Isolation And Enumeration Of Micro-organisms Associated With Spoiled Tomatoes Sold In Abakaliki, Ebonyi State
Isolation And Enumeration Of Micro-organisms Associated With Spoiled Tomatoes Sold In Abakaliki, Ebonyi State
Tomatoes are among the vegetable products cultivated world wide that has the greatest nutritional value (Thompson, 1998). To place an order for the Complete Project Material, pay N5,000 to Then text the name of the Project topic, email address and your names to 08060565721.
Account Name – Chudi-Oji Chukwuka
Account No – 0044157183
To place an order for the Complete Project Material, pay N5,000 to
Then text the name of the Project topic, email address and your names to 08060565721.Because they are seasonal crops, has made their microbiological study necessary so as to identify their spoilage organisms. Added to their nutritive value, is their medical importance, inspectional effect as well as economic important (Frederick, 1983).
Tomatoes fruits can be consumed raw and fresh, though mostly, it is cooked. It is consumed world wide there is knowledge of the spoilage organism in only developed countries, little in developing countries (Eckert Summer, 1976). These organisms are detrimental to human health.
Spoilage of tomatoes is defined as those adverse changes in quality of tomatoes, which are brought about by action of predominantly biological factors and physical factors. These change may include; change in the taste, smell and appearance or texture of the tomatoes fruits (Hooker, 1967).
The importance of tomatoes in food industries and its nutritional importance cannot be over emphasized, and the need for microbiological examination of tomatoes is very importance as it contributes to a large extent to economic development.
1.2 LITERATURE REVIEW
Tomatoes (Lycopersicon esculentum) belong to the family solanacea (night shade family). Wild type tomatoes species are said to originate from western South Africa and specially in the coastal desert of Peru. First historical reference in Europe to tomatoes was a yellow-fruited type in Italy in 1544 and cultivated in Germany in 1553 (Chambers, 1979).
The Italians were said to have acquired it from Turks. Tomatoes was first grown in Spain, then taken to Italy where it was called “pomd” “oro” or golden apple, because of its yellow colour.
The French called it “poma aman’s” or apple of love. The African Indians used to name it “tomall”. The tomatoes were not very popular until the 10th century mainly because of rumor that labelled it as a poisonous fruit. It was suggested that certain famines during this century forced people to try this plant (Stary, 1983).
The tomatoes was introduced into the united states in 1710 and by 1779, it was used in cat sup in new orleans. Thomas Jattenson grew tomatoes in virgina. But as late 1900 the tomato was still considered poisonous (Hill, 1952).
George Washington cancer was known for standing before crowds and eating tomatoes, trying to introduce tomatoes into the diet. The tomato originated in South American due to the mild species formed from Equador to Chile. In summary, tomato traveled from South America to Europe before arriving the united states (Chief, 1984).
Lycopersicon esculentum is probably derived from lycopersicon priminllitollum which is indigenous to Peru and Ecuador, Lycopersicon Esculentum var cavsiforme (Cherry tomato) is considered a wild plant in the tropics and subtropics (Uphof, 1999).
There is also evidence that tomatoes originated in Mexico (Bianchini, Corbetta and Postoria 1987). These tomatoes show great diversity in size and shape including all colour types, such as pink, red and yellow. The cherry tomato is widely used in Mexico and has many India names the Nah watt language of Mexico used the name “tomatt which has followed the plant through Europe and South America. Mexico would be a logical origin since cortex conquered Mexico in 1519, tomatoes could have easily found their way to Italy by 1544 (Chief, 1984)
1.3 VARIETIES OF TOMATOES
There are hundred varieties of available tomatoes than other vegetable which includes; Lycopersicon esculentum, Lycopersicon lycopersicum and Lycopersicon hirsutum. Most tomatoes are normal but some appear in pear shapes, elongated, flattened and heart shape. Tomatoes vary in their acid composition, with white and yellow ones being less acidic. Sizes ranges cherry tomatoes less than an inch across to whoppers that can weigh more than two pounds and exceed 6 inch (15CM) in diameter (Bianchini, 1979).
Tomatoes varieties can be either determinate or in-determinate. Determinate tomatoes plants are small and spinally have just one fruiting or vent. The indeterminate types continue to grow and produce fruit as long as conditions are favourable. They require staking a caging and their fruits usually are higher in sugar than determinate types. Lycopersicon pimp-inellifolium (the new discovered specie produces small (half-inch in diameter) five engine red fruits in profuse grapelike clusters on long trailing vines (Chief, 1984). They have a distinctive less acidic flavour and a more watery texture but are nonetheless quite delicious as well as very showy (Facciola, 1990).
1.4 DESCRIPTION OF THE VARIETIES
Species Shape Size Colour
elongated 15 cm (6 in diameter) Orange
Lycopercicum Flathened 4cm (1.5 in diameter) Pink
hirsutum humb Deeply ribbed Not definite Various combination
1.5 DIFFERENCES AMONG THE MOST AVAILABLE SPECIES
1.5.1 (A) LYCOPERSICON LYCOPERSICUM
This species produce small (Chalf-inch in diameter) five engine red fruits in profuse grape like clusters on short trailing vines. They have a distinctive less acidic flavour and have more watery texture but are nonetheless quite delicious as well as very showy they have just fruiting event and do not grow continuously.
1.5.2 (B) LYCOPERSICOM ESCULENTUM;
This species produces elongated or heart-shape (5 inch in diameter) tomatoes fruits on grape like clusters on long, trailing vines, they also have a distinctive, less acidic flavour and watery texture, not withstanding, quite decisions. They require staking, trellising or caging and their fruits usually are lighter than the determinate fruit types. They grow continuously and produce fruit as long as conditions are favourable.
1.5.3 (C) LYCOPERSICON HIRSUTUM HUMB AND BONPL
This species produces round or deeply-shaped (four inch in diameter) striped with various combination fruits sparsely distributed (not clustered) nor moderately trailing vines. They are less acidic, flavoured with a more watery texture but quite delicious as well as attractive. They have just one fruiting event (do not grow continuously). This type is usually produced in large quantity due to its high productive ability and widely distributed, it is the most consumed type of tomatoes. They also require staking, trellising or caging for high yield its high productive ability and widely distributed, it is the most consumed type of tomatoes.
1.6 PLANT CHARACTERISTICS
1.6.1 ROOT SYSTEM
The root zone of tomatoes can extend outward and downward up to 5 feet the depth of the tap root depends on the soil (nature) and cultural practice (harvesting tomato transplants for replanting generally damage the tap root making these tomato plants depend upon a fibrous root system in the upper 6 to 10 inches of the soil). In cross section, the xylem forms a cylinder in the center of the root, with two lateral wins roots arises from. The peri-cycle cells and grows through the cortex. The xylem is usually hard (Hill, 1952). The Phloem completes the vascular tissue, filling out the space between the wings and forming cylinder. This is surrounded by an endodermis, a three layered central and epidermis lateral roots.
The stem is typically about 4cm in diameter at the base and is coloured with glandular and non-glandular horns that when touched stain the hands greenish black and give a characteristics tomato smell. There are phloem strands both inside and outside a tube of xylem fibers, which surrounds a core center of the tomatoes cells. A secondary phloem develops, the outer phloem forms a more continuous cylinder secondary xylem is more developed in the slow growing stems containing larger core centre of the tomato cells. The whole vascular structure and the peri-cycle form the siphemastelel are Dermis, collenchymatus cells and photosynthetic cells, Immediately under the epidermis at the top of the main stem is the apical new stem. It is dome-shaped and is protected by newly formed leaves, this is where new leaves are formed before. The apex becomes a terminal inflourescence every three leaves, where as determinate cultivers produce a limited number of inflourescence on each axis and strong axillaries buds develop on the base of the stem producing a bushy appearance. (Chittendon, 1951)
The size of the compound leaf is variable. Small leaflets are interspersed with large leaflets. The lowest leaves are small with few leaflets leaves thereafter can be up to 0.5 meters long with up to light lateral leaflets. The leaves are covered with hairs of the same types as the stem.
Botanically, a tomato fruit is berries consisting of seeds within a fleshy pericarp development from an Ovary Fruits of Lycopersicon esculentum have two to several carpals. There is extreme variability of fruit characters such as size, shape exterior colour or of mature and immature fruit and interior flesh colour.
The seed of a commercial cultivers is a flattened avoid, up to 5mm long 4mm and 2mm deep, consisting of the embryo, endosperm and testa the embryo consist of the radical hypocotyls, two cotyledons and the short apex. The testa is covered with large soft hairs which tend to bind with other seeds.
1.7 PROPAGATION METHODS
Tomatoes can be direct seeded or transplanted and seeding is used if soil moisture and temperature favoures rapid germination and if mechanical Harvesters are used.
Tomatoes require a lot of fertilizer for the best production, before planting.
Precision seeders place (3) three to five seeds at desired spacing. One of the (3) three of the seedlings generally develops in one chump. These seeds are either Thinned or a left to develop. Thinning generally dose not effect yield per acre. Seedlings rate per acre varies from 56,000-192,000.
Tomato growers with a shorter growing season and most home gardens employ transplant seedling. These are mostly grown in the south cast (Hedrick, 1972). These transplants are four to six niches tall and are four to six weeks old before being harvested for transplanting. (Hedrick, 1972)
Since tomato is a warm season perenial cultivated as an annual plant, it requires a day to night temperature change of at heart 10% during long days to be productive. In other words tomatoes should be grown in full sum.
Tomatoes should be watered before the soil dries out completely. Irregular watering can cause the fruit to spit of crack and can also contribute to blossom –end not.
1.8 SUMMARY OF PROPAGATION METHODS
Tomatoes are actually perennials in tropical climates but graders in temperate climates grow them as warm season, frost- tender annuals. Tomato foliage is damaged by frost and freezing temperature will kill the plant. Most varieties will not set fruit if nighttime temperature fall below 500f (100C) or if daytime temperature says above 900f (320C). (Hill, 1952).
1.8.1 PRODUCTION AREA
Since 1900, the tomato has become the second most important vegetable crop with the white potato being the number one. In the U.S. over eight million tons produced annually. Seven million tons-were processed (soups, juice catsup, source, whale tomatoes and prepared foods) and over one million tons were sold as fresh tomatoes. This does not include considerable amount of tomatoes grown in home gardens. Ninety-three percent of the 33 million gardens in America grow tomatoes with this number of homes cultivating gardens increasing each year.
The leading commercial flesh market producing states are florida California and south Carolina. The leading processing states are California, Ohio and Indiana. California produces 84% of tomatoes fruit used in processing. Both processing and fresh market tomatoes can either be determinate or indeterminate. A determinate with set fruit at the terminal bud and growth stops. Harvest time far determinate is short; sometime only a week to ten days.
An indeterminate does not set fruit at the terminal bud. The growing point produces leaves and more stems. Harvest time may last for months and the vine will travel extensively over the ground if allowed to grow in this manner.
1.9 SOIL AND MINERAL REQUIREMENT FOR TOMATO CULTIVATION
Tomatoes do well on moist soils but they prefer deep, well-drained sandy loams. Deep tillage can allow for adequate root penetration on heavy clay soil which allows for production in these types, soils extremely high in organic matter are not recommended due to high moisture content of the medium and nutrient differences. But as always, the addition of organic matter to mineral soil will increase yields (Bianchini, 1987)
Tomato is moderately tolerant crop to a wide PH range. A PH of 5.5-68 as preferred though tomato plants will do well in more acidic soil with adequate nutrient supply and availability. Calcium availability is also very important to control soil PH and nutrient availability.
Mono aluminum phosphate (MAP) may be used as starter fertilizes to supply adequate phosphorous during germination and seedlings stage. Sandy soil will require a higher rate of fertilizer and mere frequent applications of these fertilizers due to increase leaching of essential nutrient tissue analysis of a nutritionally sufficient plant will show the following:
Macro Elements N P K CA MG S
% 0.4-5.6 0.30-0.60 3.0-4.5 1.25-3.5 .0-0.65 0.65.1.4
Micro Elements MU FE B CU ZR
Ppm 30-400 30-300 20-60 5-15 30-90
Probably the most common nutritional problem with tomatoes is calcium deficiency. Blossom end not is caused by a lack of calcium, water or both.
This heathery scar on the blossom end of the berry is a product of insufficient calcium in the berry. Irrigation is the great importance as water is required to move the calcium into the berry. Adequate calcium, early in berry development is essential to tomato production. Ninety percent (90%) of the calcium requirement for adequate fruit development must be in the fruit prior to the formation of a wax covering over the surface of young fruit.
Irrigation also plays importance role since tomatoes have been observed to withdraw water from depth up to 18 feet in a well structural soil. Tomatoes are heavy water user and require frequent irrigation to delay maturity and prolong plant productivity. Erratic moisture conditions can cause radial and concentric cracking on fruit. This is a serious physiological and quickly defoliating (Huxley, 1992).
1.10.1 INSECTS AND DISEASES OF TOMATOES
Tomatoes are subjected to a large number of pest and diseases from the time plants first emerge to harvest. Aphids, flea beetles, leaf miners, spiders, mites are a problem from plant bed to tomatoes. Flea beetles, aphids, leafminers, sink bugs and fruit worms cause foliage damage in the yield. But their fruit damage and diseases spreading problems can be very serious.
1.10.2 THESE PEST ARE DIVIDED INTO TWO GROUPS
Pest that feeds on the upper plant and pest that feed on the lower plant. Upper plant feeders either chew holes on leaves or are sapsuckers. The pest that mine leaves or bore with fruits and buds are fruit worm, bud worm, pinworm and leaf miner. The pest that chew holes in leaves are the olister beetles the norm worms. The sap-sucking insects transmit diseases and ease fruit drop. The low plant and root feeders are the cutworm and wire worm. The controls vary from state to state and region to region. The local agricultural chemical mammal will list control and rates for each pest (Philbrick and Gregg 1978).
There are many disease pest of tomato including bacteria, fungi and many viruses. The bacterial group (Pseudomonas Solana ceapum) is a soil borne bacterium which infects the root and stem of the plant causing sudden disease. Crop rotation and sanitation are control measures for this.
Bacterial canker, (Corynebacterium michiganerise) is a seed borne bacterium is which cankers form on the sterile and petioles light halos may form on the berry.
The use of disease free seed is the best control from canker. Bacterial speak (Pseudomonas singe -7) is a problem is most weather. Dark green halos on fruit characterize this disease.
1.11.1 FUNGAL DISEASE INCLUDES
Fusarium (Fasarium oxyporum F. lycopersici)
Verticullum (Verticullum dahliae)
Fungal vascular disease
Verticillium is most severe in the north and Fusarium is most severe in the south. The use of resistant cultures are the most effective controls of these diseases. Early and late lights (Alternarra salani and phytophtova infections respectively) have similar problems as with potato. Both are controlled with fungicides.
Anthracnose (Geotrichum phomoides) is shown by its sunker spot of the fruit.
Rotation is the major control measures for this disease. Virus disease such as tobacco mosaic only top and spotted unit are problems that only sanitation, weed and insect control can keep in check. There is resistance for tobacco mosaic and curly top viruses (Hatfield, 1977).
The harvest of tomatoes is dependent on the closeness to the market, because if the market far from the point of harvest, overippening of the fruit will set in, thereby creating a conducive environment for microbial growth which in turn leads to spoilage of the fruits. Local sale of tomatoes may be vine ripened to a firm ripe or a full red colour before harvesting. Those picked to be shipped are picked at mature green stage and sprayed with ethylene 48 hours before the shipping. Tomatoes for the fresh market are generally hand picked. Processing tomatoes are picked fully ripe. The harvest is sometimes mechanized and sometimes hands picking is used. The plants are cut at the ground level and mechanically shaken to remove the fruit. It is then washed and processed.
1.11.3 POST HARVEST
Full ripe tomatoes can be stored at a temperature of 550f for up to several days. Temperature cooler than this will cause chilling injury, producing poor colour and off flavours. In processing, tomatoes must have a pH below 4.5 to retain contamination with botulism causing agents.
Mature green fruit are bulk packed in ventilated containers for shipment. Upon arrival they are repacked and covered with cellophane and sold (Huxley, 1992).
1.12.1 (A) EDIBLE USES
Fruit, raw or cooked can be used as savoury or flavouring in cooked foods, or can be eaten out of hand as a fruit. It is much used in salads and as a flavoring in soups and cooked foods.
A juice made from their fruit is often sold in food shops. The fruit can be dried and ground into pancakes etc… edible oil is obtained from seed.
Suitable for culinary purposes; the seed is small and it would be very fiddly to utilize. It is only viable to use the seed as source large quantities of the plants are being grown for their fruits and is not wanted.
1.12.2 MEDICAL USES
The pulped fruit is an extremely beneficial skin-wash for people skin, in other words, the pulped fruit has been said to be a very active ingredient used in soup making (Carruthers 1987). Sliced fruits are quick and easy used to treat burns, scalds and sun burn. A concotion of the root is ingested in treatment of toothache. The skin of tomato fruits is a good form of lycopine, a substance that has been shown to protect people from heart attacks (Anon 2001). It seems to be more effective when it is cooked and to be obtained from food products such as tomato ketch up and tin tomatoes. Lycopine has also been shown to have a very beautiful effect upon the prostrate and is being used increasingly to treat prostrate and difficulties in urination that accompany this disorder (Huxley, 1992). Homeopathic remedy is also made from the plant. It is used in the treatment of rheumatism and severe headache (Caruthers, 1987).
1.12.3 OTHER USES
The strong aroma of this plant is said to repel insect from near. A semi-drying oil is obtained from the seed. It can be used in soup making it is a very effective insecticide (Hooker, 1967). It is especially effective against ants but should be used with canton because it will also will beneficial insects, ingestion is toxic to humans. The pulp of the fruit is used no covering face-packs.
1.13 SPOILAGE OF TOMATOES
To spoil is to deprive of good or effective qualities. Tomatoes are said to be spoiled, when it’s characteristic are changed so that it is no longer good for consumption. Such changed may not always be micro biological in origin, a product may become unacceptable as a result of insect damage, drying out, discoloration or stating for instance, but by and large most food spoilage is as a result of microbial activity. Which is our primary concern in this context.
A general features of microbial spoilage is its related sudden onset. It does not appear to develop gradually, day by day a little worse, but more often as an unexpected and unpleasant revelation. This is a reflection of the exponential nature of microbial growth and its consequence that microbial metabolism can also proceed at on exponentially increasing rate (Brackett, 1992).
1.14 FACTORS AFFECTING SPOILAGE
So many factors affect to large extent the spoilage of tomatoes (creating a conductive environment for the growth of micro-organisms in the tomato) some of the factors includes.
1.14.1 (A) NUTRIENT CONTENT
Micro-organisms make use of the food content of tomatoes as a source of nutrient and energy. From tomato they denre the chemical elements that constitute microbial biomass, those molecules essential for growth that the organism cannot synthesize and a substrate that can be used as an energy source. The inability of an organism to utilize a major component of a food national will limits its growth and put it at a complete disadvantage condition with those that can. This ability of the organism to synthesize the available nutrient (minerals). Enzymes favour the growth of the organisms on the tomato.
The concentration of key nutrient can, to some extent, determine the rate of microbial growth.
1.14.2 (B) PH AND BUFFERING CAPACITY
The acidity or alkalinity of an environment has a profound effect on the activity and stability of micro-molecules such as enzymes, so it is not surprising that growth and metabolism of micro-organisms are influenced by pH
In general, bacteria grow fastest in the pH range 6.-8.0, yeast 4.5-6-0 and filamentous fungi 3.5-4.0. the higher pH value of the tissues of the tomato makes them more susceptible to bacterial invasion than fruits although there are also a number of important spoilage fungi or stored tomatoes. The onset and rate of spoilage will depend on the intruders between and the philological changes occurring on the tissue after harvest and changes on microbial activity.
1.14.3 (c) WATER ACTIVITY
Water is a remarkable compound. Considered as a hydride of oxygen (H20) it was good exceptional properties when compared with hydride of new many element on the periodic table such as ammonia (NH3), methane (CH4), hydride sulfide (H2S) and hydrofluoric and (Hf) life as we know it is totally dependent on the presence of water in its liquid state. The reactions which take place in the cytoplasm do so in an aqueous environment and the cytoplasm is surrounded by a membrane which is generally permeable to water molecules which way pass freely from the cytoplasm to the environment and from the environment to the cytoplasm. The dynamic through way flow of water molecules is normally in a steady state and a living organism will be stressed if there is a net flow into cell leading to rupture of the membrane and the lather is normally prevented by the presence of a wall in the bacteria and fungi, in other words, availability of water in tomatoes creates a favourable condition for the growth of the organisms which is turn leads the spoilage of tomatoes (Brackett, 1992).
1.14.4 (D) TEMPERATURE
Microbial growth can occur over a temperature range from about 80C up to 1000C at atmosphere pressure. The most important requirement is water. Which should be present in liquid state and this available to support growth in single organism is capable of growing over the whole of this ranger bacteria are normally limited to a temperature span of around 350C and would rather less, about 300C.
Micro-organisms can be classified based on their conical temperature as shown below.
Group Minimum (0C) Optimum (0C) maximum (0C)
Thermopiles 40-45 60-90 60-90
Mesophiles 5-15 30-40 40-47
Psychrophiles 5-+5 12-15 15-20
Psychrotrophs -5-+5 25-30 30-35
As a role mesophiles grow more rapidly at their optimal, then psychrotrophs and 80 spoilage of perishable products stored in the mesophilic growth range is more rapid than spoilage under chill conditions.
Tomatoes are susceptible to spoilage by these organisms because its temperature range falls between 100C to 320C. This temperature is a factor affecting spoilage of tomatoes.
1.14.5 (E) HARVESTING
This is another factor that vividly affect spoilage of tomatoes by micro organisms as the onset, and rate of spoilage depends on the interactions between the physiological changes occurring in the tissue after harvest and changes in microbial activity. Harvesting itself will produce physiological stress, principally as a result of water loss and wilting and cut surfaces may release nutrient for microbial growth. This stress may also allow growth of the otherwise of quiescent endophicflora. Also the presence of film of water on the surface will allow across far motile bacteria such as Eriwinia and pseudomonas to cracks, wounds and natural openings such as Stomata (Brackett 1992).
The higher PH value of the tissue of tomatoes and many other vegetables makes them more susceptible bacterial invasion than fruit although there are also a number of important spoilage fungi of stored tomatoes.
The bacteria involved are usually pectriolytic specks from negative genera Erwinia Pseudomonas and xanthromonas, although pectinolytic strain of clostridium, can also be important in the spoilage of vegetable made foods, some circumstances and the non-spring gram – positive organisms.
However, the most frequent agents of spoilage are not the plant, pathogen themselves, but opportunities micro-organisms which grown across to plant tissues through wounds, cracks, insect damage a even the various caused by the plant pathogens. All freshly harvested tomatoes have a natural surface flora including low numbers of pectinolytic bacteria and it is becoming increasing evidence that healthy tissue of the infact plant may also contain very low number of viable micro-organisms (endophytic). The onset and the rate of spoilage which depends on the interactions between the physiological damage occurring in the tissue after harvest and changes and changes in microbial activity.
Harvest itself will produce physiological stress principally as a result of water loss and wilting and out surface may release nutrient for microbial growth. This stress may also allow growth of the otherwise quiescent endophytic flora. The most frequently observed form of spoilage is a softening of the tissue due to the pectinolytic activity of micro-organisms.
The presence of a film of water on the surface will allow access to most bacteria such as Erwimia and Pseudomonas to cracks, wounds and naturally openings such as stomata (Brackett, 1992).
1.15 AIMS AND OBJECTIVE
To isolate the micro-organism involved in tomato spoilage.
To enumerate the micro-organism.
To characterize the micro-organism involved in spoilage of tomatoes.
Enlighten the populace on the danger of consuming spoiled tomatoes.
2.1 MATERIALS AND METHOD
The following equipment and materials were used during the course of the project.
2.1.1 EQUIPMENT AND MATERIALS
20 samples of spoiled fresh tomatoes fruits was obtained from Abakaliki markets, namely, Ishieke and Abakpa markets was used for the analysis/ examination. The sample were collected in sterile polythene bag and transported to the laboratory immediately for analysis.
2.3 MEDIA USED
Saburiod dextrose Agar
2.4 REAGENTS AND CHEMICALS USED
Oxidase reagent (TMPPDH)
Methylene blue dye
2. Preparation of sample solution
The samples bought from different market in Abakaliki (Ishieke and Abakpa market) were washed in sterile water to reduce the microbial load, after which a sterile forcep was used to pull apart the tomatoes, at the site of spoilage, the monocarp from the advancing margin of the scar were each aseptically transferred into 10mls of sterile normal saline in various beaker according to the number of sample collected. The tomatoes were allowed to stand in the normal saline for 10 minutes, before being removed with the sterile forcep from the beakers.
2.6 SERIAL DILUTION
Six text tubes containing 9mls of sterile normal saline were placed on a rack on the bench, using test tube rack. 1ml from the sample solution was pipetted aseptically into the first test tube and mixed. It was then transferred to the second test tube and mixed and repeated up to the last tube (10-6). Then 1ml from the last test tube was discarded.
Sterile Bijou bottles were brought and the respective media, (Saburoid Dextrose Agar for fungi and nutrient agar and mackonkey Agar for bacteria growth and Isolation) and prepare. 15mls of the media were measured into the bijou bottle. It was then autoclaved at 1210C for 15 minutes. It was then allowed to cool to 450C so as not to kill the organisms 1ml of each dilution were ascetically transferred to a sterile
Petri dish. The agar was then poured and gently rotated. It was then allowed to cool and gel. The plates were transferred to the incubator and incubated at 370C for 48 hours.
2.8 MICROBIAL COUNTING
Microbial count was carried out to determine the microbial concentration in a given sample from each market and to also compare the amount of growth of micro-organisms under various conditions (Onyeagba, 2004).
This is done by counting the growth colonies based on their physiological characteristics and their number on each plate (culture plate) taking note of the serial diluted tube number. Counts were made from plates that contain between 30-300 colonies.
2.9 PREPARATION OF PURE-CULTURE
For pure culture, distinct colonies were aseptically transferred into newly prepared sterile saburiod dextrose Agar for fungi and Nutrient Agar for bacteria by streaking, using sterile wire loop, which was later transferred into sterile Agar slants in bijou bottles, the culture was preserved. Samples for identification tests were taken from the agar slants.
The bacteria culture was kept on nutrient and mackonkey agar slants while fungi cultures were stored on saburiod dextrose agar. The slants were kept in refrigerator and maintained at 40C.
CHARACTERIZATION AND IDENTIFICATION OF ISOLATES
2.10. IDENTIFICATION OF BACTERIAL ISOLATES
Bacterial Isolates were characterized and identified by staining method, macroscopic examination and biochemical tests. These tests, includes, gram stains, indole test, catalose test, motility test, citrate utilization test, oxidase test, starch hydrolysis test and sugar fermentation tests (Bradshaw, 1979).
2.10.1 GRAM STAINING
Gram staining is of great important in the recognition and identification of bacteria. Grams staining reaction has the widest application, distinguishing nearly all bacteria as gram positive or gram negative, according to whether or not they resist decolorization of crystal violet and subsequent treat with iodine.
Under this form of characterization, a thin smear of the bacterial isolates was made on a glass slide, air, dried and heart fixed by passing it over Bunsen flame for at least four seconds. The smear was covered with crystal violet stain for 30-60 seconds, washed of rapidly with clean water. The slides were tipped off all the water and the smear covered with lugol’s iodine for 30-60 seconds. The iodine washed off with clean water, decolorized rapidly (few seconds) with actone and washed of immediately with clean water. The smear was then covered with safranin for 1 minutes washed off with clean water, the back of the slides were wiped and placed in a draining rack to be air-dried. The dried smear was examined with immersion oil microscopically using the (x100) objective lens microscope.
Gram-positive cells appeared purple while the gram negative cells appeared red. (Chees brough, 2006)
2.10.2 CATALASE TEST
This test is used to differentiate those bacteria that produce the enzyme catalase. This enzyme acts as a calalyst in the breakdown of hydrogen peroxide to oxygen and water.
In this test, a lopful of each bacteria isolate was aseptically transferred to clean sterile non vented Petri dish for about two to three days after which 3% hydrogen peroxide was introduced into the Petri dish was closed and tithe it, so that the hydrogen peroxide made contact with bacterial growth.
Evolution of oxygen or effervescence as observed production of gas bubbles indicates a positive result (Cheesbroug, 2006)
2.10.3 OXIDASE TEST
This test is used in differentiating and identification of pseudomonas, Neisseria, Vibrio, Brucella, and Pasteurella species all of which produce the enzyme Cytochrome Oxidase.
At the course of carrying out this test, 1.1 gram of the oxidase reagent (tetra methyl-p-phenylene diamine dehydrochoride) Tmppeh was dissolved in 10ml of distilled water which was added into a filter paper in a Petri dish. The inoculum of test culture was smeared into the filter paper using a glass rod, a positive test was observed of purple colouration occurs within 10 seconds. (Onyeagba, 2004).
2.10.4 MOTILITY TEST
The hanging drop method was used for this test. A loopful of the bacteria isolates was introduced into a clean cover slip held between two fingers. A circle was made with vaseline on a glass slide. The slide was then placed on top of the cover slip very well without touching the isolate. The slide was then inverted and viewed under high power (x40) and oil immersion (x100) objective microscope (Onyeagba, 2004).
2.10.5 SUGAR UTILIZATION TEST
The sugar solutions used in this experiment were glucose, fructose, lactose and manitol. The media and each of the sterilization sugar solution were dispersed into different set of test tubes containing invested durham tubes. The test organism was asceptically introduced into the sugar and medium mixed. Also 0.1% phenol red indicator was added into test tubes. (Cheesbrough, 2006)
The indicator and invested Durham tubes were used to detect respectively the production of acid and liberation of gas by the isolates. Unoculated tubes were used as control.
2.10.6 INDOLE TEST
Testing for indole production is important in the identification of organism that are capable of breaking down the amino acid and tryptophan with the release of indole. The isolated organisms were inoculated into peptone water and incubated for 48 hours at 370C 0.5ml of kovac’s reagent (p-metting amion benzal dehyde) and gently shaken.
Appearance of a reddish colour indicated the presence of indole while no red surface layer indicated absence (Cheesbrough, 2006).
2.10.7 CITRATE UTILIZATION TEST
The test is used in the identification of entrobacteria. This test is based on the ability of an organism to use citrate as the only source of carbon.
At the course of carrying out this test, a loopful of the culture isolate was streak inoculated into plates of prepared simmon’s citrate agar. It was incubated at 370C for 72 hours to observe for citrate as a carbon source was indicated by a change of the agar from green to deep colour blue where as the negative citrate organism showed neither growth nor colour charge (Onyeagba, 2004).
2.10.8 STARCH HYDROLYSIS
Soluble starch was separately prepared and added to nutrient agar to a 1% soluble starch medium. The medium was sterilized at 1210C and mg/ cm2 pressure for 15 minutes. It was poured into Petri dishes and allowed to cool. The isolates were ascepetically inoculated on the starch agar plates and inoculated at 370C for 48 hours. After incubation the plates were flooded with iodine and zone of clearance or hollow around the test organism were observed (Onyeagba, 2004).
2.10.9 VOGES PROSKAUER (V-P) TEST
One milligram of 40% potassium hydroxide and three milligram of 5% alcoholic naphthol was added to the test tube of glucose and shaken well to observed for colour change within 2-5 minutes. Isolates that produce enough acid during glucose fermentation were v-p negative (Cheesbrough, 2006).
2.11 IDENTIFICATION AT FUNGI ISOLATES
The slide was prepared using methylene blue dye. The dye was dropped into the center of the clean glass slide made to stand on the staining rack. A fungi hyphae was asceptically removed from the sub cultured slants with a wire loop and leased apart on the stain. The slide preparation was carefully covered with cover slip with the exclusion of air bubbles.
Blotting paper was used to remove excess stain coming through the edge of the cover slip slides of each colony were made and observed under the low power objective (x10) and high power objective (x40) lens of compound microscope (Chandler and Craven, 1981). For the purpose of identification, the characteristics and type of spores as well as mycelia forms of the different fungal isolates were recorded.
2.11.1 USE OF POTASSIUM HYDROXIDE (KOH) TREATMENT
Fungi isolates were also identified using KOH reagent. This reagent softens and digests the debris surrounding the fungi so that hyphae and conidia (spores) can be seen.
A drop of potassium hydroxide solutions was dropped on a clean glass slide with the smear of the suspected fungi organism on it, the slide was covered with a clean cover slip. The slide was placed in a Petri dish and allowed to stand for 30 minutes to digest the debris.
The slide was examined microscopically using the 10x and 40x objectives with the condenser and iris diaphragm closed sufficiently to give good contrast (Onyeagba, 2004).
3.1.1 MACROSCOPIC EXAMINATION
Macroscopic examination of individual spoiled tomatoes showed that some of the tomatoes has the following features.
The sample has lost it’s fitness leading to the lost of its usually shape.
It has broken epicarp where its juice and seeds gushes out when compressed.
Some has black spots at its epicarp.
It has slightly unpleasant smell.
The total number of bacteria isolates was (5) five and fungi was (6) six in number. The bacteria isolates from the spoiled samples include; Salmonella species, Proteus species, Erwinia species, Shigella species and Pseudomonas species (Table 1) listed below are the fungi isolates, Cladosporium species, Aspergillus species, Geotrichum species, Mucor species, Rhizopus species and Penicillium species. (Table 2)
All the bacteria isolates were gram negative, the result of their shown also is their individual biochemical reaction were given below (Table 4) The bacteria dissimulation the Isolates were also given in (Table 3).
3.2 TABLE 1: AVERAGE CELL COUNTS OF BACTERIAL ISOLATES FROM THE SPOILED TOMATOES.
S/N Organism Abakpa mkt. Ishieke mkt.
1 Salmonella species 5.3×105 Cfu 6.7×105 Cfu
2 Proteus species 4.7×105 Cfu 5.1×105 Cfu
3 Erwinia species 2.73×105 Cfu 9.3×105 Cfu
4 Shigella species 3.1×105 Cfu 2.7×105 Cfu
5 Pseudomonas species 2.9×105 Cfu 9.5×105 Cfu
3.3 TABLE 2: AVERAGE CELL COUNTS OF FUNGI ISOLATES FROM THE SPOILED TOMATOES OF VARIOUS MARKETS.
S/N Organism Abakpa mkt. Ishieke mkt.
1 Cladosporium species 6.0×105 Cfu 1.6×105 Cfu
2 Aspergillus species 1.4×105 Cfu 2.1×105 Cfu
3 Geotrictium species 1.2×105 Cfu 3.4×105 Cfu
4 Mucor species 2.9×105 Cfu 3.8×105 Cfu
5 Rhizopus species 3.4×105 Cfu 5.4×105 Cfu
6 Penicillium species 1.5×105 Cfu 4.1×105 Cfu
3.4 TABLE 3: COLONY AND MORPHOLOGICAL CHARACTERISTICS OF BACTERIAL ISOLATES
S/N Organism Colony characteristics Morphological Characteristic
1 Salmonella species Pare coloured coloniies with black dot center Rods in scattered form
2 Proteus species raised Surface, round and creamy with waxy margin Rod in chains
3 Erwinia Milky mucoid spreading colonies Short rods
4 Shigella species Pale coloured colonies that turns to pink colour when incubated for a long time Slender rods in chains
5 Pseudomonas species Creamsy large irregular edge colonies Rods in chains
3.5 TABLE 4: DESCRIPTION OF BIOCHEMICAL PROPERTIES OF BACTERIAL ISOLATES FROM SPOILED TOMATOES
S/N Identified Biochemical Properties
A 1 2 3 4 5 6 7 8 9 10 11
1 Salmonella species – – N + – – – ND – + n –
2 Proteus speeches – – + + + + – – – AG – –
3 Erwinia species – + + + – – ND – + AG + +
4 Shigella species – N + – – – – – – A – –
5 Pseudomonas species – – + + – + + + – ND A –
+ = positive reaction G = grams reaction
– = Negative reaction 1 = Indole test
A = Acid production 2 = Catalase test
AG = Acid and gas production 3 = Motility test
ND = Not done 4 = Citrate utilization
N = Neutral in reaction 5 = Oxidize test
6 = Vogesproskaever test
7 = Start hydrolysis
8 = lactose utilization
9 = glucose utilization
10 = Fructose utilization
11 = Mannitol utilization
3.6 TABLE 5: DESCRIPTION OF THE FUNGI ISOLATE FROM THE SPOILED TOMATOES.
S/N Identified fungi Microscopic observation Colony Characteristics
1 Aspergillius Short septate, the conidiophore is on the sterigmata and it bears characteristic brown spores Brownish radial velvety mycellum grooves at tip. Takes long time to sporulate, growth is more pronounced at the end and spreads upwards.
2 Cladoporium species The conidia are one-celled and produced branched chains on conidiophores. Brown colonies fluffy and spreading flat and creamy round the edges.
3 Geotrichum species Spherical axospores which breaked up into athrospores and are cylindrical with round septate. Whitish in colour, smooth and butryous circular and raised surface.
4 Mucor species Non-septate hyphae with erect sporangiophores bearing a single globose sporangium with a large number of spherical spores White and wooly aerial mycellium which later turns darker as the spores were produced all over the mycelia
5 Rhizopus species Non-septate, long and branched mycelium with terminal club shape sporangia on columella Long hyphae, sporulated within (2) two days with initial white colour which later turned black.
6 Penicillium species The conidiosphores on septate hyphae are branched forming a brush-like conidial head Green and yellow colonies raised and rough surface powdery.
in this study, 20 spoiled tomatoes samples that was microbiologically examined, a total of five bacteria and six fungi were isolated. The Bacteria Isolates includes; Proteus species, Erwinia species, Pseudomonas species, Salmonella species and Shigella specie.
The fungi isolates are: Aspergillus species. Cladosporium spices, Geotrichum species, Mucor species, Penicillium species and Rhizopus species.
All the bacteria isolates were all gram negative. Most of the isolates identified in the spoiled tomatoes were environmental contaminants which gained access into the tomatoes fruits due to their exposure to contaminants from unclean surroundings, either by handlers, transporters, container used in carrying it, some could be by the source and nature of water used in washing these tomatoes fruits for instance, salmonella contamination could be as a result of feacal contamination of water or environment to which the sample were exposed. The result revealed that the market with higher contaminants is the Ishieke markets and this could be as a result of poor sanitation and hygiene practices observed in these localities by food handlers and their sources and nature of water used in washing these fruits.
Most of the isolates, especially bacteria’s have in one time or the other been associated with many cases of food poisoning (Beuchart, 1995) and to this effect, it is advisable that spoiled tomatoes should be disposed instead of being consumed since its consumption could be detrimental to health. In addition, food handlers on our market should handle food product with care by practicing proper hygiene in the market environment, by using clean water to wash food products to avoid contamination by micro-organisms present in the water.
The importance of tomatoes with its nutritional and other importance can not be over emphasized, as its spoilage often result to wastage of economical resources as well food poisoning, (Cooper and Johnson, 1984).
The result obtained from this work revealed that spoilage organism (mostly fungi) gained access into these fruits during the process of cultivation and harvesting. The nature of fertilizer and the water used also serve as sources of some of these contaminants. The deduction here is that many of the spoilage organism, were environmental contaminant which have in one time or the other been involved in food poisoning (Casemore, 1991).
In conclusion, spoiled tomatoes should be disposed and food handlers should be enlightened on how to handle food products with care as well as practice hygiene to keep their environment clean. These consumers are as well adviced not to consume spoiled tomatoes and should also wash the good ones properly with clean water before consumption (Beuchat, 1998).
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A clean filter paper was placed on the weighing balance and the weight of the paper is measured, after which a clean spatula was used to collect the dehydrated media onto the clean paper on the weighing balance until the media weighed 7 grams for nutrient agar was measured out. The container of the dehydrated media was closed immediately after collection. The weighed out media was dissolved in 250 mls of distilled water from clean glass. The dissolution was in water by gently rotation in the cornical flask. After complete dissolution of the media the flask was covered with cotton wool and foil before autoclave for 15 minutes at 1210c and 15 psi. After autoclaving the sterilized media where poured into Petri dishes in a sterile environment, allowed to gel. The gelled media were preserved in the refrigerator. The plates is incubated for 35 to 37.0c and kept for senility check.
SABUROID DEXTROSE AGAR
A clean fitter just as above but 15.5 grams was measured out and dissolved in 250ml, The dissolution was in water by gently rotation in the cornical flask. After complete dissolution of the media the flask was covered with cotton wool and foil before autoclave for 15 minutes at 1210c and 15 psi. After autoclaving the sterilized media where poured into Petri dishes in a sterile environment, allowed to gel. The gelled media were preserved in the refrigerator. The plates is incubated for 35 to 37.0c and kept for senility check.
Isolation And Enumeration Of Micro-organisms Associated With Spoiled Tomatoes Sold In Abakaliki, Ebonyi State
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