# Diffusion and Osmosis Through Dialysis tubing Essay

We did this experiment to test the diffusion of different substances through dialysis tubing. We used what we knew about diffusion to make predictions on what we thought the mass of the dialysis tubing to be after submerging them for 30 mins and we knew that diffuse occurs from highest concentration to lowest concentration.

Since the dialysis tubings are filled with different substances than what they are being put into then they should all gain or lose mass. If the dialysis tubing is submerged in different substance than what is in the tubing then some of the dialysis tubings will lose mass and some will gain mass.

To begin this experiment we filled 5 dialysis tubings with one of the five substances: water, egg white, NaCl, glucose, or sucrose.

The equipment used was: 5 dialysis tubing, a scale, bekers, water, egg white, sucrose, glucose, NaCl, and 5 cups. We put the five dialysis tubing filled with one of the five substance into a cup filled with one of the substance: water, egg white, NaCl, glucose, or sucrose. Then we let them sit in there for 30 mins then took them out of the cups and remassed them.

The dialysis tubing did let some of the different solutions in but not all of them. Most of the dialysis tubing gained mass and some lost some mass as well. We found that the substances go from an area of high concentration to an area of low concentration. The data supports our purpose of doing this experiment to find out what happens when a substance in a dialysis tubing is put into a cup of a difference substance to see which ones gain mass and which ones lose mass. A pattern of the data is that the control group did not gain or lose mass. Two of the substances gained mass and two of the substance lost mass. Our results proved our hypothesis that some of the dialysis tubings would gain mass and some of the dialysis tubing would lose mass. Because two of the dialysis tubings gained mass and two of the dialysis tubings lose mass it just shows that the substances went for an area of high concentration to an area of low concentration.

A few errors of our experiment is that we could have not used enough of the substances in the dialysis tubings or we could have left the dialysis tubings the cups of substance for a long amount of time which could have changed our results majorly because the more time the dialysis tubings sit in the cup of substances the more they had time to diffuse.

Osmosis through Dialysis Tubes

We performed this experiment to see how water moves across a semi-permeable membrane. We filled the dialysis tubes with different Mole concentrations of sucrose, and we used our knowledge of osmosis to figure out the different concentrations. If the dialysis tube increases both in size and weight, then that dialysis tube had the highest mole concentration of sucrose.

We learned that during osmosis, a form of passive diffusion which means that it requires no energy to move across the membrane, water will always move to the area where the water concentration is lower, so if the sucrose has a high mole concentration then the water will move into the bag since there is less water there, and if the bag has a low mole concentration then there will be little water moving into the bag since there is already a relatively high concentration in the bag.

The materials we used to conduct this experiment were: 6 Dialysis Tubes, 6 different mole concentrations of sucrose (0 M concentration, 2 x 10⁻¹M concentration, 4 x 10⁻¹ M concentration, 6 x 10⁻¹ M concentration, 8 x 10⁻¹ M concentration, 1 M concentration), 6 cups to hold the water, a scale to weigh the mass of the dialysis tubes before submerging the bags in water and after.

To begin this experiment we first filled the bags with the different mole concentrations of sucrose, the different concentrations were color-coordinated with different colors for a different concentration. Next we weighed the mass of the bags before submerging them in the water, and filled the cups with water so that we could submerge the tubes. After the bags were massed and the cups were filled with water we submerged the bags for 30 minutes. After the 30 minutes were up we took the bags out of the water and blotted them off with a dry paper towel. Finally we massed the bags and recorded our results so that we could compare them with the results from before we submerged the tubes.

After analyzing our results we concluded that the Blue substance was water, because it gained no mass,

The purple substance was the 2 x 10⁻¹ M concentration because it gained little mass, more than the blue substance.

The light green substance the 4 x 10⁻¹ M concentration because it gained some mass, more than the 2 x 10⁻¹ .concentration but not as much as the 6 x 10⁻¹ M concentration.

The red substance was the 6 x 10⁻¹ M concentration because it gained more mass than the 4 x 10⁻¹ M concentration but less than the 8 x 10⁻¹ M concentration.

The green substance was the 8 x 10⁻¹ M concentration because it gained more mass than the .6 Molar concentration but less than the 1 M concentration, and finally the Yellow substance was the 1 M concentration, because it gained the most mass.

Our results answered our question, How can you tell the molar concentration of a 0 .2, .4, .6, .8 , and 1 molar concentration of sucrose? Our results showed us that our hypothesis, If the dialysis tube increases both in size and weight, then that dialysis tube had the highest mole concentration of sucrose, was also correct because the Yellow substance gained the most mass out of all the other substances and was also the substance with the highest Molar concentration of 1, and the Blue concentration gained no mass therefore it was water, because if there is as much water on the inside as the outside then no osmosis takes place.

Some sources of error for this experiment is that we could have left the solutions in longer, possibly changing our results. Or that we could have also not used enough of the solutions.

Diffusion using Potato rounds

We are conducting this experiment in order to see what happens during the process known as diffusion across a semi-permeable membrane. Our hypothesis was that if the Molar concentration is higher in a sucrose solution then the potato will lose mass and if the Molar concentration is lower in a sucrose solution then the potato will gain mass.

We learned about diffusion and how a semi-permeable membrane only lets certain molecules pass through it. Diffusion is the act of a molecule passively passing through a semi-permeable membrane. This action of diffusion helps regulate the cell’s processes and this is done on a regular basis so that the cell can live and function.

For this experiment we needed: 36 potato sticks (18 of a sweet potato and 18 of a regular potato), 6 sucrose solutions (0M, .2M, .4M, .6M, .8M, and 1M), a scale, cups. First what we did was we massed our potato sticks, and recorded them. Next we filled the cups with the different solutions of sucrose and submerged the potato sticks for 30 minutes. After the 30 minutes we massed the potato sticks and recorded the changes and analyzed the previous data with the data we received, here are our results:

After analyzing our results, we concluded that the potato sticks in the Blue concentration gained the most mass out of all the solutions, and the yellow concentration grained not mass, but instead lost mass. This is because water will move from an area of high concentration to an area of low concentration. We also calculated the water potential of the potato sticks and found it to be -7.86 for a regular potato and -17.01 for the sweet potato.

All of the experiments that we did, the substances had a semi-permeable membrane that only let certain things through it. All of the experiments consisted of putting different substances into cups filled with different solution and seeing if they lost or gained mass dependent on if the amount of water that was in the substances and the solutions. There was a pattern throughout all of the experiments where one of the substances in the cups of solution gained nor lost mass but stayed the same and two of the substances in the cups of solution gained mass and two of the substances in the cups of solution lost mass.

# Australia’s Population Distribution and Density Essay

The physical environment has determined to some extent, the distribution and density of Australia’s population. Discuss. People do not live evenly spread through the world. Australia is one of the counties that considered has a low population density. This is due to approximately 23 million population were distributed unevenly. Besides the physical environment, there are also a variety of other factors that have actually worked together to distribute in the Australia’s population that cannot be neglected.

These included the economic and history factors.

The physical environment is one of the well-known factors that affect the distribution and density of Australia’s population. This refers to the landforms, climate, flora and fauna and of course the components including people constantly interact and affect each other. Australia is very famous with its diversity of animals and plants that are able to survive in the harsh and extreme climate such as the marsupials and eucalypts, which had, actually attracts many people to live in this unique land.

The climate is rather dry due to the little rainfall, and this caused mass migration of people to other places. Therefore, this results in the different population density in different region. Australia’s economic has also contributed to the distribution and density of its population. This is due to the reliance on maritime trade in the past. The first European settlement was located on the coast. Therefore, most of the industries were attracted to these centres. This provides a variety of job opportunities to the people.

Therefore, many people have settled down there. In the early days, coastal shipping was the main transportation to them because the land routes were difficult to construct and take time to cross. The main function of that is to transport commodities to ports for the exportation to all over the world. This has directly influenced many of the employers and they have found that it is easier to attract labour to coastal location, which has also significantly increased the number of population in that region.

The most significant point is, Australia is rich with its natural resources such as iron ore and coal. These are mainly exported to all over the world and hence planted many business and investment opportunities. Colonial historical factor tends to affect the distribution and density of Australia’s population too. In the past, the vegetation patterns and agricultural regions are the most significant points. Most of the interior of the country is uninhabitable so people mainly lived on the coast, especially on the east coast where conditions are better.

Besides that, most of the major urban cities have developed on the sites that first settled by the European invaders and settlers. The capital city is the main area of dense population while the rural areas are considered as a very low dense populated region in each state and territory. This is because people can easily get food from the coastal area, which has now been adopted to be the city of the state. Australia is meant to be a very sparsely populated country. This is due to the many factors present which actually played a role in it.

It includes the physical environment, economic and history factors. However, some statistics show that there is a sudden growth in the distribution and density of Australia’s population since 1990s as people started to migrate into Australia from all over the world. In a nutshell, there will be an ongoing growth in the distribution and density of Australia’s long-term population that will also brings a lot of goods such as financial and social benefits. (575 words)

# Nature of Communities Essay

At the beginning of the 20th century there was much debate about the nature of communities. The driving question was whether the community was a self-organized system of co-occurring species or simply a haphazard collection of populations with minimal functional integration (Verhoef, 2010). Krebs (1972) described a community as an assemblage of populations of living organisms in a prescribed area or habitat. However, according to Wright (1984), the working definitions of community can be divided into two basic categories: organismic or individualistic. The organismic approach contends that communities have discrete boundaries and that the sum of the species in an area behaves as organism with both structure and function.

In contrast, the individualistic concept regards communities as collections of species requiring similar environmental conditions (Wright, 1984).

A)Organismic versus individualistic distribution

Solomon (2005) stated that the nature of communities is discussed based on two traditional views which are Clements’s organismic model and Gleason’s individualistic model. The organismic model views community as a superorganism that goes through certain stages of development (succession) toward adulthood (climax).

In this view, biological interactions are primarily responsible for species composition, and organisms are highly interdependent. In contrast, according to individualistic model, abiotic environmental factors are the primary determinants of species composition in a community, and organisms are largely interdependent on each other.

According to organismic concept it is expected that an entire community or biome will respond as a unit and to relocate as climatic conditions change. Pleistocene biome migration in response to multiple glaciations, the accordian effect, is a classic example of this model (Wright, 1984). In contrast, Wright (1984) further explained that the individualist expects each species experiencing similar climatic changes to respond independently and thus, the community composition of an area to change via both immigration and emigration of some individual taxa while others remain in the area. Communities are not stable under this model but recognize in response to changing local conditions.

According to Clements’ organismic hypothesis, species that typically occupy the same communities should always occur together. Thus, their distributions along the gradient would be clustered in discrete groups with sharp boundaries between groups (Russell et al., 2011).

In the 1920s, ecologists; Frederic Clements and Henry A. Gleason developed two extreme hypotheses about the nature of ecological communities (Russell et al., 2011). Clements championed an interactive (organismic) view describing communities as “superorganism” assemblages of species bound together by complex population interactions. According to this view, each species in a community requires interactions with a set of ecologically different species, just as every cell in an organism requires services that other types of cells provide.

In contrast, Gleason proposed an alternative, individualistic view of ecological communities. He believed that population interactions do not always determine species composition. Instead, a community is just an assemblage of species that are individually adapted to similar environmental conditions.According to Gleason’s hypothesis, communities do not achieve equilibrium; rather, they constantly change in response to disturbance and environmental variation. According to Gleason’s individualistic hypothesis, each species is distributed over the section of an environmental gradient to which it is adapted. Different species would have unique distributions, and species composition would change continuously along the gradient. In other words, communities would not be separated by sharp boundaries.

B)Stochastic Versus Equilibrium Schools

The stochastic school believes that most communities exist in a state of equilibrium, where competitive exclusion principle is prevented by periodic population reductions and environmental fluctuations (Crawley, 1997). More generally, stochastic effects can cause a population to shift from one type of dynamic behavior to another (Turchin, 2003). In addition, stochastic school maintains that physical and temporal factors are dominant influences of community composition. This view argues that species abundance varies and is largely determined by differential responses to unpredictable environmental changes (Levin, 2009).

In contrast, the equilibrium explanations assume that community composition represents the stable outcome of interspecific interactions (set of species abundances reached when the rates of change in population is zero) and also assume that the community will return to an equilibrium after those populations are perturbed (Verhoef, 2010). For instance, the traditional equilibrium model assumes that the probability of an individual fish larva surviving to reproduce is limited in a density-dependent manner by the abundance of the adult fish. Alternatively, stochastic model predicts that recruitment to the adult phase is independent of the density of the adults (Chapman et al., 1999).

Equilibrium model states that species richness is entirely determined by ongoing immigration and extinction (Kricher, 2011). Therefore, equilibrium model can be said to be deterministic process which is important in shaping community structure through competition and predation on native species over short temporal scales (Thorp et al., 2008). For example, Chapman et al., (1999), stated that coral reefs communities are at equilibrium showing precise resource partitioning in response to the competition between the various fish species.

However, in contrast, the community may also be more susceptible to stochastic processes. For example, the number of fish species on coral reefs is kept high largely by stochastic processes. According to Naiman et al., (2001), stochastic processes are unpredictable and operate in a relatively density-independent fashion. This is the opposite of the traditional, equilibrium hypothesis which emphasizes density dependent competition between species.

# Reproductive Isolating Mechanisms Essay

In the 1940s, Ernst Mayr coined the term Biological Species Concept that was subsequently widely embraced by the scientific community. The definition stated that “Species are groups of actually or potentially interbreeding natural populations that are reproductively isolated from other such groups”. Certain mechanisms are in place to prevent species from interbreeding with others and these are referred to as reproductive isolating mechanisms, which are biological incompatibilities. There are many mechanisms acting on natural populations and these are broadly grouped into two categories namely prezygotic and postzygotic mechanisms.

Prezygotic isolating mechanisms are those mechanisms that isolate species before fertilisation i.e. before a zygote is formed. They include geographical, ecological, temporal, ethological, mechanical, morphological and gametic isolation. These mechanisms lesson the possibility of gametes from different species coming into contact and hence forming a zygote.

One prezygotic isolating mechanism is ecological isolation, also referred to as habitat isolation. Ecological isolation prevents different species that live in the same territory but different habitats from interbreeding.

These species are referred to as sympatric species since they occur in the same territories. Individuals mate in their preferred habitat, and therefore do not meet individuals of other species with different ecological preferences. An example of ecological isolation occurs within the Rana genus. R. grylio, the pig frog and R. areolata, the gopher frog both occur around New Orleans, Louisiana. The exceptionally aquatic pig frog lives in deep ponds, lakes and marshes amongst surfacing vegetation and breeds in deep water so has no contact with the gopher frog which lives in burrows during the day, and in the swamp margins at night and breeds in shallow water. This way the possibility of interbreeding between the two species is eliminated.

Temporal isolation, also known as seasonal isolation is a prezygotic mechanism that prevents interbreeding between species. Temporal isolation is the isolation of species by changing the time at which they release gametes. A particularly strong example of temporal isolation occurs in three tropical orchid species of the genus Dendrobium. The species only flower for a single day, opening at dawn and withering by nightfall. The flowering of each species is in response to the same environmental stimuli, such as a sudden storm on a hot day, but the lag time between the occurrence of the stimulus and the flowering is eight, nine, and ten or eleven in the different species.

Since they flower for a single day, inter-species fertilization is made impossible because when one species has flowered the others are either not yet mature or already withered. Other examples of temporal isolation in natural populations are not as pronounced such as in cases were species are isolated due to breeding during different seasons like Sciurus carolinensis, the gray squirrel which mates in July and August; and Sc. niger, the fox squirrel which breeds in May and June.

Another prezygotic isolating mechanism is ethological isolation or behavioural isolation. This mechanism prevents species, mostly animals from interbreeding based on their different behavioural patterns. It affects animals that occur within the same territory and habitat but with different mating behaviours. In most animals, mates are chosen in a species-specific approach often after species-specific mating rituals of some form. Matings follow these rituals which can be ended if at least one of the mating parties decides that the process leading to the mating is not as expected. Ethological isolation can be strong reproductive isolating mechanism in animals also between closely related species.

An effective example of ethological isolation occurs in certain frog species of the Hyla species. H. versicolor, the gray tree frog and the closely related H. femoralis, pine wood tree frog often breed in the same ponds. Both species are physically very similar but their male mating calls, which last about three seconds and sound the same to humans can be differentiated by the female tree frogs and thus insures species-specific mating. This is also the case when considering why dogs and wolves don’t mate as frequently as expected because of their different behavioural patterns.

Mechanical isolation is a mechanism that prevents copulation between different animal species because of incompatible shape and size of the genitalia. This occurs in species are sympatric and live in a common habitat and have overlapping breeding seasons without any ethological isolating mechanisms. In plants, variation in flower structure can inhibit cross species pollination. In California, two sage species, namely Salvia mellifera and S. apiana exhibit this form of prezygotic isolation. Two- lipped S. mellifera has stamens and style in their upper lip, whereas S. apiana has long stamens and style and a specialized floral configuration. Small and medium sized bees that carry pollen on their backs pollinate the two-lipped flowers, and large carpenter and bumble bees that carry pollen on their wings and other body parts pollinate S. apiana. This mechanism thus ensures that pollen cannot be transferred between the species, as only the corresponding pollinators are able to transfer pollen to the style of each flower.

Another prezygotic isolation mechanism is morphological isolation, which in some cases overlaps with mechanical isolation mechanisms. This isolating mechanism prevents mating due to differences in size and shape between species. Morphological isolation prevents the spread of genes between the oak toad, Bufo quercicus and the Gulf Coast toad, Bufo vallicpes due to the size variation between the two species. The female oak toad has a maximum length of approximately 3 centimetres whereas the smallest Gulf Coast males are about twice as long. This way the size differences between the spaces make copulation extremely unlikely as male oak toads are too small to grasp the female Gulf Coast toad and male Gulf Coast toads are large enough to, and generally do, eat the female oak toads.

The last prezygotic isolating mechanism is gametic isolation also known as gametic mortality. Gametic isolation is the mechanism in which fertilization cannot occur between species generally due to chemical incompatibilities between their gametes. In animals that practice internal fertilization, sperm may not be able to survive in the female’s sperm receptacles. Some plant species, pollen grains of one species usually cannot germinate on the stigma of another species thus preventing fertilization between species. Since many aquatic animals disperse their ova and sperm into the water, gametesof different species do not have affinities for each other. This was demonstrated between the sea urchins Stronglocentrotus purpuratus and S. franciscanus when after induction of simultaneous realease of eggs and sperm, all resultant fertilizations were between eggs and sperms of the same species.

Postzygotic isolation mechanisms are those reproductive isolating mechanisms, which are effective after the union of gametes of different species i.e. after fertilisation. Postzygotic isolating mechanisms include hybrid inviability, hybrid sterility and hybrid breakdown. Postzygotic isolating mechanisms reduce the viability or fertility of hybrids or their progeny. These mechanisms come into play when hybridzygotes are formed either naturally or unnaturally.

Hybrid inviability is the postzygotic isolating mechanism that occurs between fertilisation and birth. This mechanism prevents the full development of zygotes and thus leads to their subsequent death. An example in animals is the death of artificially created sheep-goat hybrid embryos in early developmental stages before birth. Hybrid inviability also occurs in plants, most commonly resulting in hybrid seeds either failing to germinate or dying shortly after germination.

Hybrid sterility or hybrid infertility is a postzygotic reproductive isolating mechanism which affects hybrids that survive to adulthood. This mechanism creates sterile hybrids i.e. hybrids that cannot produce offspring. The most well known example is the case of the mule. When a horse and a donkey mate they are capable of producing offspring called mules. But mules are sterile, leaving them ‘out’ of the gene pool thus maintaining species integrity.

Hybrid breakdown is the last postzygotic isolating mechanism in place to maintain species differentiation. It occurs in cases of crossbreeding were hybrids of inter species matings are viable and fertile. Hybrid breakdown occurs commonly and results in the hybrid’s offspring or the F2 generation being unviable or significantly weaker and unable to contribute genes to a next generation. Certain cotton plants show this form of reproductive isolation such as Gossypium barbadense, G. hirsutum and G. tomentosum which produce hybrids that seem viable and fertile, but their offspring die either as seeds, early during development or develop into weaker plants unable to compete and reproduce.

Reproductive isolation combines traits that reduce gene flow, such as mate choice or fertilization barriers, with traits that select against genes that have flowed, such as hybrid incompatibility. Reproductive isolating mechanisms are ultimately mechanisms evolved in species to prevent interbreeding with other species and thus allow for scientists to differentiate between species using the biological species concept.

Bibliography

Futuyma, DJ. 1998. Evolutionary biology, 3rd ed., Sinauer Assoc. Inc., USA

Griffiths, A.J.F., Miller, J.H. Suzuki, D.T., Lewontin, R.C. and Gelbart, W.M. 1999. Introduction to Genetic Analysis. W.H Freeman & Co. New York.

Hale, W. and Margham, J. 1988. Collins Dictionary of Biology. Collins, UK.

Mayr, E. 1942 Systematics and the Origin of Species. Columbia University Press, New York, USA.

Mayr, E. (1970) Populations, Species, and Evolution. Harvard University Press, Cambridge, Massachusetts, USA.

Starr, C. and Taggart, R. (2001). Biology, The Unity and Diversity of Life, 9th ed. Brookes/ Cole, USA.

www.abacus.gene.ucl.ac.uk/jim/Sp/isolmech.html

www.library.thinkquest.org/ 19926/java/library/article/17a.ht

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## Buffering Region of Histidine Monohydrochloride

The objective of this experiment is to determine the buffering region of histidine monohydrochloride by titrating histidine with a base, NaOH. By plotting a suitable graph, the pKa values of histidine can be observed. Normally, a titration curve is constructed to illustrate the relationship between the pH of the mixture and the number of moles of base added to it. However in this experiment, the graph of pH against the number of moles of NaOH per mole of histidine is plotted. This is to ensure that the graph is independent of the volume and concentrations of the solutions used. After determining the pKa values of histidine, the maximal buffering capacity of the histidine-NaOH mixture, as well as the effective buffering range can be determined.

## Materials and Methods

To prepare 20mM solution of histidine monohydrochloride, 0.196g of histidine monohydrochloride was dissolved in 46.8mL of water, according to the calculations below:

No. of moles of histidine =

=

9.35 10-4 mol

=

46.8 mL

Upon complete mixing of the 20mM histidine monohydrochloride solution using a magnetic stirrer, 20mL of the solution was transferred into a beaker. The burette was washed with distilled water followed by NaOH and subsequently filled with 0.05M NaOH. The original pH of histidine solution was measured using the pH meter before proceeding with titration. Titration was carried out by adding NaOH to the histidine solution at 0.5mL increments. After each increment, the pH value of the resulting acid-base mixture was recorded. Titration was stopped when the acid-base mixture reached pH 11.5.

=

=

## Plotting graph of pH against no. of moles of NaOH per mol of histidine

Table: pH of histidine-NaOH solution with every 0.5mL of NaOH added

## Determining pKa values of histidine

(i) Based on Graph 1, the two rectangles indicate the two regions where the curve approaches the point of inflection. The maximum and minimum points of the regions are marked with the yellow circle. By finding the average values of each set of maximum and minimum points, the respective pKa values can be determined.

pKa1 =

= 6.12

pKa2 =

= 9.45

(ii) pKa1 is the point where = 0.5

pKa2 is the point where = 1.5

Based on Graph 1, pKa1 and pKa2 are points marked with the red cross.

pKa1 = 6.16

pKa2 = 9.30

## Maximal buffering capacity & Effective buffering range

Based on Graph 1, the acid-base mixture shows maximal buffering capacity at pH 6.12 and pH 9.45. The effective buffering range of a buffer is between ±1 of the maximal buffering capacity. Thus, the effective buffering range of histidine is pH 5.12 to pH 7.12 and pH 8.45 to pH 10.45.

If NaOH has not been accurately prepared, method used in (c)(i) will give a more reliable estimate of the pKa values.

If NaOH has not been accurately prepared, the number of moles of NaOH will be different, changing the ratio of number of moles of NaOH per mole of histidine. Method (c)(ii) depends on this ratio to determine the two pKa values. Hence, inaccurate ratios will cause the resulting pKa values to vary, leading to less reliable estimate of pKa values.

On the other hand, method (c)(i) does not depend on the ratio between number of moles of NaOH and histidine. Thus, an inaccurate ratio will not affect the pKa values being determined. Instead, method (c)(i) relies on the point of inflection of the graph, which plots pH against the number of moles of NaOH per mole of histidine. Plotting the graph in this manner ensures that it is independent of the volume and concentrations of the solutions used. In other words, even if NaOH has been inaccurately prepared, changing the concentration of the NaOH solution, the shape of the curve remains similar. Since the shape of the curve does not change, the point of inflection will be almost at the same point. pKa values obtained by method (c)(i) will be similar to the original values when NaOH was prepared accurately.

## 5mL of NaOH

No. of moles of NaOH added = Ã- 0.05 = 2.5 x 10-4 mol

NaOH ‰¡ Histidine

No. of moles of histidine reacted = 2.5 x 10-4 mol

Initial no. of moles of histidine = 4 x 10-4 mol

No. of moles of histidine left = 4 x 10-4 – 2.5 x 10-4 mol

= 1.5 x 10-4 mol

pH = pKa + log

pH = 6.12+ log

= 6.34

## (ii) 12mL of NaOH

No. of moles of NaOH added = Ã- 0.05 = 6.0 x 10-4 mol

No. of moles of NaOH left = 6.0 x 10-4 – 4 x 10-4

= 2.0 x 10-4 mol

NaOH ‰¡ Histidine

No. of moles of histidine reacted = 2.0 x 10-4 mol

Initial no. of moles of histidine = 4 x 10-4 mol

No. of moles of histidine left = 4 x 10-4 – 2.0 x 10-4 mol

= 2.0 x 10-4 mol

pH = pKa + log

pH = 9.45 + log

= 9.45

(i) Three ionisable groups are present in histidine at the initial pH of the experiment. The three groups are: carboxyl group, amino group and the R group (imidazole group).

(ii) The amino group is responsible for the observed pKa value of 6.12 and the imidazole group is responsible for the pKa value of 9.45.

## Discussion

Histidine is an amino acid that acts as a buffer and it has three ionisable groups: carboxyl group, amino group and imidazole group. In this experiment, the focus is on the dissociation constant of the amino and imidazole group. The titration curve (as shown in Graph 1) has two ‘steps’, or two points of inflection because the amino group dissociates first followed by the dissociation of imidazole group. Hence, the amino group is responsible for the observed pKa value of 6.12 and the imidazole group is responsible for the pKa value of 9.45. Two methods were used to determine the pKa values of histidine. However these calculated values are only estimates and may deviate from the actual values due to the following experimental errors:

Parallax error occurs during the reading of the burette, resulting in inconsistent increment of NaOH added to the histidine solution. In other words, each increment of NaOH was not maintained at 0.5mL. This directly affects the precision of the experiment.

Possible solution to minimise error:

To avoid parallax error, ensure that the burette reading is taken from eye level at the bottom of the meniscus. The burette should also be placed in an upright position, perpendicular to the table. For a more precise burette reading, a black burette reading card can be placed behind the burette so as to get a clearer view, especially when colourless solutions are used.

The beaker containing the histidine-NaOH mixture is placed on the magnetic stirrer throughout the titration to ensure a homogenous mixture for more accurate pH readings. After every 0.5mL of NaOH added to the mixture, the pH of the resulting mixture is recorded by using the pH meter. However, it takes time for the pH meter to generate a final pH reading that does not fluctuate. If the pH value is recorded too quickly after the addition of NaOH, the pH reading may be inaccurate.

Possible solution to minimise error:

To obtain greater accuracy in pH reading, ensure that an appropriate waiting time (about 2min) is maintained between the addition of NaOH and the recording of pH value.

## Conclusion

From this experiment, it can be concluded from the titration curve that the amino group of histidine is responsible for the observed pKa value of 6.12 and the imidazole group is responsible for the pKa value of 9.45. These two pKa values correspond to the pH at which the acid-base mixture shows maximal buffering capacity. The effective buffering range of histidine is pH 5.12 to pH 7.12 and pH 8.45 to pH 10.45.

## Introduction

Buffers are solutions that are able to maintain a fairly constant pH when a small amount of acid or base is added. This experiment examines the effect of buffer’s pKa on buffering capacity by studying how well the two buffers of different pKa resist pH changes when acid or base is added. In scientific experiments, it is advisable to choose a buffer system in which the pKa of the weak acid is nearer to the pH of the interest. It will be ineffective for a buffer to resist pH changes if its pKa value is more than 1 pH unit from the pH of interest. Thus the study of the effect of pKa on buffering capacity is important in making a suitable choice of pH buffers for a specific experiment.

## Materials and Methods

We study the effect of buffer’s pKa on buffering capacity by using 2 different buffers, potassium phosphate buffer and Tris-HCl, with pKa value 6.8 and 8.1 respectively. 3mL of 0.01M potassium phosphate buffer was pipetted into two test tubes, labelled A and B. 3mL of 0.01M Tris-HCl was also pipetted into two test tubes, labelled C and D. Three drops of universal pH indicator were added into each test tube, causing the solutions to turn green in colour (pH 7.0). HCl was added to test tubes A and C until the solutions turned pink (pH 4.0). KOH was added to test tubes B and D until the solutions turned purple (pH 10.0). The number of drops required for the solutions on each test tube to turn pink or purple in colour is recorded. The pH colour chart is used as it shows the colours of the solution at each pH level.

## Results & Questions

Table : Number of drops of acid or base needed for buffer solution to deviate from its initial neutrality (pH 7.0)

pH Buffer

pKa of buffer

Initial pH

No. of drops of HCl required to become acidic (pH 4.0)

No. of drops of KOH required to become alkaline (pH 10.0)

0.01M potassium phosphate buffer

6.8

7.0

5

11

M Tris-HCl

8.1

7.0

2

20

## Conclusions drawn from experiments

According to Table 2, potassium phosphate buffer requires five drops of HCl to reach pH 4.0, compared to Tris-HCl which requires only two drops of HCl to reach pH 4.0. This shows that potassium phosphate buffer is a more effective buffer against acids. Potassium phosphate buffer requires eleven drops of KOH to reach pH 10.0 while Tris-HCl requires twenty drops of KOH to reach pH 10.0.

Based on the results, Tris-HCl behaves as a more efficient buffer under basic conditions as it requires more amount of KOH than that of potassium phosphate to reach pH 10.0. This means that Tris-HCl has greater ability to resist increases in pH but not decreases in pH. On the other hand, potassium phosphate buffer is a more efficient buffer under acidic conditions as it requires lesser amount of HCl to reach pH 4.0. Similarly, this means that potassium phosphate buffer has greater ability to resist decreases in pH but not increases in pH.

It can be deduced that a buffer with greater pKa value is a more efficient buffer in basic conditions while a buffer with smaller pKa value is a more efficient buffer in acidic conditions.

## Choosing a suitable buffer to study the properties of a phosphatase which functions optimally at pH 7.2

I would use the 0.01M Tris-HCl to study the properties of a phosphatase.

It is more appropriate to use a buffer with effective buffering range nearer to the pH of phosphatase. Tris-HCl has an effective buffering range of pH 7.1 to 9.1 while potassium phosphatase buffer has an effective buffering range of pH 5.8 to 7.8. Simply by considering the effective buffering range of the two buffers, it can be concluded that both buffers can be used to study the properties of phosphatase which functions optimally at pH 7.2.

However, considering the effective buffering range of the buffers is not sufficient to come to a sound conclusion. In this case, phosphatase is an enzyme that functions to hydrolyse phosphate groups. By adding potassium phosphate buffer to phosphatase, phosphatase will break down the phosphate group in the potassium phosphate buffer. This changes the chemical properties and hence the buffering capability of the potassium phosphate buffer.

Therefore, Tris-HCl is a more suitable buffer for the studying of phosphatase.

## Discussion

In Experiment 1, the endpoint of the reactions is determined using a pH meter and construction a titration curve. However in this experiment, the endpoint is visually observed by the help of a pH colour chart. Possible sources of experimental errors arising from this method and ways to improve the experiment are discussed below:

In this experiment, only two types of buffers, Tris-HCl and potassium phosphate buffer, were used. The experiment can be improved by using more types of pH buffers to obtain more data. This will allow more accurate evaluation of the relationship between the pKa value and the buffering capacity, and thus the effect of pKa value on the buffering capacity.

Although the pH colour chart is used to compare the colours of the solutions, personal judgment comes into play when determining the colour change in the chemical reactions.

Possible solution to minimise error:

Be consistent in deciding the point of colour change and the endpoint of the experiment.

## Conclusion

From this experiment, it can be concluded that a buffer with greater pKa value is a more efficient buffer in basic conditions and a buffer with smaller pKa value is a more efficient buffer in acidic conditions. Though a buffer’s pKa can affect its buffering capacity, however when choosing a suitable buffer for an experiment, we cannot simply rely on the pKa of a buffer. It is also crucial to consider the chemical properties and structure of the buffer and other reagents to be used in the experiment.

## Introduction

The aim of this experiment is to examine the effect of temperature on the pH of a buffer. This can be done by observing the changes in pH of two different buffers when temperature of the buffer solution decreases from room temperature to 4°C. pH of the buffers that are used to maintain the pH of the lab samples can change during changes in temperature due to cooling process. Changes in pH of buffers upon temperature changes can be explained by the Le Chatelier’s Principle. The study of the effect of temperature on pH of a buffer is crucial in choosing the right pH buffer that is able to show minimum changes in buffer pH, to maintain the properties of the biological samples that requires specific pH environment.

## Materials and Methods

We study the effect of temperature on the pH of a buffer by using two different buffers, 0.01M potassium phosphate buffer and 0.01M Tris-HCl. 3mL of each buffer solution were pipetted into two separate test tubes. The initial pH values of the two buffers at room temperature are measured using the pH meter and recorded. Subsequently, both test tubes were placed into the ice box to cool to 4°C. After 20 minutes, the test tubes were taken out of the ice box and placed in an ice bath to maintain the temperature of the buffer solutions at 4°C. The pH of the cooled buffer solutions were measured again and recorded to obtain the results as seen in Table 3. By evaluating the pH changes (either increase or decrease) and the extent of these changes from the original pH value, we can observe the effect of temperature on the pH of a buffer.

## Results & Questions

Table : The changes in the pH of the buffer solution as temperature is decreased to 4°C

Buffer

pH at room temperature

pH at 4°C

Difference in pH change (unit)

0.01M potassium phosphate buffer

7.03

7.49

0.46

0.01M Tris-HCl

7.01

8.16

1.15

## Effect of temperature on the pH of Tris-HCl and potassium phosphate buffer

According to Table 3, at low temperature of 4°C, both buffer solutions become more alkaline. As temperature decreased from the room temperature to 4°C, the pH potassium phosphate buffer increased from 7.03 to 7.49, with a difference in pH change of 0.46. With the same change in temperature, the pH of Tris-HCl increased from 7.01 to 8.16, with a difference in pH change of 1.15. This shows that Tris-HCl exhibits greater changes in pH than potassium phosphate buffer, upon a given change in temperature. In conclusion, temperature has a greater effect on the pH of Tris-HCl compared to potassium phosphate buffer.

HA A» + Hº Î”H = -ve

As illustrated by the chemical equation above, the dissociation of buffers are endothermic processes. Being an endothermic process, heat is being absorbed and temperature decreases. Based on Le Chatelier’s Principle, when temperature decreases, the system will react to result in an increase in temperature. Hence, decreasing temperature to 4°C favours the backward reaction, which is an exothermic reaction that produces heat. The position of equilibrium shifts to the left, more Hº reacts with A» to form HA. Thus, the concentration of Hº decreases and causes the pH of the buffer to increase.

## Discussion

Based on the experimental results, it is clear that temperature changes the pH of the buffer. Though this is not a complicated experiment, it is still subjected to experimental errors and can be improved by the following ways:

Only two types of buffers, Tris-HCl and potassium phosphate buffer, were used in this experiment. The experiment was also conducted at only one temperature. Using several buffers over a range of temperatures will allow us to observe the pH of a variety of buffers at different temperatures. In addition, both buffers used in this experiment showed an increase in alkalinity. Hence, including more variety of buffers will allow us to evaluate which type of buffer has tendency to become more alkaline or acidic with the changes in temperature.

This experiment was conducted without the use of a thermometer, hence there was uncertainty in determining the temperature of the buffer solutions. It was assumed that by placing the test tubes in the ice box for 20 minutes and then transferring into an ice bath, the buffer solutions would be maintained at 4ËšC. However, it is difficult to maintain ice baths at 4ËšC for a long period of time due to heat gain from the surroundings.

Possible solution to minimise error:

Keep a thermometer in the ice bath and consistently check the temperature of the ice bath. Add in more ice when the ice melts.

It was difficult to identify the endpoint of the experiment. Even after a long period of time (about 30 minutes), the pH reading shown on the pH meter still continued to increase slowly. Hence, stopping the experiment too early may result in an inaccurate pH reading.

Possible solution to minimise error:

Since it is difficult to identify the endpoint of the experiment, it is perhaps more logical to standardise the duration of the experiment for both buffer solutions. For example, 30 minutes for each buffer solution.

## Conclusion

From this experiment, it can be concluded that a decrease in temperature will cause a change in pH of a buffer. However, the pH of the buffer does not always increase when temperature decreases. This depends on whether the dissociation process is endothermic or exothermic. In the case of an endothermic dissociation process, pH of the buffer will increase when temperature decreases. This can be explained by Le Chatelier’s Principle which states that the backward exothermic reaction will occur so as to counteract the change. Hence, the Tris-HCl and potassium phosphate buffers become more alkaline as temperature decreases.

## The Most Effective Antibiotic On Bacteria Biology Essay

Before bacteria can multiply and cause symptoms our immune system can usually destroy them. We have special white blood cells that attack harmful bacteria. Even if symptoms do occur, our immune system can usually cope and fight off the infection. There are occasions, however, when it is all too much and our bodies need some help – from antibiotics.

The first antibiotic was penicillin. Such penicillin-related antibiotics as ampicillin, amoxicillin and benzylpenicilllin are widely used today to treat a variety of infections – these antibiotics have been around for a long time. There are several different types of modern antibiotics and they are only available with a doctor’s prescription in industrialized countries.

An antibiotic is given for the treatment of an infection caused by bacteria. They target only bacteria – they do not attack other organisms, such as fungi or viruses. If you have an infection it is important to know whether it is caused by bacteria, and not a virus or fungus. Most upper respiratory tract infections, such as the common cold and sore throats are generally caused by viruses – antibiotics do not work against viruses.

Antibiotics are overused or used incorrectly there is a chance that the bacteria will become resistant – the antibiotic becomes less effective against that type of bacterium.

A broad-spectrum antibiotic can be used to treat a wide range of infections. A narrow-spectrum antibiotic is only effective against a few types of bacteria. There are antibiotics that attack aerobic bacteria, while others work against anaerobic bacteria. Aerobic bacteria need oxygen, while anaerobic bacteria don’t.

Antibiotics may be given beforehand, to prevent infection, as might be the case before surgery. This is called ‘prophylactic’ use of antibiotics. They are commonly used before bowel and orthopedic surgery.

## Bacteria

The word bacteria is the plural of bacterium. Grammatically the headline should just say “What are bacteria?” The incorrect usage has been included in the headline to remind readers that it is wrong – and hopefully help correct an increasingly common mistake in the English language. Bacteria are tiny living beings (microorganisms) – they are neither plants nor animals – they belong to a group all by themselves. Bacteria are tiny single-cell microorganisms, usually a few micrometers in length that normally exist together in millions.

A gram of soil typically contains about 40 million bacterial cells. A milliliter of fresh water usually holds about one million bacterial cells.

Planet Earth is estimated to hold at least 5 nonillion bacteria. Scientists say that much of Earth’s biomass is made up of bacteria.

5 nonillion = 5,000,000,000,000,000,000,000,000,000,000 (or 5×1030)

(Nonillion = 30 zeros in USA English. In British English it equals 54 zeros. This text uses the American meaning)

Bacterial cell walls

Bacterial cell walls consist of layer of peptidoglycan which is made up of many parallel polysaccharide chains with short peptide cross-linkage forming an enormous molecule with net-like structure. However, there are two different types of bacterial cell wall, which can be distinguished by Gram staining, a staining technique developed by Christian Gram in 1984 and still in use today. Before staining, bacteria are colorless. The cell walls of Gram-positive bacteria have a thick layer of peptidoglycan containing chemicals such as teichoic acid within their net-like structure. The crystal violet in the stain binds to the teichoic acid and resists decolouring in the rest of the process, leaving the positive purple/blue color.

The cell walls of Gram-negative bacteria have a thinner layer of peptidoglycan with ni teichoic acid between the two layers of membranes and then an outer membrane-like layer made up of lipopolisaccharides. Any crystal violet which does not bind is readily decolourised and replaced with red safranine in the Gram stain. So cells appear red.

## PROBLEM STATEMENT

Which antibiotic is the most effective on bacteria?

## APPARATUS

200 ml of micropipette, conical flask, forceps, 100 ml beaker

## MATERIALS

Petri dish, sample of E.coli and staphylococcus, 3 different types of antibiotic which are carbenicilin, streptomycin and tetracycline, distilled water, Dettol handwash, 75 % of ethanol, agar, tissue paper

## VARIABLES

Fixed variable : volume of bacteria

Manipulated variable : types of antibiotic

Responding variable : area of inhibition zone

## HYPOTHESIS

The most effective antibiotic to inhibit bacteria growth is ampicillin

## PROCEDURE

First of all, wash out hands with the soap or handwash. The working area is sprayed thoroughly with the disinfectant spray. It is left for at least 10 minutes, and wiped with a paper towel.

An agar plate seeded with is prepared. The petri dish is labeled on the base at the edge out of name, the date and type of bacterium it is inoculated with by usng permanent marker pen.

After finishing marking the petri dish, the Esterichia coli bacteria is poured by using micropipette into the petri dish on the marked labeling and distribute it evenly.

The agar is taken out from 60 ÌŠC oven. The mouth of the conical flask containing the agar is warmed in the flame to prevent any different bacteria from surrounding grow inside it.

The agar is left for 10 minutes for it to solidify before putting the sterile disc dip into three different antibiotics.

The forceps are flamed and used them to pick up disc or Mast ring. It is dip into the antibiotic solution and is placed firmly in the centre of the agar.

The dish is taped securely with two pieces of adhesive tape and kept it upside down at room temperature for 24 hours.

Step 1 until steps 7 are repeated by using another type of bacteria which is staphylococcus.

Our hands are washed with soap or handwash and the bench is cleaned again using the 75 % of ethanol.

After the incubation, the plate should be looked at carefully but do not open it. Where bacteria have grown the plate will look opaque, but where the antibiotics have inhibited growth, clear zones called inhibition zones will be seen.

The diameter of the inhibition zones is measured in milimetres and the information is used to decide which antibiotic is most effective at inhibiting the growth of the bacterium.

The data is collected from other members of the class who used the other bacterial cultures.

## PRECAUTIONS

When carrying out the experiment, we should work very closed to the Bunsen burner to prevent any impurities in each apparatus that is used.

Do not seal completely the upper and lower part of petri dish.

Both petri dishes contain different bacteria need to be inverted to prevent the water vapour from forming at the upper site of the agar. It might affected or overshadow the inhibition zones that are formed inside the dish.

Make sure our hands are constantly being wash with disinfectant before touching any apparatus.

Before using any apparatus, they should be sterile to prevent any unwanted impurities to grow in the petri dish.

Do not open the petri dish after incubation to prevent from infection.

## RESULTS

Antibiotic

Diameter of inhibition area/ cm

Inhibition area / cm²

E. Coli

Staphylococcus

E.Coli

Staphylococcus

Tetracyclin

2.6

3.0

5.3

7.0

Streptomycin

1.6

2.0

8.0

3.1

Carbenicillin

1.5

3.5

4.9

9.6

Ampicillin

3.6

3.8

10.2

11.3

Control

0

0

0

0

## Explanation of the data

Table above shows the area of inhibition zone of the bacteria growth on two different bacteria. Four types of antibiotics are used to be investigated which is the most effective on a particular bacteria. They are tetracycline, streptomycin, carbenicillin and ampicillin. Whereas two types of bacteria are used which are staphylococcus and Esterichia Coli.

For E.coli, ampicilin showed the greatest inhibition area of bacteria growth with 10.2 cm². Another antibiotic that showed the closest reading is streptomycin. Tetacyclin and carbenicilin showed 5.3 cm² and 4.9 cm² respectively. This result clearly shown that ampicilin is the most effective antibiotic to inhibit the growth of E.coli. Besides that, E.coli is a gram negative bacteria. The cell walls of Gram-negative bacteria have a thinner layer of peptidoglycan with no teichoic acid between the two layers of membranes and then an outer membrane-like layer made up of lipopolisaccharides.

In addition to that, Staphylococcus also had a higher inhibition zone of bacteria on ampicilin. Other bacterias have no effect as great as ampicilin which can be consider as a strong antibiotic. Staphy is a gram positive bacteria. The cell walls of Gram-positive bacteria have a thick layer of peptidoglycan containing chemicals such as teichoic acid within their net-like structure. The crystal violet in the stain binds to the teichoic acid and resists decolouring in the rest of the process, leaving the positive purple/blue color. That is why ampicilin is said to be the most effective antibiotic on both bacterias.

## Limitations

There are some limitations that cannot be avoided when carrying out the experiment. The first one was every apparatus that we used had been sterile by the laboratory assistant. Thus we had to wash our hands thoroughly before touching anything. We even could not talk during the preparation. The problem was we carried out the experiment on the lab’s table, instead of using the cupboard fume to maintain the sterile apparatus. Thus, there might be some of the apparatus that we were using had been contaminated. This condition might affect the reliability of the experiment hence the expected results might not get at the end of the experiment.

Besides that, we had to wash our hands by using the dettol hand soap before we start and end the experiment. We had to remove all the impurities and bacteria and that was the purpose of washing hand. The problem rose as we did not know whether we had washed our hand to the maximum cleanliness. Consequently, all the apparatus and materials that we used might be affected by our hand. This is done so that there are no infections or contaminations on the agar that might give problems later.

## Sources of error

There are some sources of error when carrying out this experiment. Firstly, we prepared the experiment at room temperature. It was supposed to carry out in sterile medium to prevent anything from affecting during the preparation of bacteria and antibiotics.

Next, the major problem that could be seen during the experiment was most of the groups could not be able to solidify their agar to the maximum hardness. Consequently, when inverting the petri dish, all its contents would be messy inside the petri dish and the disc had mixed to one another. Thus, the antibiotics on each disc also had mixed that could affected the whole result. To overcome this problem, students should let the agar solidify before inverting the petri dish. Another problem was some of the groups do not invert their petri dish before keeping it inside the incubator. This would cause the water vapour to form at the upper part of the petri dish. It would also affect the growth of the bacteria in agar.

Fourth, the layer of agar inside the petri dish was too thin that caused it fell to the bottom when inverting it. Besides ruining the shape of the agar and the position of the discs in the agar, the thin layer of agar also inhibits the growth of bacteria because it lacked of nutrients. To prevent this, the layer of agar should be half of the petri dish.

## Further work

To get more reliable and accurate result, this experiment should be repeated by varying the temperature to investigate the activity of antibiotic on bacteria. This means that increase the temperature would cause the antibiotic to act more rapidly compared to the temperature that we used before. Thus we do not have to wait for 24 hours to see the inhibition zone. This manipulated variable also do not waste the time consuming for the experiment. Moreover, as the antibiotic shows more faster effect, thus the bacteria and antibiotic should be put in separately different petri dish to prevent it from overlapping.

## CONCLUSION

The most effective antibiotic is ampicilin.

The hypothesis is aceepted.

## Reaction Between Sodium Thiosulphate And Hydrochloric Acid Biology Essay

The aim of this experiment is to study the rate of reaction and the different parameters that affect it. In this experiment, we will be investigating the effect of temperature on the reaction between Sodium Thiosulphate and Hydrochloric Acid.

Theory:

Chemical reactions involve collisions between reactant molecules or atoms to from bonds. For this, the molecules or atoms are required to come close to one another since new bonds can form only when the reactants are close enough to share electrons or facilitate a transfer of electrons. Collisions that lead to products are referred to as effective collisions which results when the collisions occur with enough speed energy and force to break bonds of the reactants.

The minimum energy reactants need to possess to break bonds and cause a reaction is known as activation energy. Therefore there are two ways of increasing the rate of reaction:

increase the number of collisions

increase the amount of movement (kinetic) energy so that more collisions lead to a reaction.

These depend on a number of factors:

Size of particles

Concentration

Temperature

Pressure of a gaseous reactant

This experiment deals with the effect of one of these factors-concentration of reactants.

Increasing concentration, increases the probability of collisions between the reactant as there are more of them available for interaction in the same amount of the solution mixture. Since the probability of the collisions is higher, the probability of effective collisions increases too.

The aim of this experiment to study and investigate the effect of this factor, on the rate of reaction between sodium thiosulphate and hydrochloric acid.

The reaction between sodium thiosulphate and hydrochloric acid proceeds to the following chemical equation:

## Na2S2O3(aq) + 2HCl(aq) ® 2NaCl(aq) + SO2(aq) + H2O(l) + S(s)

This equation can also be represented in the ionic-equation form in the following manner:

## S2O32- + 2H+ ® H2O + SO2 + S

The sulphur dioxide thus formed is dissolved in water as sulphurous acid. The partially dissolved sulphur makes the solution turbid due to the formation of a colloid. Because of this, the solution turns more dirty and opaque as the liberated sulphur increases in quantity. This property is used in the experiment to determine the rate of the reaction.

## Hypothesis:

It can be hypothesized that an increase in the concentration of the sodium Thiosulphate would increase the rate of the reaction. Quantitatively, if the concentration of Na2S2O3 is increased by a factor of two, then the time taken for the reaction to occur will

decrease by a factor of two. This is equivalent to increasing the reaction rate by a

factor of two.

## Variables

Controlled: Factors like pressure, temperature, surface area of particles and the proportion of particles with respect to each other, that affect the rate of the reaction must be maintained.

Independent: Concentration of the reactants

Dependant: The rate of the reaction as the time taken for the reaction to take place would depend on the concentration.

## Requirements:

Chemicals:

1 M sodium thiosulfate solution

1 M HCl solution

distilled or deionized water

Equipment:

250-mL beakers

stirring rods

stopwatch

## Procedure

Prepare 5 beakers with different concentrations of Sodium Thiosulfate amd Hydrochloric Acid by preparing 25ml solutions in the following manner:

## (mL)

1

25

0

2

20

5

3

15

10

4

10

15

5

5

20

Make a small “x” on a sheet of white paper with a pencil. Place the 1st beaker containing the sodium thiosulfate solution over this “x” and add 5 mL HCl solution and immediately begin timing the reaction.

Stir the contents of the flask and record the time taken for the cross to be obscured by the sulphur precipitate formed and record this time in your data table.

Repeat this procedure for the remaining samples.

Make a graph of the data obtained by plotting the time (in sec) for each reaction on the y-axis against the volume (in mL) of sodium thiosulfate on the x-axis.

1.

1

25

0

18

0.0560

2.

2

20

5

23

0.04340

3

3

15

10

31

0.03220

4.

4

10

15

56

0.02000

5.

5

5

20

112

0.00087

## Data Analysis:

The graph above corresponds to our hypothesis as the relation between the rate of reaction and the concentration of reactants is proportional to the concentration of one particular reactant. This is due to the increased chances of fruitful collisions resulting in products. in this case, the reciprocal of the reaction time, 1/time, is being used to measure the speed of the reaction and represents how long it takes for a certain concentration of sulphur to form when the hydrochloric acid is added to the sodium Thiosulphate. The rate of reaction i.e. the time taken for the reaction to occur therefore is seen to increase with an increase in the concentration of one of the reactants.

## Limitations:

The graph above is best fitted to our results which means that the experimental results had a degree of inaccuracy. This could have been due to the following reasons:

Impurities in the reactants can affect the rate of reactions and inaccurate time periods.

The least count of the measuring cylinder used to measure solution is 0.05cm3. the volumes measured are therefore not precise. Due to this, the volumes of solutions measured might be inaccurate leading to inaccurate concentrations.

Since our laboratory was air-conditioned, the reaction mixture could have undergone uneven and sudden cooling.

The reaction time when stopping and starting the stopwatch also added to the inaccuracy especially when the time periods are really small.

## Modifications:

To overcome the limitations and give more accurate results, we can modify the experiment in the following ways:

A burette or a pipette could be more accurate in measuring the volumes of solutions.

The experiment must be carried out at a distance from the air-conditioner to prevent rapid cooling of the solutions and maintain a constant temperature.

A purer solution can be made by using better quality of the sodium Thiosulphate.

Every individual’s definition of the disappearance of the cross could be different. To overcome this, an indicator could have been used to indicate the completion of reaction.

## Precautions:

To minimize inaccuracy, the following precautions were incorporated in the experiment:

The water bath is very hot and so the beaker with the hot acid must be handled extra carefully or it could be dangerous.

The acid must be handled with care.

It is best that one person time during the whole of the reaction to reduce the error due to reaction time.

## Conclusion:

At the end of this experiment, it can be concluded that the concentration does have an effect on the time taken for the reaction and consequently on the rate of reaction. More specifically, an increase in concentration leads to an increase in the rate of reaction. The following conclusions can therefore be drawn:

Concentration µ / ____________ 1________________

time taken for reaction to be complete

Concentration µ Rate of reaction

## Genetically Modified Plants And Selective Breeding Biology Essay

The words “genetically modified” constitute a serious misnomer. Humans have bred plants and animals for thousands of years. In selective breeding, we have built into animals and plants gene combinations that are not normally found in nature and that probably would not survive without human intervention. The term “genetically modified” is currently applied to plants and animals that results from adding genes, in particular genes from completely unrelated organisms, to preexisting plants or animals. Genetically modified plants are commonly grown today in the United States. Most of the corn and soyabean-and conala derived products sold in this country are the result of plants engineered with recombinant genes. The most common genetic modifications are those that confer resistance to certain insect pests and those conferring resistance to certain herbicides. Let us first see how genetic modification of plants is achieved.

What follows is a brief potted account of techniques of genetic engineering of plant varieties and their degree of commercialization. This is penned, not from the standpoint of trying to satisfy an introduction to microbiology, but to give some idea of the technical background that is relevant to understanding environmental debates about GM crops.

The first step is to extract the piece of DNA that has the desired characteristic. The two most widely utilized (in commercial terms) genetically ‘transplanted’ characteristics have been herbicide tolerance and insect resistance. (Anderson, L, 1999)

Herbicide tolerance means that the GM crop can be sprayed with a broad range herbicide that will kill most types of weeds. The main types of this are ammonium glyphosate, marketed as ’roundup’ by Monsanto and ammonium gluphosinate used by Bayer (which incorporated Aventis). Both are regarded by US and European regulators as being reasonably benign compared to other herbicides, although, of course, for fans of organic food, there is no such thing as a benign herbicide. Many types of herbicide tolerant crop are available including corn (maize), soya, canola (oil seed rape) and sugar beet. Herbicide tolerant soya has been the most successful, being grown by the USA and Argentina, the first and third biggest global soya producers. Brazil, the second biggest, is still non-GM as are other important soya producers such as China and India. Herbicide tolerant canola (oil seed rape) is grown in the USA and Canada. (Anderson, L, 1999)

GM insect resistant crops exude a toxin in their pollen that kills insects which would otherwise eat the crop. The insect resistant crops are actually referred to by the name of the bacteria, bacillus thuringiensis. The most popular Bt crop is cotton, grown in the USA, South Africa, India, China and Australia. Its relatively rapid spread to these countries must be explained partly by the fact that it is not a food crop. This makes the product much less controversial than GM food crops. Bt versions of food crops such as corn (maize) and potato are also available and are widely grown in the USA and Argentina. (Anderson, L, 1999)

Despite the take-up of GM food crops in the USA, Canada and Argentina, the expected spread of GM food technology has not (yet) occurred. People often mistake reports of ‘trials’ for commercialization. In China, for example, there have been a large number of trials of a number of GM crops (Huang et al. 2002:675), but, apart from the case of Bt cotton, the trials do not seem to have resulted in widespread commercialization. Trials do not automatically lead to commercial planting since GM plants which are the subject of trials may not be suitable for commercialization, they may not be licensed, and they may simply not be grown (as in Europe) because there is no market for their products.

Both herbicide tolerant and insect resistant characteristics are associated with bacteria which can be found in the soil. The required DNA sections are extracted from the chromosomal string of these bacteria by so-called ‘restriction enzymes’, which are used by micro-organisms to resist attack from viruses. The enzymes act as biochemical ‘scissors’. Having separated the vital genes these genes are then spliced onto a ‘plasmid’. Plasmids are self-contained bundles of genetic information which supplement the principal chromosomal bundles held in bacteria, and the desired characteristic is added to the DNA chain in the plasmid by the help of ‘ligase enzymes’. These enzymes are normally used in organisms to repair accidental breaks in DNA strands. The assembly of these plasmids are key events, for the plasmids are the agents which carry the required genetic information for implantation into the target plant cells.

At this point it is necessary to mark the bundles of DNA material which are being transferred to the plant in such a way that you can isolate the plant cells that contain the desired new DNA recombination (hence the term ‘recombinant DNA’). Herein lies a particularly acute controversy because it was for a long time the usual practice, because of the criteria of sheer convenience, for antibiotic resistant marker genes to be spliced into the plasmids containing the genes being transferred. Genes which confer antibiotic resistance are especially convenient for the purpose of marking the plasmids that contain the DNA that is to be transferred because it is a very simple procedure to isolate the plant cells containing the recombinant by dousing the cell culture with that antibiotic. The cells that do not carry the antibiotic resistant genes die. This appeared to the GM pioneers, to be an elegant way of isolating the cells containing the recombinant DNA. Unfortunately many years later people started to worry that if antibiotic resistant genes were carried into the guts of animals or humans then the DNA might pass into the genes of pathogens. This might promote the proliferation of diseases that were resistant to those antibiotics. (Pusztai, A, 2002)

The practice of using antibiotic resistant marker genes in GM plants became a major item of controversy when it involved an antibiotic that was still in use, that is ampicillin, that was used in a variety of Bt corn. In fact other types of marker gene are available today, including genes which allow the cells which have combined with the desired genetic characteristics to grow in the presence of mannose, a type of sugar. In this case the cells that grow are taken for further processing and the unrecombined genes are discarded.

Before this happens the plasmids containing the genes with the new characteristics and the marker genes have to be transferred to the target plant cells. There are different ways of doing this. One way, suitable for ‘broad leaved plants (such as sugar beet, soybean and oilseed rape) involves a bacterium agrobacterium tumifaciens which is used to transfer the DNA’ (Mayer 2000:97). This bacteria causes crown gall normally, and it is thus quite effective at penetrating plant cells. This is good news for genetic engineers who can place the specially prepared plasmid in an agrobacterium tumifaciens bacterium that has been neutered to stop it spreading and it will easily infect the required plant with itself, and with it, the required genes. The cells which have been recombined with the new DNA will be separated from the rest and they can then be grown into full sized plants. Then seed can be produced and the product can be tested and, much later, marketed.

Of course a lot of crops are not susceptible to infection by this bacterium, and these include rice and maize. Instead, genetic engineers take recourse to firing gold particles, coated with the plasmids, directly into the cells. This is, as with using crown gall bacteria, a hit and miss affair, but the recombinant genes can be isolated by utilizing the properties of the marker genes.

Judging from current trials going on in the USA, future developments are likely to occur in four directions. First, development of more crop strains that have advantages for farmers such as crop varieties which are resistant to various types of diseases or which are suitable for currently inhospitable conditions like high salinity, low rainfall or excessive soil aluminium. Biotechnologists point to the adoption of virus resistant GM technology which they say has saved the papaya industry in Hawaii. Second, crops which deliver ‘quality’ attributes to the consumer like the high betacarotine, Vitamin A inducing, (and much talked up) ‘golden’ rice being developed in India. Third there are ‘plant based pharmaceutical crops’ (or biopharming) that can be used to grow medical products like blood thinners, clotting agents, anti-arthritis, contraceptive products or even anti-cancer drugs. Fourth are animals modified either to deliver larger amounts of product, like the large salmon that are already being considered for commercial approval in the USA or animals that have been genetically modified to produce drugs for humans. For example, the widely used enzyme-drug trypsin can be produced from genetically engineered animals.

Certainly the downbeat assessments made by many ‘establishment’ as well as radical environmentalists in Europe provide a stark contrast to the rallying cries for the cause of biotechnology made by the scientific elite in the USA. As a keynote paper at a National Academy of Sciences colloquim put it: ‘Widespread adaptation of biotech-derived products of agriculture should lay the foundation for transformation of our society from a production-driven system to a quality and utility-enhanced system’ (Kishaw and Shewmaker 1999:5968).

Theoretically the biotechnologists will be on relatively safe ground with products where there is no fear about the consequences of ‘genetic pollution’. For example, will there be any tears if the trait giving higher levels of beta-carotene spreads to other types of rice? Perhaps not, but many anti-GM groups are still opposed to this innovation arguing that as yet unknown genetic modifications will enter ecosystems.

However, as far as future development is concerned, the biggest problem faced by agricultural biotechnology is consumer resistance. Unfortunately for the biotechnology industry, European consumers may prove not to be idiosyncratic in their skepticism about GM food. In India, where the dominant Hindu religion favors vegetarianism and where many are worried about animal genes being spliced onto vegetables, sale of GM food is still, at the time of writing, actually illegal.

Biotechnologists have long held hopes that China would prove more receptive to GM food technology, but commercialization of GM food technology seems to have stalled. Increasingly farmers around the world are becoming unwilling to grow GM crops lest their produce becomes unsaleable in a rising tide of consumer resistance to GM food. In short, the USA may win some sort of victory at the WTO, but the key battle is in the food markets, and here it is heading for defeat.

## Buffering Region of Histidine Monohydrochloride

The objective of this experiment is to determine the buffering region of histidine monohydrochloride by titrating histidine with a base, NaOH. By plotting a suitable graph, the pKa values of histidine can be observed. Normally, a titration curve is constructed to illustrate the relationship between the pH of the mixture and the number of moles of base added to it. However in this experiment, the graph of pH against the number of moles of NaOH per mole of histidine is plotted. This is to ensure that the graph is independent of the volume and concentrations of the solutions used. After determining the pKa values of histidine, the maximal buffering capacity of the histidine-NaOH mixture, as well as the effective buffering range can be determined.

## Materials and Methods

To prepare 20mM solution of histidine monohydrochloride, 0.196g of histidine monohydrochloride was dissolved in 46.8mL of water, according to the calculations below:

No. of moles of histidine =

=

9.35 10-4 mol

=

46.8 mL

Upon complete mixing of the 20mM histidine monohydrochloride solution using a magnetic stirrer, 20mL of the solution was transferred into a beaker. The burette was washed with distilled water followed by NaOH and subsequently filled with 0.05M NaOH. The original pH of histidine solution was measured using the pH meter before proceeding with titration. Titration was carried out by adding NaOH to the histidine solution at 0.5mL increments. After each increment, the pH value of the resulting acid-base mixture was recorded. Titration was stopped when the acid-base mixture reached pH 11.5.

=

=

## Plotting graph of pH against no. of moles of NaOH per mol of histidine

Table: pH of histidine-NaOH solution with every 0.5mL of NaOH added

## Determining pKa values of histidine

(i) Based on Graph 1, the two rectangles indicate the two regions where the curve approaches the point of inflection. The maximum and minimum points of the regions are marked with the yellow circle. By finding the average values of each set of maximum and minimum points, the respective pKa values can be determined.

pKa1 =

= 6.12

pKa2 =

= 9.45

(ii) pKa1 is the point where = 0.5

pKa2 is the point where = 1.5

Based on Graph 1, pKa1 and pKa2 are points marked with the red cross.

pKa1 = 6.16

pKa2 = 9.30

## Maximal buffering capacity & Effective buffering range

Based on Graph 1, the acid-base mixture shows maximal buffering capacity at pH 6.12 and pH 9.45. The effective buffering range of a buffer is between ±1 of the maximal buffering capacity. Thus, the effective buffering range of histidine is pH 5.12 to pH 7.12 and pH 8.45 to pH 10.45.

If NaOH has not been accurately prepared, method used in (c)(i) will give a more reliable estimate of the pKa values.

If NaOH has not been accurately prepared, the number of moles of NaOH will be different, changing the ratio of number of moles of NaOH per mole of histidine. Method (c)(ii) depends on this ratio to determine the two pKa values. Hence, inaccurate ratios will cause the resulting pKa values to vary, leading to less reliable estimate of pKa values.

On the other hand, method (c)(i) does not depend on the ratio between number of moles of NaOH and histidine. Thus, an inaccurate ratio will not affect the pKa values being determined. Instead, method (c)(i) relies on the point of inflection of the graph, which plots pH against the number of moles of NaOH per mole of histidine. Plotting the graph in this manner ensures that it is independent of the volume and concentrations of the solutions used. In other words, even if NaOH has been inaccurately prepared, changing the concentration of the NaOH solution, the shape of the curve remains similar. Since the shape of the curve does not change, the point of inflection will be almost at the same point. pKa values obtained by method (c)(i) will be similar to the original values when NaOH was prepared accurately.

## 5mL of NaOH

No. of moles of NaOH added = Ã- 0.05 = 2.5 x 10-4 mol

NaOH ‰¡ Histidine

No. of moles of histidine reacted = 2.5 x 10-4 mol

Initial no. of moles of histidine = 4 x 10-4 mol

No. of moles of histidine left = 4 x 10-4 – 2.5 x 10-4 mol

= 1.5 x 10-4 mol

pH = pKa + log

pH = 6.12+ log

= 6.34

## (ii) 12mL of NaOH

No. of moles of NaOH added = Ã- 0.05 = 6.0 x 10-4 mol

No. of moles of NaOH left = 6.0 x 10-4 – 4 x 10-4

= 2.0 x 10-4 mol

NaOH ‰¡ Histidine

No. of moles of histidine reacted = 2.0 x 10-4 mol

Initial no. of moles of histidine = 4 x 10-4 mol

No. of moles of histidine left = 4 x 10-4 – 2.0 x 10-4 mol

= 2.0 x 10-4 mol

pH = pKa + log

pH = 9.45 + log

= 9.45

(i) Three ionisable groups are present in histidine at the initial pH of the experiment. The three groups are: carboxyl group, amino group and the R group (imidazole group).

(ii) The amino group is responsible for the observed pKa value of 6.12 and the imidazole group is responsible for the pKa value of 9.45.

## Discussion

Histidine is an amino acid that acts as a buffer and it has three ionisable groups: carboxyl group, amino group and imidazole group. In this experiment, the focus is on the dissociation constant of the amino and imidazole group. The titration curve (as shown in Graph 1) has two ‘steps’, or two points of inflection because the amino group dissociates first followed by the dissociation of imidazole group. Hence, the amino group is responsible for the observed pKa value of 6.12 and the imidazole group is responsible for the pKa value of 9.45. Two methods were used to determine the pKa values of histidine. However these calculated values are only estimates and may deviate from the actual values due to the following experimental errors:

Parallax error occurs during the reading of the burette, resulting in inconsistent increment of NaOH added to the histidine solution. In other words, each increment of NaOH was not maintained at 0.5mL. This directly affects the precision of the experiment.

Possible solution to minimise error:

To avoid parallax error, ensure that the burette reading is taken from eye level at the bottom of the meniscus. The burette should also be placed in an upright position, perpendicular to the table. For a more precise burette reading, a black burette reading card can be placed behind the burette so as to get a clearer view, especially when colourless solutions are used.

The beaker containing the histidine-NaOH mixture is placed on the magnetic stirrer throughout the titration to ensure a homogenous mixture for more accurate pH readings. After every 0.5mL of NaOH added to the mixture, the pH of the resulting mixture is recorded by using the pH meter. However, it takes time for the pH meter to generate a final pH reading that does not fluctuate. If the pH value is recorded too quickly after the addition of NaOH, the pH reading may be inaccurate.

Possible solution to minimise error:

To obtain greater accuracy in pH reading, ensure that an appropriate waiting time (about 2min) is maintained between the addition of NaOH and the recording of pH value.

## Conclusion

From this experiment, it can be concluded from the titration curve that the amino group of histidine is responsible for the observed pKa value of 6.12 and the imidazole group is responsible for the pKa value of 9.45. These two pKa values correspond to the pH at which the acid-base mixture shows maximal buffering capacity. The effective buffering range of histidine is pH 5.12 to pH 7.12 and pH 8.45 to pH 10.45.

## Introduction

Buffers are solutions that are able to maintain a fairly constant pH when a small amount of acid or base is added. This experiment examines the effect of buffer’s pKa on buffering capacity by studying how well the two buffers of different pKa resist pH changes when acid or base is added. In scientific experiments, it is advisable to choose a buffer system in which the pKa of the weak acid is nearer to the pH of the interest. It will be ineffective for a buffer to resist pH changes if its pKa value is more than 1 pH unit from the pH of interest. Thus the study of the effect of pKa on buffering capacity is important in making a suitable choice of pH buffers for a specific experiment.

## Materials and Methods

We study the effect of buffer’s pKa on buffering capacity by using 2 different buffers, potassium phosphate buffer and Tris-HCl, with pKa value 6.8 and 8.1 respectively. 3mL of 0.01M potassium phosphate buffer was pipetted into two test tubes, labelled A and B. 3mL of 0.01M Tris-HCl was also pipetted into two test tubes, labelled C and D. Three drops of universal pH indicator were added into each test tube, causing the solutions to turn green in colour (pH 7.0). HCl was added to test tubes A and C until the solutions turned pink (pH 4.0). KOH was added to test tubes B and D until the solutions turned purple (pH 10.0). The number of drops required for the solutions on each test tube to turn pink or purple in colour is recorded. The pH colour chart is used as it shows the colours of the solution at each pH level.

## Results & Questions

Table : Number of drops of acid or base needed for buffer solution to deviate from its initial neutrality (pH 7.0)

pH Buffer

pKa of buffer

Initial pH

No. of drops of HCl required to become acidic (pH 4.0)

No. of drops of KOH required to become alkaline (pH 10.0)

0.01M potassium phosphate buffer

6.8

7.0

5

11

M Tris-HCl

8.1

7.0

2

20

## Conclusions drawn from experiments

According to Table 2, potassium phosphate buffer requires five drops of HCl to reach pH 4.0, compared to Tris-HCl which requires only two drops of HCl to reach pH 4.0. This shows that potassium phosphate buffer is a more effective buffer against acids. Potassium phosphate buffer requires eleven drops of KOH to reach pH 10.0 while Tris-HCl requires twenty drops of KOH to reach pH 10.0.

Based on the results, Tris-HCl behaves as a more efficient buffer under basic conditions as it requires more amount of KOH than that of potassium phosphate to reach pH 10.0. This means that Tris-HCl has greater ability to resist increases in pH but not decreases in pH. On the other hand, potassium phosphate buffer is a more efficient buffer under acidic conditions as it requires lesser amount of HCl to reach pH 4.0. Similarly, this means that potassium phosphate buffer has greater ability to resist decreases in pH but not increases in pH.

It can be deduced that a buffer with greater pKa value is a more efficient buffer in basic conditions while a buffer with smaller pKa value is a more efficient buffer in acidic conditions.

## Choosing a suitable buffer to study the properties of a phosphatase which functions optimally at pH 7.2

I would use the 0.01M Tris-HCl to study the properties of a phosphatase.

It is more appropriate to use a buffer with effective buffering range nearer to the pH of phosphatase. Tris-HCl has an effective buffering range of pH 7.1 to 9.1 while potassium phosphatase buffer has an effective buffering range of pH 5.8 to 7.8. Simply by considering the effective buffering range of the two buffers, it can be concluded that both buffers can be used to study the properties of phosphatase which functions optimally at pH 7.2.

However, considering the effective buffering range of the buffers is not sufficient to come to a sound conclusion. In this case, phosphatase is an enzyme that functions to hydrolyse phosphate groups. By adding potassium phosphate buffer to phosphatase, phosphatase will break down the phosphate group in the potassium phosphate buffer. This changes the chemical properties and hence the buffering capability of the potassium phosphate buffer.

Therefore, Tris-HCl is a more suitable buffer for the studying of phosphatase.

## Discussion

In Experiment 1, the endpoint of the reactions is determined using a pH meter and construction a titration curve. However in this experiment, the endpoint is visually observed by the help of a pH colour chart. Possible sources of experimental errors arising from this method and ways to improve the experiment are discussed below:

In this experiment, only two types of buffers, Tris-HCl and potassium phosphate buffer, were used. The experiment can be improved by using more types of pH buffers to obtain more data. This will allow more accurate evaluation of the relationship between the pKa value and the buffering capacity, and thus the effect of pKa value on the buffering capacity.

Although the pH colour chart is used to compare the colours of the solutions, personal judgment comes into play when determining the colour change in the chemical reactions.

Possible solution to minimise error:

Be consistent in deciding the point of colour change and the endpoint of the experiment.

## Conclusion

From this experiment, it can be concluded that a buffer with greater pKa value is a more efficient buffer in basic conditions and a buffer with smaller pKa value is a more efficient buffer in acidic conditions. Though a buffer’s pKa can affect its buffering capacity, however when choosing a suitable buffer for an experiment, we cannot simply rely on the pKa of a buffer. It is also crucial to consider the chemical properties and structure of the buffer and other reagents to be used in the experiment.

## Introduction

The aim of this experiment is to examine the effect of temperature on the pH of a buffer. This can be done by observing the changes in pH of two different buffers when temperature of the buffer solution decreases from room temperature to 4°C. pH of the buffers that are used to maintain the pH of the lab samples can change during changes in temperature due to cooling process. Changes in pH of buffers upon temperature changes can be explained by the Le Chatelier’s Principle. The study of the effect of temperature on pH of a buffer is crucial in choosing the right pH buffer that is able to show minimum changes in buffer pH, to maintain the properties of the biological samples that requires specific pH environment.

## Materials and Methods

We study the effect of temperature on the pH of a buffer by using two different buffers, 0.01M potassium phosphate buffer and 0.01M Tris-HCl. 3mL of each buffer solution were pipetted into two separate test tubes. The initial pH values of the two buffers at room temperature are measured using the pH meter and recorded. Subsequently, both test tubes were placed into the ice box to cool to 4°C. After 20 minutes, the test tubes were taken out of the ice box and placed in an ice bath to maintain the temperature of the buffer solutions at 4°C. The pH of the cooled buffer solutions were measured again and recorded to obtain the results as seen in Table 3. By evaluating the pH changes (either increase or decrease) and the extent of these changes from the original pH value, we can observe the effect of temperature on the pH of a buffer.

## Results & Questions

Table : The changes in the pH of the buffer solution as temperature is decreased to 4°C

Buffer

pH at room temperature

pH at 4°C

Difference in pH change (unit)

0.01M potassium phosphate buffer

7.03

7.49

0.46

0.01M Tris-HCl

7.01

8.16

1.15

## Effect of temperature on the pH of Tris-HCl and potassium phosphate buffer

According to Table 3, at low temperature of 4°C, both buffer solutions become more alkaline. As temperature decreased from the room temperature to 4°C, the pH potassium phosphate buffer increased from 7.03 to 7.49, with a difference in pH change of 0.46. With the same change in temperature, the pH of Tris-HCl increased from 7.01 to 8.16, with a difference in pH change of 1.15. This shows that Tris-HCl exhibits greater changes in pH than potassium phosphate buffer, upon a given change in temperature. In conclusion, temperature has a greater effect on the pH of Tris-HCl compared to potassium phosphate buffer.

HA A» + Hº Î”H = -ve

As illustrated by the chemical equation above, the dissociation of buffers are endothermic processes. Being an endothermic process, heat is being absorbed and temperature decreases. Based on Le Chatelier’s Principle, when temperature decreases, the system will react to result in an increase in temperature. Hence, decreasing temperature to 4°C favours the backward reaction, which is an exothermic reaction that produces heat. The position of equilibrium shifts to the left, more Hº reacts with A» to form HA. Thus, the concentration of Hº decreases and causes the pH of the buffer to increase.

## Discussion

Based on the experimental results, it is clear that temperature changes the pH of the buffer. Though this is not a complicated experiment, it is still subjected to experimental errors and can be improved by the following ways:

Only two types of buffers, Tris-HCl and potassium phosphate buffer, were used in this experiment. The experiment was also conducted at only one temperature. Using several buffers over a range of temperatures will allow us to observe the pH of a variety of buffers at different temperatures. In addition, both buffers used in this experiment showed an increase in alkalinity. Hence, including more variety of buffers will allow us to evaluate which type of buffer has tendency to become more alkaline or acidic with the changes in temperature.

This experiment was conducted without the use of a thermometer, hence there was uncertainty in determining the temperature of the buffer solutions. It was assumed that by placing the test tubes in the ice box for 20 minutes and then transferring into an ice bath, the buffer solutions would be maintained at 4ËšC. However, it is difficult to maintain ice baths at 4ËšC for a long period of time due to heat gain from the surroundings.

Possible solution to minimise error:

Keep a thermometer in the ice bath and consistently check the temperature of the ice bath. Add in more ice when the ice melts.

It was difficult to identify the endpoint of the experiment. Even after a long period of time (about 30 minutes), the pH reading shown on the pH meter still continued to increase slowly. Hence, stopping the experiment too early may result in an inaccurate pH reading.

Possible solution to minimise error:

Since it is difficult to identify the endpoint of the experiment, it is perhaps more logical to standardise the duration of the experiment for both buffer solutions. For example, 30 minutes for each buffer solution.

## Conclusion

From this experiment, it can be concluded that a decrease in temperature will cause a change in pH of a buffer. However, the pH of the buffer does not always increase when temperature decreases. This depends on whether the dissociation process is endothermic or exothermic. In the case of an endothermic dissociation process, pH of the buffer will increase when temperature decreases. This can be explained by Le Chatelier’s Principle which states that the backward exothermic reaction will occur so as to counteract the change. Hence, the Tris-HCl and potassium phosphate buffers become more alkaline as temperature decreases.

## The Most Effective Antibiotic On Bacteria Biology Essay

Before bacteria can multiply and cause symptoms our immune system can usually destroy them. We have special white blood cells that attack harmful bacteria. Even if symptoms do occur, our immune system can usually cope and fight off the infection. There are occasions, however, when it is all too much and our bodies need some help – from antibiotics.

The first antibiotic was penicillin. Such penicillin-related antibiotics as ampicillin, amoxicillin and benzylpenicilllin are widely used today to treat a variety of infections – these antibiotics have been around for a long time. There are several different types of modern antibiotics and they are only available with a doctor’s prescription in industrialized countries.

An antibiotic is given for the treatment of an infection caused by bacteria. They target only bacteria – they do not attack other organisms, such as fungi or viruses. If you have an infection it is important to know whether it is caused by bacteria, and not a virus or fungus. Most upper respiratory tract infections, such as the common cold and sore throats are generally caused by viruses – antibiotics do not work against viruses.

Antibiotics are overused or used incorrectly there is a chance that the bacteria will become resistant – the antibiotic becomes less effective against that type of bacterium.

A broad-spectrum antibiotic can be used to treat a wide range of infections. A narrow-spectrum antibiotic is only effective against a few types of bacteria. There are antibiotics that attack aerobic bacteria, while others work against anaerobic bacteria. Aerobic bacteria need oxygen, while anaerobic bacteria don’t.

Antibiotics may be given beforehand, to prevent infection, as might be the case before surgery. This is called ‘prophylactic’ use of antibiotics. They are commonly used before bowel and orthopedic surgery.

## Bacteria

The word bacteria is the plural of bacterium. Grammatically the headline should just say “What are bacteria?” The incorrect usage has been included in the headline to remind readers that it is wrong – and hopefully help correct an increasingly common mistake in the English language. Bacteria are tiny living beings (microorganisms) – they are neither plants nor animals – they belong to a group all by themselves. Bacteria are tiny single-cell microorganisms, usually a few micrometers in length that normally exist together in millions.

A gram of soil typically contains about 40 million bacterial cells. A milliliter of fresh water usually holds about one million bacterial cells.

Planet Earth is estimated to hold at least 5 nonillion bacteria. Scientists say that much of Earth’s biomass is made up of bacteria.

5 nonillion = 5,000,000,000,000,000,000,000,000,000,000 (or 5×1030)

(Nonillion = 30 zeros in USA English. In British English it equals 54 zeros. This text uses the American meaning)

Bacterial cell walls

Bacterial cell walls consist of layer of peptidoglycan which is made up of many parallel polysaccharide chains with short peptide cross-linkage forming an enormous molecule with net-like structure. However, there are two different types of bacterial cell wall, which can be distinguished by Gram staining, a staining technique developed by Christian Gram in 1984 and still in use today. Before staining, bacteria are colorless. The cell walls of Gram-positive bacteria have a thick layer of peptidoglycan containing chemicals such as teichoic acid within their net-like structure. The crystal violet in the stain binds to the teichoic acid and resists decolouring in the rest of the process, leaving the positive purple/blue color.

The cell walls of Gram-negative bacteria have a thinner layer of peptidoglycan with ni teichoic acid between the two layers of membranes and then an outer membrane-like layer made up of lipopolisaccharides. Any crystal violet which does not bind is readily decolourised and replaced with red safranine in the Gram stain. So cells appear red.

## PROBLEM STATEMENT

Which antibiotic is the most effective on bacteria?

## APPARATUS

200 ml of micropipette, conical flask, forceps, 100 ml beaker

## MATERIALS

Petri dish, sample of E.coli and staphylococcus, 3 different types of antibiotic which are carbenicilin, streptomycin and tetracycline, distilled water, Dettol handwash, 75 % of ethanol, agar, tissue paper

## VARIABLES

Fixed variable : volume of bacteria

Manipulated variable : types of antibiotic

Responding variable : area of inhibition zone

## HYPOTHESIS

The most effective antibiotic to inhibit bacteria growth is ampicillin

## PROCEDURE

First of all, wash out hands with the soap or handwash. The working area is sprayed thoroughly with the disinfectant spray. It is left for at least 10 minutes, and wiped with a paper towel.

An agar plate seeded with is prepared. The petri dish is labeled on the base at the edge out of name, the date and type of bacterium it is inoculated with by usng permanent marker pen.

After finishing marking the petri dish, the Esterichia coli bacteria is poured by using micropipette into the petri dish on the marked labeling and distribute it evenly.

The agar is taken out from 60 ÌŠC oven. The mouth of the conical flask containing the agar is warmed in the flame to prevent any different bacteria from surrounding grow inside it.

The agar is left for 10 minutes for it to solidify before putting the sterile disc dip into three different antibiotics.

The forceps are flamed and used them to pick up disc or Mast ring. It is dip into the antibiotic solution and is placed firmly in the centre of the agar.

The dish is taped securely with two pieces of adhesive tape and kept it upside down at room temperature for 24 hours.

Step 1 until steps 7 are repeated by using another type of bacteria which is staphylococcus.

Our hands are washed with soap or handwash and the bench is cleaned again using the 75 % of ethanol.

After the incubation, the plate should be looked at carefully but do not open it. Where bacteria have grown the plate will look opaque, but where the antibiotics have inhibited growth, clear zones called inhibition zones will be seen.

The diameter of the inhibition zones is measured in milimetres and the information is used to decide which antibiotic is most effective at inhibiting the growth of the bacterium.

The data is collected from other members of the class who used the other bacterial cultures.

## PRECAUTIONS

When carrying out the experiment, we should work very closed to the Bunsen burner to prevent any impurities in each apparatus that is used.

Do not seal completely the upper and lower part of petri dish.

Both petri dishes contain different bacteria need to be inverted to prevent the water vapour from forming at the upper site of the agar. It might affected or overshadow the inhibition zones that are formed inside the dish.

Make sure our hands are constantly being wash with disinfectant before touching any apparatus.

Before using any apparatus, they should be sterile to prevent any unwanted impurities to grow in the petri dish.

Do not open the petri dish after incubation to prevent from infection.

## RESULTS

Antibiotic

Diameter of inhibition area/ cm

Inhibition area / cm²

E. Coli

Staphylococcus

E.Coli

Staphylococcus

Tetracyclin

2.6

3.0

5.3

7.0

Streptomycin

1.6

2.0

8.0

3.1

Carbenicillin

1.5

3.5

4.9

9.6

Ampicillin

3.6

3.8

10.2

11.3

Control

0

0

0

0

## Explanation of the data

Table above shows the area of inhibition zone of the bacteria growth on two different bacteria. Four types of antibiotics are used to be investigated which is the most effective on a particular bacteria. They are tetracycline, streptomycin, carbenicillin and ampicillin. Whereas two types of bacteria are used which are staphylococcus and Esterichia Coli.

For E.coli, ampicilin showed the greatest inhibition area of bacteria growth with 10.2 cm². Another antibiotic that showed the closest reading is streptomycin. Tetacyclin and carbenicilin showed 5.3 cm² and 4.9 cm² respectively. This result clearly shown that ampicilin is the most effective antibiotic to inhibit the growth of E.coli. Besides that, E.coli is a gram negative bacteria. The cell walls of Gram-negative bacteria have a thinner layer of peptidoglycan with no teichoic acid between the two layers of membranes and then an outer membrane-like layer made up of lipopolisaccharides.

In addition to that, Staphylococcus also had a higher inhibition zone of bacteria on ampicilin. Other bacterias have no effect as great as ampicilin which can be consider as a strong antibiotic. Staphy is a gram positive bacteria. The cell walls of Gram-positive bacteria have a thick layer of peptidoglycan containing chemicals such as teichoic acid within their net-like structure. The crystal violet in the stain binds to the teichoic acid and resists decolouring in the rest of the process, leaving the positive purple/blue color. That is why ampicilin is said to be the most effective antibiotic on both bacterias.

## Limitations

There are some limitations that cannot be avoided when carrying out the experiment. The first one was every apparatus that we used had been sterile by the laboratory assistant. Thus we had to wash our hands thoroughly before touching anything. We even could not talk during the preparation. The problem was we carried out the experiment on the lab’s table, instead of using the cupboard fume to maintain the sterile apparatus. Thus, there might be some of the apparatus that we were using had been contaminated. This condition might affect the reliability of the experiment hence the expected results might not get at the end of the experiment.

Besides that, we had to wash our hands by using the dettol hand soap before we start and end the experiment. We had to remove all the impurities and bacteria and that was the purpose of washing hand. The problem rose as we did not know whether we had washed our hand to the maximum cleanliness. Consequently, all the apparatus and materials that we used might be affected by our hand. This is done so that there are no infections or contaminations on the agar that might give problems later.

## Sources of error

There are some sources of error when carrying out this experiment. Firstly, we prepared the experiment at room temperature. It was supposed to carry out in sterile medium to prevent anything from affecting during the preparation of bacteria and antibiotics.

Next, the major problem that could be seen during the experiment was most of the groups could not be able to solidify their agar to the maximum hardness. Consequently, when inverting the petri dish, all its contents would be messy inside the petri dish and the disc had mixed to one another. Thus, the antibiotics on each disc also had mixed that could affected the whole result. To overcome this problem, students should let the agar solidify before inverting the petri dish. Another problem was some of the groups do not invert their petri dish before keeping it inside the incubator. This would cause the water vapour to form at the upper part of the petri dish. It would also affect the growth of the bacteria in agar.

Fourth, the layer of agar inside the petri dish was too thin that caused it fell to the bottom when inverting it. Besides ruining the shape of the agar and the position of the discs in the agar, the thin layer of agar also inhibits the growth of bacteria because it lacked of nutrients. To prevent this, the layer of agar should be half of the petri dish.

## Further work

To get more reliable and accurate result, this experiment should be repeated by varying the temperature to investigate the activity of antibiotic on bacteria. This means that increase the temperature would cause the antibiotic to act more rapidly compared to the temperature that we used before. Thus we do not have to wait for 24 hours to see the inhibition zone. This manipulated variable also do not waste the time consuming for the experiment. Moreover, as the antibiotic shows more faster effect, thus the bacteria and antibiotic should be put in separately different petri dish to prevent it from overlapping.

## CONCLUSION

The most effective antibiotic is ampicilin.

The hypothesis is aceepted.