TAXONOMY AND DIVERS ITY OF MIOCROBES SEMESTER

INTRODUCTION:

Decaying organic matter or putrefaction is the decomposition of organic matter whereby heterotrophic organisms, including some bacteria, fungi, saprophytic plants, and lower animals, utilize the remains of once -living tissue as a source of nutrition. Microorganisms such as bacteria and fungi involved by breaking down the polysaccharides, lipids, nucleic acids, and proteins of dead tissue and converted into simpler molecules with the help of enzymes secreted into external environment.

Source of energy which bacteria and fungi absorbed will be used in respiration process, fermentation and also protein synthesis.

This decomposition process takes place in the presence of oxygen in wet environment. Fungi, acting as decomposers, a re important in today's terrestrial environment. Colonization of most fungi preferably in dark and moist conditions. Fungi play a crucial role in the balance of ecosystems.

In a way to identify the bacteria, a few measures should be carried out. Fir st, we must know the type of leaves that is known to be the site for recognition of bacterial degradation to become decayed.

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Our center of attraction would be bacteria which lies on the epidermis of leaves and intercellular spaces (apoplast) of the mesoph yll. Frequently, leaves occupied at the surface of epidermis with pathogenic bacteria at the apoplast are higher than commensal organisms.

We collect the sample at the Fraser's Hill Forest Reserve which relatively a primary canopy of pristine highland retreat in Peninsular Malaysia consisting of tree species origin of Dipterocarpaceae. Primary rainforest dominated with large tree trunks and there isn't any disturbance from logging activities while secondary forest have abundant of pioneer trees.

We want to observe if the pathogenic bacteria might be different in such habitats for leaves to decompose.

Furthermore, the occurrence of rotting leaves are due to some aspects such as humidity, air pollution and presence of pathogen. Contaminated air wil l disturb the production of chlorophyll, results of dead plants. Leaf spots generally need hi gh humidity to initiate an infection. Plus, wind and rain could be the cause of leaf spots appears on the leaves.

OBJECTIVE:

1. To obtain organic decay matte r from Hutan Simpan Bukit Fraser.
2. To isolate bacteria and fungi that contain in the sample.
3. To identify and classify bacteria and fungi in the sample.
4. To prepare pure culture for each of the isolated bacteria and fungi .

METHODOLOGY/ MATERIALS:

SAMPLING

BACTERIA ON LEAVES AND BARK AND TREE BRANCHES /ROOTS :

LEAVES: Infected leaves will be obtained or collected from the forest which was from different location as well as different type of tree and geographical properties of the area . Sampling wi ll be collected by choosing leaves that show sign of infection by using scarpel. Infected leaves would be placed in the sterile container or zip lock bag . The sample will be grinded into small particles then will be soak ed in 0.35% of calcium hypochloride for 10 minutes. The sample then recovered and rinsed by distilled water and inoculated into agar.

BARK: Sample that was identified as cankers can be seen clearly by shaving off or cut through the outer bark of plant stem or the necrotic area up to 1cm de ep. Then, the sample was placed in the dry paper towels or sterile container or zip lock bag.

TREE BRANCHES /ROOTS :The infected tree branches or roots will be identified .The tree branches or roots will be rinsed off from the soil that still adhere or att ached to the tree branches by using tap water rinsed.

FUNGI ON LEAVES /ROOTS AND BARK:

BARK: Sample that was identified as cankers can be seen clearly by shaving off or cut through the outer bark of plant stem or the necrotic area up to 1cm. The sample will be wrap ped in the dry paper towels and will be placed in polyethylene bag or zip lock beg before brought to the laboratory.

LEAVES /ROOTS : The leaves or roots that has fungi growing on it will be identified and cut into small segment with a sterilized blade, surface sterilized in 1% hypochlorite for 2min

GROWTH AND ISOLATION OF BACTERIA AND FUNGI

BACTERIA ON LEAVES , TREE BRANCES /ROOTS AND BARK:

MATERIAL:

LEAVES:

• Scalpel or shears
• Sterile Water
• Nutrient agar plates or other bacterial media pe r sample
• Rose Bengal agar plate
• Tissue homogenizer (Dounce or Potter -Elvehjem type)
• Inoculating loop

PRIMARY ISOLATION:

A piece of symptomatic tissue of plant from the forest was selected . Then, the section was cut into small pieces. Some amount of water will be added and the tissue will be grinded using pestle and mortar. The sample will be transferred into the section of plate.

SECONDARY ISOLATION:

By using inoculating loop and aseptic technique, streak plate on the first quar ter of plate, crossing over the initial streak area one time. Before streaking another part, sterilize the loop between sections .The step will be repeated on second quarter, crossing over the first quarter only once and repeat it on the third also fourth q uarter only once. Incubate d the plates at 30C for 48 hours. Growth of fungi and bacteria on plate will be observed . Individual c olonies will be choosen to streak one more time ROOT: put the infection side on the nutrient agar

FUNGI LE AVES , ROOTS AND BARK:

BARK: The sample will be dissipating on the surface of Rose bangle agar that mix with chlorophenicol by using sterile forceps. And incubate d for 5days on 28C.

LEAVES / ROOT :plated on the surface of Rose bangle agar that mix with chlor ophenicol and then incubated at 28°C for 5 days.

IDENTIFYING

BIOCHEMICAL TEST FOR BACTERIA IDENTIFICATION

All unknown bacteria will be subjected to biochemical test and microscopic as below.

The following test are to be done according t o the results of Gram staining:

1. Culture in differential, selective and enriched media
2. Carbohydrate fermentataion
3. IMViC test (indole,Metyl red,Voges -Proskauser and citrate)
4. Nitrate reduction test

BIOCHEMICAL TEST FOR F UNGI IDENTIFICATIO N

The fungi identification will be carried out by microscopic and macroscopic approach as listed below:

a. Microscopic technique

To differentiate the fungi according to their microscopic traits.

1. A drop of lactophenol Blue will be placed on a clean microscope slide.
2. Using an inoculating needle,gently a small portion of growth midway will be removed between the colony centre and edge.
3. Then, the growth from that needle in the drop of Lactophenol Blue will be placed on the slide.
4. The mycelium that has been transferred onto the slide is then will be spread ed finely on the slide to enable clearer visualization of fungal structures.
5. A cover slip edge of the lactophenol will be placed and slowly lower it but avoid trapping air bubbles und er coverslip.
6. Structure of the fungi under microscope will be observed .

Material : glass slide and Lactophenol Cotton Blue

Petri dishes with fungal cultures, Lactophenol cotton blue, Glass slides with cover slips, Wire loop/hook and Compound light microscope

1. Zygomycota: coenocytic, sporangium, sporangiospores, zygospores,sporangiophore.
2. Ascomycota: septate, conidium, conidiophores, ascospores, ascus.
3. Basidiomycota: septate, basidiospores, basidium
4. Deuteromycot a: Imperfect fungi.

b. Observation through macroscopic traits

Identification of fungi by macroscopic differentiation such as listed below:

1. Morphology of the colony
2. Mycelium colour
3. Colour on the back o f the petri dish
4. Spore colour
5. Pigmentation
6. Zonin

REFERENCE

1. The Columbia Electronic Encyclopedia, 6th ed. Copyright © 2012, Columbia University Press. Decay of organic matter -and - technology/biochemistry/biochemistry/decay -of -organic -matter
2. Yit Chin Chuan, BSC. Mar 19, 2015. Why do organic compounds decay? -do -organic -compounds -decay
3. Decomposition of Soil Organic Matter. AgriInfo.in © 2015. All rights reserved. Web site Development, Web site Designing, SEO by OnClick
4. McKinley, V.L.; Vestal, J.R.; Eralp, A.E. (1985). ""Microbial activity in composting"". Biocyle. 26 (10): 47 -50.
5. Gill -King, Harrell (199 7). ""Chapter Chemical and Ultrastructural Aspects of Decomposition"". In Hagldund, William D. Forensic Taphonomy: The post -mortem fate of human remains. CRC Press. pp. 93 -108. ISBN 978 -0-8493 -9434 -8.
6. Vass, A.A.; Barschik, S.A.; Sega, G.; Caton, J.; Skeen, J.T.; Love, J.C. (2002).
7. "Decomposition chemistry of human remains: a new methodology for determining post -mortem interval"". Journal of Forensic Sciences. Wiley -Blackwell. 47 (3): 542