BDV 4801 FIELD WORK GROUP 17
ISOLATION & IDENTIFICATION OF PROTOZOA PROT OZOA AMOEBAE AMOEBAE IN WATER & SOILS AT SEKAYU RECREATIONAL RAIN FOREST
SUPERV SUP ERVISE ISED D BY BY : Prof Prof.. Madya Madya Dr Dr.. Nakis Nakisah ah bin binti ti Mat Amin
INTRODUCTION
Protozoa are single-cell organisms that behave like animal because they mobile and heterotrophs. Presence of protozoa indicates the quality of certain certain ecosyste ecosystem(W m(Windt,1 indt,1995) 995).. Protozoa are classified into four groups; which are amoeb moeba ae, flag flagel ella late tes s, cilia iliate tes s and and spor sporoz ozo oa.
OBJECTIVES •
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To identify the species of amoebae in water and soil at Sekayu Recreational Forest. To determine the distribution of amoebae in relation to water quality. quality.
JUSTIFICATION
For the first time, the research on protozoa amoebae is done in Sekayu Recreational Forest. We can determine the importance of free living amoebae at that area.
MATERIALS •
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Pages Amoebae Saline solution (PAS) Non-nutrient agar Heat Kill E.coli Membrane Filtration Unit YSI Multiparameter Vortex Autoclave Light Microscope Carmine stain Blue Cap Bottle
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Inverted Microscope Analytical balance Petri dish Boot Rubber glove Sampling bottle Plastic bag Dropper pipette Spatula Glass slide Cover slip
SAMPLING LOCATION
Soil samples
Stagnant Water (5 sampling points)
METHODOLOGY MEDIA PREPARATION -Page’s Amoebae saline -Non-nutrient agar
SAMPLING
STERILIZATION
-Soil
-Autoclave
-Water
-Aseptic Technique
-Physical parameter measurements
ISOLATION -Soil-Vortex and drop in non-nutrient agar -Water-Filtration membrane unit and place the filter membrane on non-nutrient agar
CULTIVATION -Incubate at 30°C -Fed with Heat Kill E.coli
IDENTIFICATIONS -Inverted microscope -Taxonomic key
IDENTIFICATIONS
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•The protozoa were cultured on the non-nutrient agar and fed with Heat Kill E.coli every 3 days. •It was observed daily for the presence of amoebae.
•The amoebae were sub-cultured to obtain single species in a plate. •The amoebae were identified based on Page’s descriptions (1988). •The breadth of amoebae, movement, morphology of trophozoites were used to identified the species.
•Pictures were captured by using Image analyzer microscope.
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RESULTS
Vanella sp. 20.53µm
1000x magnification
20.05µm
1000x magnification
22.53µm Vanella sp. : Breadth in range 24-46µm,hyaloplasm commonly extending around sides. Vanella sp . have floating form for movement and have pointed pseudopodia 1000x magnification
Thecamoeba sp. 54.71µm
1000x magnification
40.00µm
1000x magnification
Thecamoeba sp : General surface fairly smooth, often with several parallel folds extending far interiorly, surface also highly wrinkled , becoming smoother in locomotion. Range in size 35100µm. Normal locomotions.
Sappinia sp. 36.93µm
1000x magnification
36.43µm
1000x magnification
Sappinia sp. : Look like smooth Thecamoeba sp.. Breadth in 1337µm,. Normal movement of Sappinia sp.
Platyamoeba sp. 30.46µm
1000x magnification
16.66µm
1000x magnification
Platyamoeba sp. : Outline usually oval, often broadly elongate, usually in range 15-35µm. Moving by elongating the pseudopodium.
Acanthamoeba sp. 23.80µm
1000x magnification
35.98µm
1000x magnification
Acanthamoeba sp. : Acanthamoeba sp . is differentiated by it movement that produce many acanthapodia. Acanthamoeba has various size. Moving by the acanthopodia.
Vahlkampfia sp. 9.11µm
1000x magnification
6.81µm
1000x magnification
Vahlkampfia sp . : General surface fairly smooth, have big contractile vacuole, eruptive movement. Range in size 6-19µm,
Amoeba A 18.68µm
Have many contractile vacuole. The trophozoites is similar to Rosculus sp. but the size is not in range of Rosculus sp.. (5-17µm).
1000x magnification
Amoeba B 26.73µm Have many contractile vacuole. Like Rosculus sp. but the size is not in range of Rosculus sp. 1000x magnification
Amoeba species In Relation to water Quality Parameters POINT
pH
Temper ature (°C)
Dissolved oxygen (mg/L)
Water Turbi conducti dity vity (ms/cm) 0.024 0.017
Species
W1a
7.2
24.65
6.5
W1b
6.81
24.66
6.30
0.031
0.018
Vanella sp., Amoeba Type B,
W2a
6.48
24.54
6.92
0.025
0.015
W2b
6.66
24.52
6.52
0.023
0.015
W3a
6.39
24.52
6.72
0.024
0.016
W3b
7.11
24.57
6.81
0.025
0.016
Vanella sp., Vahlkamphia sp., Sappinia sp., Vanella sp., Sappinia sp., Thecamoeba sp., Vanella sp., Platyamoeba sp., Amoeba Type B, Vanella sp., Acanthomaeba sp.,
W4a
6.43
24.56
6.84
0.025
0.016
Vanella sp., Rosculus sp.,
W4b
6.36
24.63
7.03
0.026
0.017
Vanella sp., Platyamoeba sp.
W5a
6.45
24.63
6.80
0.021
0.015
W5b
67
24 62
6 67
0 024
0 015
Vanella sp., Thecamoeba sp., Platyamoeba sp., Vanella Sappinia
Vanella sp., Acanthamoeba sp.,
Amoeba species in soil POINT
Species
S1a
Vanella sp.
S1b
Vanella sp.
S2a
Vanella sp., Thecamoeba sp., Sappinia sp., Amoeba Type A
S2b
Vanella sp., Sappinia sp, Thecamoeba sp.
S3a
Vanella sp., Platyamoeba sp.
S3b
Vanella sp.
S4a
Vanella sp.
S4b
Vanella sp.
S5a
Vanella sp., Platyamoeba sp., Thecamoeba sp.
S5b
Vanella sp., Sappinia sp., Thecamoeba sp.
DISCUSSION
Vanella sp. is the dominant amoeba in Sekayu Recreational Forest, in both water and soil. ◦ Easily reproduce and active. This make it easily to get the nutrients. ◦ No study about vanella sp. Species
Water
Soils
Vanella sp. Thecamoeba sp. Sappinia sp. Platyamoeba sp. Acanthamoeba sp. Vahlkamphia sp.
The dominant species of amoeba in both soil and water are the same because the soil samples are taken near to the river, and the soil is moist.
Water physical parameter affect the amoeba
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◦ The water parameter is consistent, thus distribution amoeba species affected (Windt,1995) ◦ If there are changes in water quality and not suitable to amoebae, they will encyst. ◦ And the cyst will transform into trophozoite when environment is suitable to the amoebae.
Importance of amoeba in ecosystem ◦ Control the microorganisms in the ecosystem like bacteria. Amoebae consumed the bacteria, control bacteria populations. Some of them are pathogenic. ◦ In our sample, only Acanthamoeba sp. have potential to become pathogenic. ◦ For other amoeba species obtained in this study might also important, but no study has been done.
Two genera of amoebae cannot be identified and just give a type name. ◦ Limited instruments and time. ◦ Does not fit to Page’s descriptions.
POINT
pH
Temperat ure (°C)
Dissolved oxygen (mg/L)
Water conductivi ty (ms/cm)
Turbidity
W1a
7.2
24.65
6.5
0.024
0.017
W1b
6.81
24.66
6.30
0.031
0.018
W2a
6.48
24.54
6.92
0.025
0.015
W2b
6.66
24.52
6.52
0.023
0.015
W3a
6.39
24.52
6.72
0.024
0.016
W3b
7.11
24.57
6.81
0.025
0.016
W4a
6.43
24.56
6.84
0.025
0.016
W4b
6.36
24.63
7.03
0.026
0.017
W5a
6.45
24.63
6.80
0.021
0.015
W5b
6.7
24.62
6.67
0.024
0.015
All parameter shown the small differences range for each point.
SUGGESTIONS
This is first time in amoebae research at Sekayu Recreational Forest. For further study, research can be done about amoebae especially Vanella sp., which are dominant at Sekayu Recreational Forest.
CONCLUSIONS
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There are at least 8 species of amoebae exist in both water and soil at Sekayu Recreational Forest. The amoebae can tolerate with different water qualities. However, they can encyst when the conditions are unfavourable.
REFERENCES
Darbyshire, J. F. (1994). Soil protozoa. United Kingdom: Bindles ltd. Isolation of Amoebae. (2002, August 23). Retrieved January 22, 2011, from THE UNIVERSITY OF EDINBURGH: http://www.bms.ed.ac.uk/research/others/smaciver/Protoc ols/AmoebaProts/isolation_of_amoeb ae.htm Patterson, D. (1998). Free Living Freshwater Protozoa . University of Wales press ltd. P.Hicker, Larry. S.Robert, S.L Keen, A.Larson, D.J Eisenhour. 2009. Animal Diversity. New York, Mc Graw-Hill Publishing Company. Eldra P Solomon, Linda R.Berg, Diana W.Martin. 2008. Biology 8th Edition. Belmont, USA, Thomson Corporation. F.C.Page.1988. A new Key to Freshwater and Soil Gymnamoebae. Ambleside, Cumbria,Freshwater Biological Association.
