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Bacterias are found everyplace ( Campbell & A ; Reece, 2005 ) . They have the ability to colonise about all home grounds which means they come in broad assortments of signifiers. Understanding bacterial construction can therefore do it possible to understand their maps and endurance. For illustration, merely by analyzing the capsule bed of bacteriums, one can acquire an thought of the pathogenicity of the bacterium. But they grow among diverse and assorted populations, where it is impossible to analyze a individual species of bacteriums. Thus it is necessary that the pure civilization of bacterial settlements be obtained for analyzing. Pure civilization is defined as a mass or group of cells originating from the same parent cell. Pure civilization techniques, such as streak home base, pour home base and spread home base, insulate bacterial settlements from mixtures so that settlements consisting of the indistinguishable beings can be studied. Isolation of pure civilization is critical for qualifying a individual species of bacteriaA­ otherwise presence of contaminations can take to inaccurate observations ( Prescott et al. , 2005 )

Then the stray bacterial settlements undergo incubation at 370C for 24 hours for all the three home bases, after which, a settlement can be selected and isolated once more to obtain pure civilizations. Another measure in the experiment is to execute a differential staining method known as Gram staining, which chiefly differentiates the bacteriums into two classs: Gram positive and Gram negative based on their cell wall constructions ( Campbell and Reece, 2005 ) .

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The species of bacteriums present in the given broth civilization were Escherichia Coli, Staphylococcus Aureus and Bacillus Cereus. The visual aspects of these bacteriums known in theory province that Escherichia Coli are Gram negative and bacillar ; Bacillus Cereus are Gram positive and bacillar while Staphylococcus Aureus are Gram positive and coccus.

So the experiment was carried out to see if it was possible to obtain pure civilizations from assorted broth by using run, spread and pour home base techniques.

Purposes

The chief aims of this experiment were to understand the intent, rules and techniques of Gram staining every bit good as the isolation of pure civilizations. The experiment farther aimed to compare the different isolation techniques, viz. streak home base, spread home base and pour home base in footings of bring forthing pure civilizations, and besides compare the morphological characteristics of three types of bacteriums.

MATERIALS AND METHODS

The process was carried out harmonizing to the instructions stated in the pages 32-36 of the Microbiology lab manual ( MIC2011, Class Notes, 2011 ) .

RESULTS ( combined with Morning Lab, Bench 1, Group A3 )

The full experiment was carried out in three different Sessionss.

Session 1

A broth civilization, labelled as Culture 2, was provided and it was known to incorporate a mixture of Staphylococcus aureus, Escherichia coli and Bacillus Cereus. Sample from the broth civilization was heat-fixed and so gm stained for observation under the light microscope. Three different types of bacteriums were seen and their identifiable features were matched with the three sorts of bacteriums known to be present in the mixture.

Table 1 – Observation of Culture 2 ( the broth civilization ) and word picture of the types of bacteriums seen under the microscope

Speciess

Cellular Morphology

Type of Bacteria

Gram Stain

Shape

Agreement

Type 1

( Gram +ve ) Purple

Coccus

Bunch

Staphylococcus aureus

Type 2

( Gram -ve ) Pink

Rod ( shorter )

Single/Pair

Escherichia coli

Type 3

( Gram +ve ) Purple

Rod ( longer )

Chain

Bacillus Cereus

After the presence of all three types of bacterium was confirmed, loopfuls from the broth civilization were taken in order to fix pure civilizations utilizing three isolation techniques – run home base, spread home base and pour home base. Once the three isolation home bases were ready, they were incubated overnight at 37A°C for the following session.

Session 2

After nightlong incubation at 37A°C, the settlements which formed on the run, spread and pour home base were observed under the microscope to see the distribution and the morphology of bacterial settlements. These isolation techniques helped find which bacteriums were responsible for the settlement morphology.

Table 2 – Distribution and observation of bacterial settlements present in run home base, spread home base and pour home base, after one-day incubation at 37A°C

Appearance of Bacterial Colonies

Streak Plate

Spread Plate

Pour Home plate

Distribution

Primary inoculant and initial runs were overlapping while the concluding runs were distinguishable and separate

Overlaping and could non be differentiated

Overlaping and could non be differentiated

Density

High

Higher

Highest

Location

Few big settlements present on the surface of the agar

Overlaping settlements present on the surface of the agar

Distributed on the agar surface every bit good as within the agar medium

Other Features

Assorted characteristics were noted among different settlements

Opaque, guerrilla, smooth and level

White and opaque

Since distinguishable and well-isolated settlements were non found from the spread home base and pour home base techniques, choice of distinguishable and clear settlements of all three types of bacteriums were made from the run home base and observed under the microscope.

Table 3 – Morphology of the bacterial settlements

Appearance

Colony 1

Colony 2

Colony 3

Shape

Round

Round

Irregular

Size

Small

Medium

Large

Surface

Smooth and glittering

Smooth and glittering

Rough and dull

Elevation

Flat

Convex

Raised

Color

Pale yellow

Yellow

White

Edge

Rippling

Smooth

Rippling

Opacity

Opaque

Transparent

Opaque

Identity

Staphylococcus aureus

Escherichia coli

Bacillus Cereus

Session 3

The stray settlements were incubated overnight at 37A°C and inoculated onto new alimentary agar ( NA ) plates, after which pure home bases of each type of bacteriums were prepared and Gram stained followed by the observation of individual settlements of bacteriums under the microscope.

Table 4 – Observation of cellular and colonial morphology

Type of Bacteria

Colony Morphology

Cellular Morphology

Gram Stain

Shape

Arrange-ment

Other Features

Staphylococcus aureus

Smallest in size ; round ; level ; smooth surface and undulating borders ; pale xanthous and opaque

( Gram +ve ) Purple

Coccus

Bunch

Pure civilization was extracted. No taint.

Escherichia coli

Medium in size ; round ; convex ; smooth surface and borders ; yellow and transparent

( Gram -ve ) Pink

Rod ( shorter )

Single/

Pair

Pure civilization was extracted. No taint.

Bacillus Cereus

Largest in size ; irregular ; raised ; unsmooth surface and undulating borders ; white and opaque

( Gram +ve ) Purple

Rod ( longer )

Chain

Pure civilization was extracted. No taint.

Discussion

( 1 ) As seen in the table-4 of consequences, Bacillus Cereus had the largest settlement with irregular form, raised lift, unsmooth and dull surface, undulating borders, opaque and white in coloring material. They are Gram positive and bacillar, which are arranged in ironss.

The medium-sized settlement was the Escherichia coli with a round form, convex lift, smooth and glittering surface, smooth borders, transparent and yellow in coloring material. They are Gram negative and are seen under the microscope as short bacillar constructions bing in braces or individual cells.

The smallest of all settlements belonged to the Staphylococcus aureus, with a round form, smooth and glittering surface, undulating borders, opaque in pale yellow in coloring material. They are Gram positive coccus arranged in grape-like bunchs and they do non organize spores ( Mahon et al. , 2007 ) . The cellular morphology of bacterium was observed by Gram staining while the settlement morphology was seen utilizing streak home base technique. The morphology of each bacteria observed in the pure cultures matched with those in the original mixture, but the settlement size in pure civilization was much bigger than those in assorted civilization. The likely ground for this is that multiple bacterial settlements experience competition for foods while settlements in pure civilization had sufficient nutrient and infinite. No taint was found and single bacterial settlements were separated on an agar surface.

( 2 ) Among the three techniques used, streak home base is the most efficient manner to bring forth well-isolated settlements, or in other words, pure settlements. In streak home base technique, the micro-organisms incorporating many feasible cells, were straight plated. However, in order for the technique to work good, there must be big populations of the being in the original mixture ( Sumbali and Mehrotra, 2009 ) . Otherwise, the concluding runs consequence in less or no settlements because in the run home base method, the bacterial suspension is diluted more and more as it proceeds from the primary inoculants towards the latter run. Streak home base is a cost-efficient and rapid-active method for dividing bacteriums in assorted civilizations of high denseness and can besides be repeated for accomplishing the coveted pureness. For these grounds, streak home base technique is widely used in research labs ( Pommerville, 2010 ) . One disadvantage of streak home base is that the hazard of taint is higher since the home base is exposed to air for several times ( Sumbali and Mehrotra, 2009 ) . For more diluted populations, spread home base and pour home base are considered since they are easy to turn up settlements among low denseness of equally distributed populations ( Willey et al. , 2011 ) . Consecutive dilutions cut down the microbic population ( Willey et al. , 2011 ) . For dispersed home base, 0.1 milliliter of the bacterial suspension was taken from 10-2 dilution and spread over the agar whereas for pour home base, 1 milliliter of the suspension was taken from the 10-2 dilution. Although dispersed home base can divide a bacterial settlement and it is more sterile than streak home base, it is non executable for insulating settlements from a mixture because the method is time-consuming and the settlements are non easy differentiated. However streak home base can be used to number microbic populations ( Prescott et al. , 2005 ) . Similarly, pour home bases are besides used for numbering microbic populations. Although it has the least hazard of taint, the procedure is time-consuming and the settlements are difficult to separate or number since the settlements besides grow inside the agar, for which this technique is non used to stray settlements from a mixture. Pour home base is used for insulating and numbering anaerobiotic bacteriums since these bugs can non last in atmospheric degrees of O and are hence merely found within the agar and non on the surface of the agar ( Hogg, 2005 ) .

( 3 ) Gelatin is colorless, brickle and semitransparent medium ( Willey et al. , 2011 ) . In this experiment, agar was used as the civilization medium. Agar is preferred over gelatin for doing solid media because of several grounds. First of all, unlike gelatine, agar contains complex polyoses that can non be nutritionally digested or degraded by most micro-organisms to organize precipitates, since the incubation temperature of agar is about 20A°C, which is below the optimal temperature of most beings and this enables the agar to stay solid when micro-organisms are turning on it ( Hogg, 2005 ) . Second, agar sets steadfastly and strongly, supplying a stable civilization medium ( Willey et al. , 2011 ) . Furthermore, agar has a broad scope of pH from 5 to 8 and besides solidifies below 40A°C, for which liquid agar can be poured over the temperature sensitive foods without damaging them. Furthermore, the runing point of solid agar is above 100A°C and hence can be used to civilization thermophiles ( Bauman, 2006 ) . Finally, agar, being porous, can besides demo motility of the bacteriums ( Prescott et al. , 2005 ) .

( 4 ) Unlike bacterial settlements with heavy growing, the well-separated settlements do non hold deficit of nutrient or infinite, which prevents congestion and competition for resources and therefore the bacterial growing rate is higher, for which they appear larger. Growth rate of bacterial cells in well-separated countries is farther enhanced by the higher diffusion rate of the O consumption by the cells and releasing of toxic metabolites out of the cell ( Willey et al. , 2011 ) . On the other manus, bacteriums in countries of high growing face competition for nutrient and infinite for which their growing rate every bit good as survival rate is lower. This is likely the ground why Staphylococcus aureus, grew ab initio, but so stopped.

In this experiment, one time the bacteriums were isolated, they were Gram stained and checked to guarantee that no taint had taken topographic point and that each settlement had individual species of bacteriums. ( 5a ) An single settlement might incorporate more than one type of bacteria due to taint. Common grounds for taint are improper sterilisation of inoculating cringle or absence of sterile techniques or sometimes even under sterilized environments. Contaminants are besides present in the extracellular sludge bed of bacteriums and besides in those bacteriums which are joined in a web of ironss. Another ground for non holding the same type of bacteria in a settlement, although really rare, is mutant and this consequences in the presence of multiple mutant strains of bacteriums being present in a individual settlement ( Pommerville, 2010 ) . ( 5b ) Bacteria reproduce asexually by a procedure called binary fission where a parent cell divides into and subsequent divisions take topographic point. In this manner, a settlement can originate from multiple cells of same or different species of bacteriums which are deposited together on a solid medium and this is how a settlement may non ever have the same parent cell. Even when bacteriums exist in bunchs, non needfully are they from a individual parent cell ( Hogg, 2005 ) .

( 6 ) Passing the dried vilification through the Bunsen fire is known as heat repair. By making so, the micro-organisms are killed and attached steadfastly to the slide due to the curdling of the proteins ; otherwise they would rinse off with the discolorations. Heat repairing besides alters the construction of the micro-organisms, continuing the general morphology, so that they become permeable to discolorations ( Sumbali and Mehrotra, 2009 ) .

( 7 ) A whole settlement would ensue in a thick vilification, which non merely look overlapping and crowded under the microscope, but besides prevent the diffusion of the dyes across the cell, for which the intoxicant would non be able to efficaciously bleach the cells, doing bulk of the cells to be stained purple. Besides different types of bacterial cells have different staining methods which may acquire confounding. Therefore, it would acquire really hard to place single cells.

( 8 ) There is no association between bacterial cell form and reaction to Gram discoloration. Gram staining depends on the thickness of the bacterial cell wall which is composed of peptidoglycan. Peptidoglycan consists of abundant teichoic acid, a thin bed of periplasmic infinite and a peptide interbridge ( Prescott et al. , 2005 ) . Both Gram positive and Gram negative bacteriums come in assorted forms. For case, Staphylococcus aureus ( coccus-shaped ) and Bacillus Cereuss ( bacillar ) are both Gram positive.

( 9 ) The bacterial cell wall construction determines its Gram discoloration reaction. Bacterial cell walls are composed of peptidoglycan which maintain rigidness and form of the bacterial cell and besides give protection from osmotic lyses ( Prescott et al. , 2005 ) . However, in Gram positive bacteriums, 90 % of the cell wall is made up of peptidoglycan, for which the cell walls in Gram positive bacterium is thicker than in Gram negative bacteriums where the peptidoglycan content is merely 5 to 20 % . The decolourisation depends on the peptidoglycan content in the cell wall and hence determines whether the cell will be Gram stained or non. Upon add-on of intoxicant, the pores of the thick peptidoglycan beds shrink in the cell wall of Gram positive bacteriums, which consequences in desiccation of the bed and retaining of the discoloration, for which they appear violet under the microscope. As for Gram negative bacteriums, the intoxicant wash opens the pores of the peptidoglycan bed and the discoloration is non retained in the peptidoglycan bed ( Prescott et al. , 2005 ) .

( 10 ) The measure which is really important in finding the result of Gram staining is the decolourisation with intoxicant. The cell wall construction of Gram positive bacteriums allows the crystal-violet I dye composite ( CV-I composite ) to be retained within the thick peptidoglycan bed when intoxicant is added. On the contrary, the cell wall construction of Gram negative bacteriums allows the CV-I composite to be removed from the peptidoglycan bed upon add-on of intoxicant. It is for the decolourization measure that the Gram negative bacteriums appear pink when counterstained with Safranin and Gram positive 1s appear violet, otherwise CV-I composite would be present in both types of bacterium and both would be seen violet under the microscope.

( 11 ) However every measure in Gram staining is necessary to obtain the right consequence. If any of the stairss is eliminated, the consequences can alter and are misdirecting. The tabular array below shows the assorted results when the stairss in Gram staining are changed.

Table 5 – Consequences from altered stairss in the Gram staining procedure:

A

Bacillus

C

Calciferol

Staphylococcus aureus

( Gram positive )

Tap

Tap

Dark purple

Dark purple

Escherichia coli

( Gram negative )

Tap

Tap

Dark purple

Colourless

Bacillus Cereus

( Gram positive )

Tap

Tap

Dark purple

Dark purple

When crystal violet wash is eliminated in measure A, none of the cells will acquire the violet coloring material and therefore all cells are stained pink when washed with Fuchsin. In measure B, when the I wash is eliminated, the CV-I composite does non organize and the crystal violet molecules are non big plenty to be retained in the peptidoglycan. Thus they are washed off with the H2O and intoxicant, for which even the Gram positive cells appear pink. In measure C, with the riddance of intoxicant wash, decolourization is non done, for which all cells, including the Gram negative one where the CV-I complex masks the pink coloring material, are stained violet ( Willey et al. , 2011 ) . When Fuchsin wash is eliminated in measure D, no alteration is brought on the Gram positive cells, but the Gram negative cells appear colourless, the ground being that after rinsing off the crystal violet with intoxicant, no counterstain is added to the Gram negative cells ( Willey et al. , 2011 ) .

A restriction in this experiment is non executing the Gram discoloration when the species of bacteriums were identified on the run home base and once more streaked on the alimentary agar home bases. The bacterial species were of taken based on their settlement morphology. However a Gram discoloration could hold confirmed if the settlements comprised of the expected bacteriums or non.

Decision

It was compulsory that sterile techniques be followed when experimenting with micro-organisms. Compared to distribute home base and pour home base techniques, streak home base was found to be the most efficient and besides the easiest manner to insulate bacterial settlements. Pure civilizations of three types of bacteriums that were obtained included Staphylococcus aureus ( bacillar and Gram positive ) , Bacillus Cereus ( cocci-shaped and Gram positive ) and Escherichia coli ( bacillar and Gram negative ) . Other than that, importance of heat repair and Gram staining was understood. Gram staining proved to be a really good method for detecting the bacterial cellular characteristics and hence, differences between Gram positive and Gram negative bacteriums were clarified. It was besides understood that reaction of a bacterial cell to the Gram discoloration is determined by the decolourization measure.

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