also grow inside the agar, for which this technique is not used to isolated colonies from a mixture. Pour plate is used for isolating and counting anaerobic bacteria since these microbes cannot survive in atmospheric levels of oxygen and are therefore scarce found within the agar and not on the surface of the agar (Hogg, 2005).(3) Gelatin is colourless, brittle and translucent medium (Willey et al., 2011). In this experiment, agar was used as the culture medium. Agar is preferred over gelatin for qualification solid media because of several reasons. First of all, unlike gelatine, agar contains knotty polysaccharides that cannot be nutritionally digested or degraded by most microorganisms to form precipitates, since the incubation temperature of agar is about 20C, which is below the optimum temperature of most organisms and this enables the agar to remain solid when microorganisms are growing on it (Hogg, 2005). Secondly, agar sets securely and strongly, providing a stable culture medium (Willey et al., 2011). Moreover, agar has a wide range of pH from 5 to 8 and also solidifies below 40C, for which liquid agar can be poured over the temperature sensitive nutrients without detrimental them. Furthermore, the melting point of solid agar is above 100C and therefore can be used to culture thermophiles (Bauman, 2006). Finally, agar, being porous, can also show motility of the bacteria (Prescott et al., 2005).(4) Unlike bacterial colonies with weighed down growth, the well-separated colonie s do not have shortage of food or space, which prevents overcrowd and competition for resources and thus the bacterial growth rate is higher, for which they appear larger. harvest rate of bacterial cells in well-separated areas is further enhanced by the higher diffusion rate of the oxygen uptake by the cells and releasing of harmful metabolites out of the cell (Willey et al., 2011). On the other hand, bacteria in areas of high growth face competition for food and space for which their growth rate as well as survival rate is lower. This is probably the reason why Staphylococcus aureus, grew initially, but then stopped.In this experiment, once the bacteria were isolated, they were Gram stained and checked to ensure that no contamination had taken place and that each colony had single species of bacteria. (5a) An individual colony cleverness contain more than one type of bacterium due to contamination. green reasons for contamination are improper sterilization of inoculating loop or absence of unfertile techniques or sometimes even under sterilized environments. Contaminants are also present in the extracellular slime layer of bacteria and also in those bacteria which are joined in a network of chains. Another reason for not having the same type of bacterium in a colony, although very rare, is mutation and this results in the presence of multiple mutant strains of bacteria being present in a single colony (Pommerville, 2010). (5b) Bacteria reproduce asexually by a process called binary fission where a parent cell divides into and posterior divisions take place. In this way, a colony can arise from multiple cells of same or different species of bacteria which are deposited together on a solid medium and this is how a colony whitethorn not always have the same parent cell. Even when bacteria exist in clusters, not necessarily are they from a single parent cell (Hogg, 2005).(6) Passing the dried smear through the Bunsen ardor is known as heat fixing. By do ing so, the microorganisms are killed and attached severely to the slide due to the coagulation of the proteins otherwise they would wipe off with the stains. light fixing also alters the structure of the microorganisms, preserving the general morphology, so that they become pervious to stains (Sumbali and Mehrotra, 2009).(7) A whole colony would result in a three-ply smear, which not only appear overlapping and crowded under the microscope, but also prevent the diffusion of the dyes across the cell, for which the alcohol would not be able to effectively decolorize the cells, causing majority of the cells to be stained regal. Also different types of bacterial cells have different staining methods which may get confusing. on that pointfore, it would get very difficult to identify individual cells.(8) There is no association between bacterial cell shape and reception to Gram stain. Gram staining depends on the thickheadedness of the bacterial cell wall which is composed of pe ptidoglycan. Peptidoglycan consists of abundant teichoic acid, a thin layer 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 determines its Gram stain reaction. Bacterial cell walls are composed of peptidoglycan which maintain rigidity and shape of the bacterial cell and also give protection from osmotic lyses (Prescott et al., 2005). However, in Gram positive bacteria, 90% of the cell wall is made up of peptidoglycan, for which the cell walls in Gram positive bacteria is thicker than in Gram negative bacteria where the peptidoglycan content is only 5 to 20%. The decolourisation depends on the peptidoglycan content in the cell wall and therefore determines whether the cell result be Gram stained or not. Upon addition of alcohol, the pores of the thick peptidoglycan layers shrink in the cell wall of Gram positive bacteria, which results in dehydration of the layer and retaining of the stain, for which they appear purple under the microscope. As for Gram negative bacteria, the alcohol wash opens the pores of the peptidoglycan layer and the stain is not maintained in the peptidoglycan layer (Prescott et al., 2005).(10) The step which is very crucial in ascertain the outcome of Gram staining is the decolourisation with alcohol. The cell wall structure of Gram positive bacteria allows the crystal-violet iodine dye multifactorial (CV-I complex) to be hold within the thick peptidoglycan layer when alcohol is added. On the contrary, the cell wall structure of Gram negative bacteria allows the CV-I complex to be aloof from the peptidoglycan layer upon addition of alcohol. It is for the decolourization step that the Gram negative bacteria appear pink when counterstained with Safranin and Gram positive ones appear purple, otherwi se CV-I complex would be present in both types of bacteria and both would be seen purple under the microscope.(11) Nonetheless every step in Gram staining is necessary to obtain the correct result. If any of the locomote is eliminated, the results can change and are misleading. The table below shows the various outcomes when the steps in Gram staining are changed.Table 5 Results from altered steps in the Gram staining processABCDStaphylococcus aureus(Gram positive)PinkPink swarthiness purpleDark purpleEscherichia coli(Gram negative)PinkPinkDark purpleColourlessBacillus cereus(Gram positive)PinkPinkDark purpleDark purpleWhen crystal violet wash is eliminated in step A, none of the cells will get the purple colour and thus all cells are stained pink when wash with Fuchsin. In step B, when the iodine wash is eliminated, the CV-I complex does not form and the crystal violet molecules are not large enough to be retained in the peptidoglycan. Thus they are washed off with the water and alcohol, for which even the Gram positive cells appear pink. In step C, with the elimination of alcohol wash, decolourization is not done, for which all cells, including the Gram negative one where the CV-I complex masks the pink colour, are stained purple (Willey et al., 2011). When Fuchsin wash is eliminated in step D, no change is brought on the Gram positive cells, but the Gram negative cells appear colourless, the reason being that after washing off the crystal violet with alcohol, no counterstain is added to the Gram negative cells (Willey et al., 2011).A limitation in this experiment is not performing the Gram stain when the species of bacteria were identified on the streak plate and again streaked on the nutrient agar plates. The bacterial species were of taken based on their colony morphology. However a Gram stain could have confirmed if the colonies comprised of the expected bacteria or not.CONCLUSIONIt was mandatory that aseptic techniques be followed when experimenting with microorganisms. Compared to spread plate and pour plate techniques, streak plate was found to be the most efficient and also the easiest way to isolate bacterial colonies. Pure cultures of three types of bacteria that 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). Other than that, importance of heat fixing and Gram staining was understood. Gram staining proved to be a very good method for observing the bacterial cellular features and hence, differences between Gram positive and Gram negative bacteria were clarified. It was also understood that reaction of a bacterial cell to the Gram stain is determined by the decolourization step.