Morphological and biochemical Identification of microorganisms causing fruit & vegetable spilage

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Morphological and biochemical Identification of common microorganisms that cause spoilage in selected fruits and vegetables.

Food is basic necessity of human life for survival. There by food materials should be clean and healthy. Food is said to be spoiled if they are injured. Various fruits and vegetables, and their products may be spoiled by one or more factors like, chemical changes oraction of microorganisms, tissue enzymes, insects, rodents or improper methods of processing, under processing. Microbiology is considered a safety of food for consumption by human beings. Despite of the improvement in the methods of production, handling and processing the microbiological quality still remains most important factor. This aspect assumes the importance of the causative agent of spoiling foods and their characteristics with microbiological identification(Verma et al, 2000).

In order to understand the spoilage agents of fruits containing Vitamin C rather than reduced contents of vitamin C foods, this work will be carried out. Tomato, potato lemon, brinjal, sweet orange, onion and carrot will be purchased in local market. Their surface will be used and extract will be prepared after storing few days to get spoiled. As vitamin C prevents cancer progression and cardiovascular disease people should intake those foods a healthy state. As some microorganisms are resistant for acidic environment they grow in vitamine C foods..Xanthomonas(Xanthomonas, 2011), Salmoneela(Ashton, 2013), Escherichia coli(Ashton, 2013),Erwinia(Adel, 2011), Candida albicans(Thomas, 1983)and Pencillium(Smuel,2005)are commonly suspected microorganisms that are aimed to isolate from vitamine C foods. Vitamin C contains ascorbic acid(Han, 2004). Thereby it makes acidic environment for microorganism which require. This kind of species can live in acidic environment. Some species specifically affect this kind of foods causing spoilage while some are not specific and not harmful to fruits but human (Prombelom, 2002). The causative organisms of rotten fruits can be classified into Bacteria, Mold, Yeast (Rao, 2006).Postharvest infection of food spoilage is mainly noted and clarified in those foods even though soil is considered as a medium for some (Sharma and Singh, 2000). So cure this problem, fruits and vegetables should be transported, separating and packing individually with safer manner.

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Vitamin C is found in high concentrations in immune cells, and is consumed quickly during infections. So people who are infected may suffer from vitamin C deficiency. Vitamin C deficiency results many symptoms (Lippincott and wilkins, 2009). Symptoms include fatigue, depression, and connective tissue defects (eg, gingivitis, petechiae, rash, internal bleeding, impaired wound healing).

The amount of the vitamins of some in 100mg are:Papaya-60mg, srawbery-60mg, Orange-53mg,lemon-53mg, Tomato-13.7mg. Fruits and vegetables are selected according to the most consumption for this task.

In this examination, six microorganisms are suspected as causal agent of spoilage(Adel, 2011). Spoiled foods would be purchased and the outlook would be marked. According to the type of the rotting, causative agents will be nominated.

Salmonella entericais rod shaped bacteria. They are non spore forming micro organism. Motile enterobacteria are predominant with 0.7-1.5µm in diameter with from 2-5µm. Colonies are red in colour of macConkey agar.. Those are Gram negative bacteria and positive for catalase test. Oxidation, reduction produce hydrogen sulphide that can be detected by ferussulfate. These live in warm and cold condition and the neutral PH (Labbe and Garica, 2001). These are faucaltative anaerobics. Erwiniacarotovora is rod shaped enterobacteria, non spore forming and pertichously flagellated. These live alone or pairs and chains. These are gram negative bacteria, negative result for catalase test and faucaltative anaerobic bacteria. These Live in 27-30Ëšc (Helias et al, 1998). E- Coli is a rod shaped bacteria.,2µm long and motile flagella have petrichousarrangements. These are gram negative bacteria and Positive for catalase test Chris, 2004). These live in 37ËšC. Xanthomonascampestris is strait rod and 1.2- 3µ long. These motile by a single polar flagellm, these are Mucoid, convex, yellow colonies. These areGram negative bacteria and Catalase positive(Kenneth, 2008).Pencilliumdigitatum is brush like head. greenish colony. Typically consist of a highly branched network of multi nucleated septae, usually colourlesshypae. Conium and phialidae are present. These are gram positive and catalase positive (Brown, 2003). Candida albicans is a fungi. Grow both as yeast and filamentous. During culturing grow as yeast. Temperature or PH changes cause morphological shift to pseudohyphal growth. These are defined anaerobic.Yeast live in 34Ëš, PH- 5.4. Hyphae grow well in 40Ëšat PH- 7.4. These are catalase positive.

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The preservation of fruits and vegetables allows for the year-round supply and consumption of these seasonal stuffs. Healthy materials should not contribute any flavor or odor changes to the product. Lower the microorganism produce higher the quality of foods. While freezing inhibits the growth of microorganisms, biochemical and enzymatic spoilage may still occur. Important chemical reactions in frozen fruits and vegetables include oxidation of vitamin C(Diane at al, 2004). Some contents cause loss of flavor, color, and nutritive value of the frozen product. While many of these reactions are catalyzed enzymatically, most fruits and vegetables are blanched prior to freezing to inactivate these deleterious enzymes. However, chemical spoilage can take. Postharvest methods should be developed to minimize the food contaminations.

General Objective: Isolation of microorganism form the surface of the selected, rotten fruits and vegetables

Specific objective: Microscopic identification of colonies from nutrient agar medium

Differentiation of gram negative and gram positive microorganisms.

Bio chemical identification of microorganisms

Materials

Glassware

Chemical reagents

Fruits and vegetables

Autoclave machine

Amonium oxalate

Brinjal 2

Bunsen burner

Crystol violet

Carrot 2

Gloves 1 box

Ethanol 95%

Lemon 2

Grease pencil

H2O2

Onion 1

Inoculum sticks

HCl

Potato 1

Microscope

Iodine crystal

Orange 1

Pipette 11

Pottasiumiodite

Tomato 1

Slides 14

Macconkey agar

Testtubes7

NaOH

Potato dextrose agar

(Nutrient agar)

Sufranin

Distilled water

Methodology.

Collection of selected fruits and vegetables

Spoiled sweet Orange, Lemon, Tomato, Potato, Onion, Brinjal would be purchased in Aluthmawatha local market Colombo 15. Rotten fruits and vegetables would be placed in the laboratory.

Nutrient media would be prepared. Approximately 0.4-0.5mm depth of agar plate would be prepared. Inoculum would be prepared using a direct colony suspension. Suspension would be used within a 60minutes. Streaking method should be used. Using a sterile cotton microorganism would be spread in the macConkey agar plate.

Clony formations will be observed. Smear will be prepared according to the guideline (subhash, 2009). By heating slide in Bunsen burner heat fixation will be performed. Microscopic observation will be launched at 10X, 40X. Oil immersion will be performedat 100X. (Barbara and norma, 2000). Gram staining technique will be launched according to guideline.(Subhash, 2009). Drop of Crystal violet mixture will be applied. After 30 seconds it will be rinsed with tap water. This will be flooded with Laugols iodine for 30 seconds. Then the slide will be rinsed with water. Rapid decolorizing will be launched by adding ethanol for 3 seconds. It will be washed with water. Saffranin will be added. Red/pink color will be noticed for negative result.H2o2 will be added for further analysis of the new slides prepared. Bubble formation will be noted for the positive results. Using inoculating loop colonies will be transferred in to tube containing water. Afterwards those will be grown on macConkeyagar at 37Ëšc for 2 days in neutral PH(Mendez, 2001).Using inoculating loop microrganisms will be streaked into macconkey agar to produce single colonies(Jane, 2001).Single cell colonies, a one type of microorganism in one colony, will be identified. Microorganism will be removed and applied on slide. Further visualization will be launched.

This will be performed as an individual. Along the couple of month this project workwould be carried out. The purpose of the examination is to create knowledge of the microorganisms that can grow well in Vitamin C fruits and vegetables that are already being spoiled by storing for 14 days.

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Expected result: Tomato, Lemon, Sweet orange and carrot are suspected to infection with above mentioned microorganisms rather than potato, onion, and brinjal. Gram negative bacteria would expose pink/red color after adding saffranin whilst gram positive bacteria remains in violet color. Catalase positive microorganisms would form bubbles on slide where as catalase negative would not.

Appendix

Crystal violet will be prepared. 2g of crystal violet powder will be added to 20ml ethanol. 0.8g of Amonnium oxalate will be added to 80ml water. Both of the solutions will be mixed. Lugaol’s Iodine will be prepared. 2g of KI will be added to 100ml of water.1g of iodine crystal will be added. It will be stored in brown or light blocking container. For saffranin preparation, 0.25g of saffranin powder will be added to 10ml ethanol.It will be added to 90ml of water. Solution will be mixed.

References

Barbara, H. E., Anna, P. R., Norma, J. W. (2000) Basic medical laboratory techniques. 4th edn. Health care: USA.

Brown, G. E. (2003). Green mould: Causal organism and disease cycle. Plantpathology department.Florida cooperative department service.Institute of food and agricultural sciences university of florida. Gainville. FL. http://edis. Ifas.ufl.edu.

Chris, H. (2004) E-Coli. Newyork.Rosen puplishing group.

Helias, V., Roux, . C. Le., Berheau, Y., Andrivon, D., Gautheir, J. P., Jouan, B. (1998)

Characterisation of Erwiniacarotovorasupsp. European Journal of plant pathology, 104.

Labbe , R.G., Garica, S. (2001) Guide to foodborne pathogens. US: Jhon Wiley and Sons.

Lippincott, W., Wilkins. (2009) Vitamine C Deficiency. 9th edn. New Delhi: Wolters Kluwer.

Kenneth, R. H. (2008) Westcotts Plant disease handbook. 7th edn. Newyork: Springer.

Perombelon, M. C. M. (2002). Potato disease caused by soft rot erwinias.An overview of pathogenesis.Plant pathol. 51, 1-12.

Rao, A. S. (2006) Introduction to micro biology. Prentice hall of India private limited: Delhi

Sharma, R. M and Singh, R. R. (2000) Harvesing, Postharvest Handling and Physiology of fruits and vegetables. New Delhi: Indus Publishing.

Subhash, K. M. (2000) Gram stain:Looking beyond bacteria to find a fungi in gram stained smear Alaboratory guide for medical microbiology. Authorhouse: USA.