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Cocoa Cultivation Practices in Nigeria

Cocoa is a member of the family Sterculiaceae in the genus Theobromae. It is a tree crop of the hot humid tropics in areas lying between latitudes 15 o north and south of the equator.
Cocoa originated in the Amazon basin in South America from where it spread to Mexico in Central America and other areas where it is cultivated. The crop is grown principally in West Africa, Central and South America and Asia.
Major world cocoa producing countries in order of volume of output are Cote d’Ivoire, Ghana, Indonesia, Nigeria, Cameroon, Brazil, Ecuador and Malaysia. These countries account for about 90% of the total world production. The total global output of cocoa beans in 2010-2011 crop year was 3.938 million metric tonnes out of which Nigeria contributed 12.7% of the production figure.
Cocoa production is largely carried by small holder farmers in developing countries but mostly consumed in industrialized countries with Europe as the largest global consumer, consuming about 37.1% of the global crop.
Cocoa was introduced into Nigeria between 1804 and 1808 and the country is currently the fourth largest world producer after Cote d’Ivoire, Ghana and Indonesia.
In Nigeria, the bulk of cocoa beans is produced in the south-western states (Ekiti, Ondo, Osun, Osun, Oyo and Ogun) which account for 70% of the country’s annual production of between 300,000 – 350,000 metric tonnes on a total land area of about 445,000ha.
Cross River State ranks second among the 14 cocoa growing states in Nigeria (Abia, Adamawa, Akwa Ibom, Cross River, Delta, Edo Ekiti, Kogi, Kwarra, Ogun Ondo, Osun, Oyo and Taraba ) after Ondo. The highest production of cocoa comes from Ondo (23.41%), while Cross River and Edo contribute 19.3% and 8.9% respectively of the nation’s production.
Major areas of cocoa production in Cross River State are in Obubra, Etung, Boki, and parts of Obudu and Obanliku Local Government Areas.
Cocoa is by far the most important tree crop in Nigeria. It provides livelihood and constitutes
ECOLOGICAL REQUIREMENT OF COCOA Cocoa should be planted in where climatic conditions favour best growth. The crop grows well in the under the dense shade in the warm rain forest or primary forest. The trees are affected by strong winds, drought or wide temperature fluctuations. The site should therefore be free of these environmental hazards. The rainfall should be well – distributed throughout the year with annual and monthly totals of 1000 – 1500mm and 100mm respectively. This will promote fruit production all year round.
The average annual temperature should be 250C. Generally, a temperature range 0f 180C to 320C is good for cocoa. Cocoa is a lowland crop and the altitude should not exceed 900m above sea level. The best soil is for cocoa is forest soil which is humus. The soil should be deep for easy penetration of roots, well drained and capable of retaining moisture during the dry season. The PH of the soil should be 4.0 – 7.5. Clay loams, loams and sandy loams are suitable for the growth of the cocoa tree. Shall and infertile soils should be avoided. Steep and convex slopes are unsuitable for cocoa cultivation.
Cocoa requires adequate shade for optimum growth. While changing the forest retain some loose trees to provide shade for the young seedlings. If the site is open or the forest, plant shade trees.
VARIETIES OF COCOA There are three large and distinct groups of cacao within the species Theobroma cacao. These groups are:
Criello Group (Cacao dulce or sweet cocoa)
Native to central America. It is mostly grown in Mexico, parts of the Caribbean, Venezuela, Papua New Guinea (PNG), the West Indies, Sri Lanka, East Timor and Java. Criello cacao typically has:
Slender trees with smaller leaves,
Green, purple or light or a combination of pods or coloured due to the presence of anthocyanin pigments. Finely rapid pods are yellow.
Plum and cylindrical seeds which may be purple or light purple and are easily fermentable.
Soft and easy to break pods due to the absence of woody lager.
Light brown cotyledons (beans) when dry.
Beans have low fat content
High susceptibility to cocoa pests and diseases.
Best flavour beans with spicy and fruity taste.
Yield potential of 1.0 – 1.5t/ha.
Most of the chocolate produced in the world is forastero cocoa.
Forastero group (Cacao amargo or bitter cocoa)
This group of cacao is native to Venezuela and Upper Amazon basin (Ecuador). It constitutes the bulk of cocoa commercially cultivated – Brazil, Central America, the Caribbeans and West Africa particularly in Nigeria. The main characteristics of Forastero group of cacao are:
Green immature pods which become yellow when ripped. There is absence of anthocyanin pigments.
Smooth pods with rounded or pointed ends
Thick woody (lignified) pods which are hard to break
Plum and flattened cotyledons/beans which are deep purple when fresh.
Vigorous and hardy trees which are more resistive to cocoa pests and diseases than criollo.
Highly favoured by farmers due to high disease resistance.
It does not have superior flavour and spicy/fruity taste unlike criollo.
Beans have high fat content.
Trees are vigorous with larger leaves and higher yield than criollo.
Trinitario or hybrid group: They arose from crosses between Criollo and Forastero. Trinitario cocoa includes a wide range of hybrids which originated in Trinidad. This group of cacao exhibits characteristics which are a combination of attributes of both Criollo and Forastero groups.
Mature pods are yellow, orange or red while unripe pods maybe green, whitish, red or purple.
Pods have variable shapes with thick walled pods due to heavy lignification.
Pods maybe smooth or deeply furrowed.
Beans maybe plum or flat
Cotyledons maybe white or dark pigmentation
Dwarf and vigorous trees with variable resistance to pests and diseases.
It has finer flavour than Criollo and is used to provide flavour for chocolate made from “Bulk beans” as well as used to create super – premium chocolate when used alone.
COCOA PLANTING MATERIALS IN USE IN NIGERIA The cocoa Research Institute of Nigeria (CRIN) has developed cocoa clones that have high – yield and are resistant to the black pod disease of cocoa that is prevalent in all cocoa – producing areas of Nigeria. The varieties include T9/15, T12/11, T19/9, T24/12, T50/32 and T86/2.
The Institute has also developed and distributed 15 progenies Virus (CSSV). These are popularly known as CRIN Elites and they include:
T7/12 X Na321 (C64)
T12/5 X Pa35 (C65)
T17/11 X Na32
T20/21 X Na32
T30/10 X Na32
T65/7 X Na32
T85/5 X Pa35
ICS1 X Na32
ICS7 X Na32
C77 X C23 (CSSV tolerant)
C77 X C64 (CSSV tolerant)
C77 X C87 (WACRI series 11F)
C75 X C14 (WACRI series 11KC)
C75 X C25 (WACRI series 11D).
Source: Raw Material Research and Development Council (RMRDC), Abuja. Report on survey of selected agricultural raw materials in Nigeria, 2004 October.
The main varieties of cocoa in current production are the F3 generation of “Amazon” cocoa and the hybrid cocoa. These are high-yielding and resistant to most pests and diseases of cocoa.
RAISING COCOA/SEEDLINGS Selection of nursery site
Select gently sloping ground that is well drained, not susceptible to flooding. The site must be close to permanent water source and close to sufficient quantities of good earth (i.e. forest top -soil rich in organic matter) or any lighter loam soils. The site must be as close as possible to the track suitable for motor vehicles.
Cocoa nursery operations usually start in September in the Southwest while in October in Southern part of Nigeria.
Preparation of the nursery site
In preparing the nursery:
All trees and shrubs should be removed
The site must be levelled out
A wire netted fence must be provided
A fire trace of 3.5m should be constructed round the nursery
Clear site of old materials.
Erecting the nursery shed
Cut poles of 3.1m (100ft) length
Map out the site
Erect poles to a depth of 0.7m (2ft.) with 2.45m (8ft) above the ground.
Erect poles at intervals of 3.1m such that each nursery block is 3.1m x 3.1m (100ft by 100ft)
Space between blocks should be 0.75 – 85m (2ft – 2²/³) wide. This space should serve as path between blocks
Cover the shed at the top, and three sides, with the fourth side open.
Erecting platforms in nursery sheds
Nursery beds can be on the ground or on raised platforms. The beds serve as base for the polythene bags
Beds can be made from split bamboos. The split bamboo should face downwards
Other materials like planks or wood can be used
The polythene bags are arranged on the beds in rows and columns. It is best to arrange the polybags in rows of 5 bags (big size) or 10 bags (medium size) and length of your choice. The row size is indicated so that effective watering of the seedlings in the bags can be done.
Erecting cross bars at intervals of every 10 polybags for the side – support of the polybags
Nursery beds should be erected across the slope of nursery site.
Selection of planting materials
Cocoa is mainly propagated by seed and vegetable propagation is also possible but not popular. The seeds will germinate and produce good plants. Select well ripped pods of not more than 15 days from trees with desired characteristics – high yield early maturity and high resistance to pests and diseases as well as good quality produce. It is very important to use ripe healthy matured disease resistant pod got from the seed garden since pods got from the seed garden are of known parentage and proven performance. Use viable improved hybrid for vigour and higher yield.
Filling the polybags
Use black polythene bags 5? (12.5cm) x (30cm) x 70 micron filled with forest top- soil arranged in hundreds and a bed containing one thousand cocoa seedlings.
Fill the polybags to three – quarters with forest top-soil or any loam soil.
Set the bags on the nursery platform
Water the bags heavily after filling with good soil.
Planting into the polybags
Select the mature cocoa pod to use as planting material.
Open the cocoa pod, the damaged ones first
Remove the seed from placenta attachment in the pod
Plant the seed with the blunt end attached to the placenta downwards. Seeds must be sown as quickly as possible and at the latest two days after the pods have been harvested.
Water the filled bags before sowing the seeds.
Also water lightly after sowing the beans.
In estimating the number of pods needed to plant your nursery, remember that each cocoa pod contains an average of 30 seeds.
Propagation by other methods
Seeds – planting at stake
2 cutting –
Gives uniform growth
New plants do not grow tall
Matures early
New plants do not have tap root.
Budding – similar to cutting
Grafting – similar to cutting.
Nursery Management
Label the Nursery – such a label should contain information on:
Type of seed
Date of sowing
No. of seeds sown
Sources of seeds
Water Management: Light and frequent watering is better to avoid losing some already germinated young cocoa seedlings from dying as a result of the excessive heat emanating from the polythene bag being resultant effect from scotching sun. Too heavy water could encourage the growth of fungi, wastage of water, starvation before the next watering. Watering of the seeds is usually done in the morning and at cool evening with the use of watering cans. Don’t allow leaves of seedlings to stick the soil as such could encourage crooked seedlings.
Weeding. Weeding around the beds and picking of weeds from the potted seedlings. Weeding should be done 2-3 times before transplanting.
Shade management: Cocoa requires shade at the initial stage of development. Shade should be erected using bamboo and palm fronds. The size would depend on the population of seedlings to be produced.
Hardening up. Seedlings should be trained to the conditions on the field hence hardening up is practiced. The shade should be gradually reduced when the seedlings is 5 months old and totally removed at six months when the cocoa seedlings are ready for transplanting.
Disease control: If rainfall is heavy, fungus diseases and damping-off spread easily and fast. Spray with the appropriate chemicals and encourage adequate air circulation.
Pest control: Check and control pest population. Use insecticides if necessary.
ESTABLISHMENT OF A NEW COCOA FARM/PLANTATION Site selection and planting of shade trees
Cocoa grows well in the natural forests in the under storey and in association with shade – producing tree species like banana/plantain, cocoyam or cassava to provide temporary shade during the early stage of establishment. Fruits trees such as Dacryodes edulis(pea tree or bush butter ), Irvingia gabonensis (ogbonno), papaya, citrus and coffee are usually interplanted with cocoa to provide permanent shade in cocoa plantations. Tall trees such as Inga spp, Erytrina spp, Gliridia spp may also be planted as plant shade trees. Shade trees should be established prior to introduction of cocoa seedlings and should be well arranged to shelter the young plants shade trees should be compatible with cocoa.
Land clearing. Clearing method in sites selected for cocoa cultivation depends on the existing vegetation. In Nigeria, virgin and secondary forests are usually used for cocoa farms. It is advisable to remove commercial timber species if present and other smaller trees and then plant seedlings along traces cut through this selectively thinned forest. Alternatively, total clearing could be done and plantains planted to provide shade, at 3.0m x 3.0m spacing in sites intended for cocoa farms.
Transplanting. Seedlings are transplanted into the field when the rains become steady and regular, usually between May and June. Planting holes should be 20cm x 20cm x 20cm spaced at 3m x 3m giving a population of 1111 plants/ha. Cocoa seedlings should be carefully transplanted to permanent sites after 6-7 months of growth in the nursery. The balls of earth should be carefully held on to the seedlings when removing the polythene bags during transplanting operation and spaced at 3.1m x 3.1m apart. About 1010 cocoa seedlings are required to transplant an hectare. One week to transplanting the seedlings to the field, seedlings raised on ground bed nursery should be partially dug in situ to stimulate the development of new roots prior to transplanting into the field. Discard badly shaped and off-type seedlings.
Fertilizer application. Soil fertility is usually not a problem on new plantations. However, for good growth and high yield, the following recommendation should be followed:
For the first two years – apply 100-200kg of Urea/ha or 200kg NPK 200kg P2O5/ha. OR
3-4th year – Apply 200-250kg Urea/ha or 500-600kg NPK Mg/ha.
5th year and above – Apply 150-250kg Urea/ha or 500-6-kg NPK Mg.
These should be applied in a ring form 15cm away from the base of the seedling.
If planted on land that has been under cultivation for some years the following fertilization programme should be followed.
Fertilizer application programme for cocoa
Recommendation
Year
Soil cleared from forest
Soil clear from cocoa and arable crop
1st year
50g single super phosphate (SSP) or
20g TSP/tree.
22g urea 50g SSP 8gKCL/tree. Apply on the soil surface in a circle at least 25cm from the stem. Apply half rate in July and the other half in September.
2nd – 3rd year after planting
125g SSP or 55g TSP/tree
54g 55g SSP 179g KCl/ tree. Apply in a ring round the stem. Half rate in April and remaining half in September.
4th – 6th year.
150 – 300kg SSP/ha or 63 – 126kg TSP/ha
108 – 216g tree 150g SSP 50g KCl/tree. Broadcast half the rate in April/May and the remaining half in August/September.
Matured ‘Amelonado’ Cocoa
305kg Urea 250kg SSP
40kg KCl/ha
305kg Urea 250kg SSP 40kg KCl/ha.
Broadcast and split annual rate into two for April/May and September.
Matured Amazon Cocoa
374kgUrea 375kg SSP 97kg KCl/ha
Boron at 30g/tree
374kg Urea 375 kg SSP 97 kg KCl/ha. Boron at 30 g/tree. Broadcast Nitrogen fertilizer in split doses, half in August. Apply P and K or Boron once in April/May.
FARM MAINTENANCE OPERATIONS: Provision of shade: Provide shade during the first 2-3 years of field transplanting before the canopy closes. An ideal shade tree should be easy to establish with a short period of time and should compete as little as possible for water and nutrients with cocoa and should be easily removed when not needed
Mulching: Newly transplanted cocoa seedlings should be mulched around October before the rains cease. This will help conserve soil moisture and enable the seedlings survive the first dry season in the field.
Weed control: Clean weeding is economical on smallholdings up to 2ha. In large plantations, row weeding thrice a year supplemented with slashing of avenue is adequate. Weeding improves air circulation and reduces the relative humidity thereby reducing the incidence of Blackpod disease. Intercropping newly established cocoa plantation with annual crops up to the third year reduces weed infestation. Suitable intercrops include:
Cocoa/yam/maize/cowpea
Cocoa/cassava/maize/melon
Cocoa/cocoyam/melon/okro.
Use of Herbicides:
This is economical and more effective on large plantations, but care should be taken not to affect young cocoa seedlings.
Use mixed formulations of Asulox 40 plus Actril DS at the rates of 7.5 x 1.5 and 8.5 x 1.5 litres/ha on old plantations.
Pruning: This is done after 2-3 years of growth to maintain regularly shaped trees. Remove all unwanted branches, Chupons and flushes on the stems or branches. Use a sharp machete. Pruning should be done in such a way to promote lateral growth for easy harvesting later on. Treat all pruned surfaces to prevent u=entry of pathogens/diseases. Light pruning maybe done at any time of the year, while heavy pruning should b done in February/march or May/June.
COMMON DISEASES OF COCOA: Swollen shoot disease:
This is a viral disease transmitted by mealy bugs. Infected plants have swellings on shoots and roots, which results to small pods, stunted growth/few leaves
Control:
Plant resistant cocoa varieties and destroy all infected plants.
Black pod disease:
It is the most serious disease of cocoa in West Africa in general and Nigeria in particular. It is a fungal disease caused by the fungus Phythopthora palmivora. The disease attacks young and matured fruits as well as young leaves and trunks of cocoa. It also causes seedling die-back. The major damage caused by the disease is the rotting of both young and matured pods. The disease is most severe in the rainy season and is spread mostly through rain drops/rain splash, pod contact, infected knives, contact with infected soil and insects/rodents.
Control:
Removal and destruction of infected pods.
Weed control.
Spray every 21 days during the rainy season with mostly copper based fungicides like Lime-Bordeaux mixture, Cupravit (Cupric hydroxide), Ridomil (Apron 35) and Rodomil plus.
Die-back disease:
This is a fungal disease caused by Calonectria regidiuscula. It is spread by Capsid and Mirid bugs which feed on tips creating wounds through which the fungus enters.
Symptoms:
Defoliation
Death of branches
Yield reduction.
Control:
Spray with Gammalin 20 to kill the mirids.
MAJOR PESTS OF COCOA: Cocoa Mirids (Capsids):
They are the most serious pests of cocoa in Nigeria. They include;
Brown Mirid (Sahlbergella singularis)
Black Mirid (Distantiella theobromae)
Cocoa mosquito (Helopeltis bergrothi)
Cocoa Mealybugs:
Planococoides njalensis
Planococoides citri
Fusarium virgata
Other important pests are Thrips, Caterpillars, Aphids and Termites.
Control:
Use insecticides like Gammalin 20, Aldrax, Aldrin, etc at recommended dosages.
HARVESTING OF COCOA: Trees start bearing after 4-5 years and remain in production for about 35 years. Cocoa fruits take about 5-6 months to ripen. Pods grow from the trunk and branches of the cocoa tree. Harvesting involves removing ripe pods from the trees and opening them to extract the wet beans. The pods are harvested manually by cutting off the stalk with a well sharpened knife or blade. Pods high on the trees should be removed with the pruning hook mounted on a long pole.
Caution:
Do not climb the trees to harvest in order not to damage the flower cushion which will produce the flowers and fruits of the subsequent harvests.
Harvest ripe pods timely to prevent germination of beans inside the pods.
Harvest only well matured fruits.
Do not tear-off cocoa fruits to avoid damage to the bark of the trees.
Breaking of Pods: Break the pods to extract beans. Use blunt machetes or sticks, pods with light shells/husks can be broken with hands or knocked together.
Caution:
Do not cut through the pods as this damages the beans
Do not delay bean extraction beyond 2-3 weeks after harvesting to prevent germination of the beans.
FERMENTATION: This is the most important operation in processing cocoa beans. Fermentation is done to;
Kill the embryo of the beans and stop germination
Reduce bitterness
Remove the mucilage/pulp to facilitate drying
Loosen the testa/seed coat for easy removal during industrial processing.
Make the embryo crisp and brittle
Develop the characteristic flavour, taste and colour.
Destruction of pathogens.
Fermentation can be carried out in three ways viz;
On small holdings, fermentation is usually done in heaps of beans enclosed by plantain or banana leaves. The heap method can handle about 25kg to 2,500kg of cocoa beans and fermentation usually last about 5-7 days.
Basket Method:
Use baskets lined and covered with fresh plantain/banana leaves to ferment the beans. It takes 3-5 days.
Box Method:
This is common in plantations and is the best method. Fermentation is normally carried out in large wooden boxes that typically hold 1-2 tonnes of beans. The beans can be covered with banana leaves or sackling to conserve th heat generated during fermentation. The use of fermentation boxes promotes aeration within the box and good drainage. Fermentation is completed in about 4 days.
DRYING AND BAGGING OF COCOA BEANS: Drying of cocoa beans stops further biochemical reactions, hardens the beans and prevents growth of mould in storage. For safe storage, the seed moisture content should be 6-7%. Sun-drying is still the practical method of drying cocoa beans in Nigeria. Slow and even drying is important to avoid wrinkling of beans and for uniform drying. Sun-drying can be done on concrete floors or raised platforms or tarpaulins. Spread the beans in thin layers and rake regularly to ensure even drying.
The seed-coat (shell) of well-dried beans should separate readily when nicked by the thumb nail and the cotyledon (nib) should crack easily when pressed. Properly dried beans exhibit a reddish colouration. Under dried beans show rubbery (pliable) cotyledons while over dried beans crumble into fragments when pressed. Both are undesirable. Dried cocoa beans are usually bagged in jute sacks in 50kg weight.
STORAGE:
Cocoa beans are bagged in jute sacks and stored in warehouses. Bags should be placed on wooden platforms and not directly on the floor. The warehouse should be well ventilated to prevent build up of relative humidity which promotes the development of fungal diseases.
COCOA QUALITY AND GGRADING:
Cocoa beans should be free from deterioration, additives, contaminants (foreign bodies) and should have neutral odour. The beans are graded based on:
Slatiness (Unfermented beans).
Mouldiness (causes permanent off-flavour)
Contaminants (chemicals, foreign bodies, damaged beans, etc)
Based on the above factors, cocoa beans are graded as:
Generally, cocoa should be;
Uniform in size
Thoroughly dry and free from
Smoky beans
Foreign bodies/odour
Adulteration/contaminants/hamful substances e.g heavy metals, insecticides, micro-organisms
Living insects (Weevils)
Broken or rotten beans
ADDING VALUE TO COCOA:
Value addition is the treatment given to cocoa to increase its economic value. Enhancing the value of cocoa beans starts from planting through harvesting, bean extraction, fermentation, drying, bagging, storage and processing into intermediate or finished products.
COCOA PROCESSING:
This includes converting the fermented beans into nibs, liquor, butter, cake or powder. Before cocoa can be made into chocolate, it must pass through;
Inspection and cleaning
Roasting
Shelling
Grinding
Products that can be obtained from processed cocoa include;
Nibs
Liquor
Cocoa butter
Cake
Powder
Chocolate
Milk chocolate
MARKETING OF COCOA IN NIGERIA:
Most cocoa produced in Nigeria is marketed as cocoa beans. Cocoa price is determined at the international market based on demand and supply. Government only ensures quality compliance/regulation.
Major cocoa buyers/importers include:
United States (32.7%)
Germany (11.6%)
France (10.3%)
United Kingdom (9.2%)
Russian Federation (7.7%)
Japan (6.4%)
Italy (4.6%)
Spain (3.8%)
Brazil (3.7%)
STAKEHOLDERS IN COCOA BUSINESS:
Cocoa farmers (producers)
Warehousing and shipping agents.
Local Buying Agents (LBAs)
Large Merchants (Middle men)
Local processors
Exporters
International processors
Final consumers/importers
Government (Agencies/Department)

Effects of Antiseptic on Bacteria Growth

This project was designed to investigate the effect of different antiseptics on bacterial growth. The antiseptics are different in their content and active ingredients. Two bacterial lawns were prepared using nutrient agar, Escherichia coli and Bacillus subtilis. Paper discs dipped into the antiseptics were put on the bacterial lawns and the diameter of clear zones around the paper discs were measured after incubating the bacterial lawns for 24 hours at 30°C. The results showed that 4.8% chloroxylenol is the most effective antiseptic in inhibiting the growth of bacteria, indicated by the greatest diameter of clear zone.
The results of this experiment can be applied to the use of specific antiseptics on specific type of bacteria. Further research can be done on cetylpyridinium chloride in the usage of the compound as an external antiseptic.
Planning A trial experiment was carried out to find out whether the antiseptics will inhibit the growth of Escherichia coli and Bacillus subtilis or not. Five fresh samples of antiseptics containing different active ingredients; which are 0.05% cetylpyridinium chloride, 4.8% chloroxylenol, 0.1% acriflavin, 70% ethanol and 0.1% chlorhexidine gluconate and sterile distilled water were put into separate sterile 50ml beakers. Distilled water was used as a control. Before conducting the experiment, the table top was wiped with alcohol and hands were washed with soap. These steps were taken to kill the bacteria present. 200µl of bacterial inoculum containing Escherichia coli was introduced into a sterile petri dish using a micropipette. The lid of the petri dish was opened as minimum as possible to avoid contamination. Molten nutrient agar was then poured into the petri dish and the petri dish was swirled gently to mix the inoculum and agar. After the mixture has solidified, a sterile filter paper disc which has been dipped into the distilled water was placed on the surface of bacterial lawn containing Escherichia coli by using a sterile forceps. This step was repeated by replacing the distilled water with the antiseptics. The paper discs were placed as further apart as possible from each other. After that, the petri dish was placed in an incubator set at 30°C. The whole procedure was repeated for bacterial inoculum containing Bacillus subtilis. After 24 hours, the petri dishes were removed from the incubator and the diameter of clear zones around each paper disc was measured using a ruler.
The whole steps in trial experiment were used in the main experiment. The experiment was repeated ten times, meaning that for each antiseptic, there will be ten readings of diameter of clear zone. Repetition is necessary to calculate the mean diameter so that the efficiency of each antiseptic is measured more accurately.
A one-way Analysis of Variance (ANOVA) test will be used to analyse the data, which will test the equality of six means at one time by using variances.[15]
[1015 words]
Risk Assessment In this experiment, I am using chemicals and bacteria. All the chemicals used are toxic if swallowed. Therefore, it is not safe to consume them. Chloroxylenol and acriflavine can cause skin and eye irritation. Therefore, gloves and goggles were worn when conducting the experiment. There was risk of infection by the bacteria during and after conducting the experiment. Aseptic measures were taken before and during conducting the experiment, such as wash hands with soap and wipe the table top with alcohol. The petri dishes must be disposed properly (do not throw into bin) and autoclaved.
Results Statistics I will use a one-way Analysis of Variance (ANOVA) test to demonstrate the data statistically. All the calculations are made using Microsoft Excel 2007 software. In ANOVA test, the means, variances, sum of squares (SS), degrees of freedom (df), mean square (MS) and F-ratio are calculated in order to get the P-value.[16] P-value less than 0.05 (5% confidence level) indicates that, there is a significance difference between the groups.
Experimental Hypothesis The diameter of clear zone around paper discs dipped into 4.8% chloroxylenol is the greatest.
Null Hypothesis There is no significant difference in the diameter of clear zone for each of the antiseptics.
P-value for both tests is less than 0.05 (1.38-10-31 and 3.83-10-42 respectively). Therefore, null hypothesis is rejected and the experimental hypothesis is accepted. 4.8% chloroxylenol shows the greatest diameter of clear zone for both bacteria.
Data Analysis The result of one-way ANOVA test showed that there is significant difference between the values of mean for each group. This is because the P-values are far lower (1.38-10-31 and 3.83-10-42 respectively) than confidence level of 0.05. The P-values are small because the values of mean and variance are very different with each other. From the P-values, it can be concluded that there is less than a 5% probability that the results occurred due to chance and more than a 95% chance that the results are significantly different. This does not agree with the null hypothesis which states that there is no significant difference between the groups, hence it is rejected and the experimental hypothesis is accepted.
Bar chart 1 shows the mean diameter of clear zone for each antiseptic tested on Escherichia coli. The effect of cloroxylenol on Escherichia coli is almost the same as cetylpyridinium chloride (mean diameter of clear zone: 1.28cm and 1.23cm respectively). As distilled water does not have any antibacterial properties, it does not have any effect on Escherichia coli. The least effective antiseptic when tested on Escherichia coli is acriflavin (0.75cm), followed by ethanol (0.95cm) and chlorhexidine gluconate (1.10cm).
[1916 words]The same trend is seen when the antiseptics were tested on Bacillus subtilis (bar chart 2). Chloroxylenol shows large difference in the effect on the bacteria compared to the other antiseptics (mean diameter of clear zone: 2.05cm). Acriflavin is the least effective antiseptic (0.73cm). There is small difference in the effectiveness of ethanol and chlorhexidine gluconate on Bacillus subtilis.
Bar chart 3 shows the comparison of the effectiveness of antiseptics on both bacteria. Generally, all antiseptics work better on Bacillus subtilis except for acriflavin, but the difference is small. Chloroxylenol shows clear difference in its effect on both bacteria (difference in mean diameter: 0.77cm), while the others show only small difference.
Chloroxylenol is a broad spectrum antibacterial substance which works by altering bacterial enzymes and cell walls.[17][18] Its mechanism of action have been little studied, but because of its phenolic nature, it would have effect on bacteria.[3] Phenol induces lysis of intracellular constituent, including the release of potassium ion (K ). It also lyses growing culture of Escherichia coli and Staphylococcus aureus.[3] E. coli is a Gram-negative bacterium while Staph. aureus is a Gram-positive bacterium. This proves that chloroxylenol can work on both type of bacteria.
Cetylpyridinium chloride is a quaternary ammonium compound which is highly sensitive to Gram-positive bacteria, but moderately sensitive to Gram-negative bacteria.[19] Its mode of action is based on interaction of basic cetylpyridinium ions with acid group of bacteria, which subsequently inhibits bacterial metabolism by forming weak ionic compound that interfere with bacterial respiration.[20] This proves that this compound works better on B. subtilis than on E. coli.
Chlorhexidine gluconate is a bactericidal agent.[3] It damages the outer cell layers, crosses the cell wall or membrane (probably by passive diffusion) and attacks the bacterial cytoplasmic or inner membrane.[3] It is active against wide range of Gram-positive and Gram-negative bacteria.[21] This proves that chlorhexidine gluconate can work both on E. coli and B. subtilis.
[2232 words]Not much is known for the mode of action of ethanol. It is believed that it causes membrane damage and rapid denaturation of proteins, with subsequent interference with metabolism and cell lysis.[3] Most hand cleansers contain between 65% to 70% of alcohol. Higher concentrations are less effective because proteins are not denatured easily in the absence of water.[22]
Acriflavin has the ability to intercalating with DNA and cause frame shift mutation.[23] It also affect DNA molecules of microorganisms.[24] Therefore, it inhibits bacterial reproduction. In this experiment, it is proven that acriflavin can affect the growth of E. coli and B. subtilis.
Evaluation The concentration for each antiseptic is different from each other. Therefore, it is not 100% confirmed that chloroxylenol is the best antiseptic in inhibiting or killing E. coli and B. subtilis. In this experiment, I am testing which antiseptic is the best, but the concentrations of all antiseptics tested are known from the label. This is concern with our daily life chemicals which we may use frequently. It is better if the pure substances of the active ingredients are available. By having the pure substance, I can test the effectiveness of same concentration of active ingredients on bacteria.
Besides that, the clear zone area around the paper discs is not regular circle in shape. This makes the reading of diameter of clear zone is not very accurate. Therefore, a few measurements of diameter have to be taken and then, the average value is calculated.
The diameter of clear zone is measured using a ruler. The smallest scale on the ruler is 1mm. In order to get more accurate reading, a more sensitive apparatus (smaller scale) can be used, such as vernier callipers.
Although aseptic steps have been taken, the environment is not 100% free of bacteria. Some bacteria might have contaminated the apparatus and materials. This is uncontrollable. Therefore, the risk of contamination is minimise as minimum as possible by conducting the aseptic steps.
[2515 words]
Sources Evaluation All the journals were taken from reliable sources. This is because the authors of the journals used wide range of sources when writing the journals and they cited where they extract the information. Besides that, careful methods and appropriate materials also made me feel that the journals are reliable. In this report, the main journal that I referred to was Antiseptics and Disinfectants: Activity, Action, and Resistance written by Gerald McDonnell and A. Denver Russell. The journal was taken from a government’s website http://www.ncbi.nlm.nih.gov. I believe that this source is reliable as a lot of experts work for them and most of them publish their work in this website.
Source 1 was taken from a book (WHO Model Formulary) published by a well known organisation, World Health Organisation. I believe that this book provides reliable information as the publisher is a world organisation. Besides that, the writers (Mark C. Stuart, Maria Kouimtzi, Suzanne R. Hill) also wrote several other books.
Source 18 was taken from another book (Browner: Skeletal Trauma, 4th ed.) published by W. B. Saunders Company. All the authors of this book are doctors or experts with at least Medical Degree. Therefore, what they wrote in the book is not bias to any side and they also cited the sources they used when writing the book.
Source 2 was taken from a website. This website is a university’s website which provides notes for their students. I believe that the writer of the article is a lecturer at the university. The university (University of South Carolina) is a well established university with a lot of achievements.
Source 10 was taken from another website administrated by a company lead by doctors (MedicineNet, Inc.). I believe that this source is reliable as the authors of the website are experts from medicine, healthcare, Internet technology and business field.
[2849 words]Source 13 was taken from a website of European Bioinformatics Institute. The organisation is part of European Molecular Biology Laboratory. This source is reliable because the organisation is a joint venture between European countries and a lot of studies have been done by this organisation.
Source 19 was taken from a corporate website. This company (Vertellus Specialties, Inc.) was established before World War 2 and mainly involve in the production of chemicals. This source seems reliable as the company involves in this field for hundred of years and has involved in many researches.
[TOTAL 2916 words]

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