BDV 4801 FIELD WORK GROUP 17

ISOLATION & IDENTIFICATION OF PROTOZOA AMOEBAE IN WATER & SOILS AT SEKAYU RECREATIONAL RAIN FOREST

SUPERVISED BY : Prof. Madya Dr. Nakisah binti 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 ecosystem(Windt,1995). Protozoa are classified into four groups; which are amoebae, flagellates, ciliates and sporozoa.

OBJECTIVES
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.

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
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 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
-Pages Amoebae
saline -Non-nutrient agar

STERILIZATION
-Autoclave -Aseptic Technique

SAMPLING
-Soil -Water -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 30C -Fed with Heat Kill E.coli

IDENTIFICATIONS
-Inverted microscope -Taxonomic key

IDENTIFICATIONS

1 2

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 Pages descriptions (1988). The breadth of amoebae, movement, morphology of trophozoites were used to identified the species.

Pictures were captured by using Image analyzer microscope.

RESULTS

Vanella sp.
20.53m 20.05m

1000x magnification

1000x magnification

22.53m
Vanella sp. : Breadth in range 24-46m,hyaloplasm commonly extending around sides. Vanella sp. have floating form for movement and have pointed pseudopodia 1000x magnification

Thecamoeba sp.
54.71m 40.00m

1000x magnification

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 35100m. Normal locomotions.

Sappinia sp.
36.93m 36.43m

1000x magnification

1000x magnification

Sappinia sp. : Look like smooth Thecamoeba sp.. Breadth in 1337m,. Normal movement of Sappinia sp.

Platyamoeba sp.
30.46m 16.66m

1000x magnification

1000x magnification

Platyamoeba sp. : Outline usually oval, often broadly elongate, usually in range 15-35m. Moving by elongating the pseudopodium.

Acanthamoeba sp.
23.80m 35.98m

1000x magnification

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.
9.11m 6.81m

1000x magnification

1000x magnification

Vahlkampfia sp. : General surface fairly smooth, have big contractile vacuole, eruptive movement. Range in size 6-19m,

Amoeba A
18.68m Have many contractile vacuole. The trophozoites is similar to Rosculus sp. but the size is not in range of Rosculus sp.. (5-17m).

1000x magnification

Amoeba B
26.73m
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 Dissolved ature oxygen (C) (mg/L) 24.65 24.66 24.54 24.52 6.5 6.30 6.92 6.52 Water Turbi conducti dity vity (ms/cm) 0.024 0.017 0.031 0.025 0.023 0.018 0.015 0.015 Species

W1a W1b W2a W2b

7.2 6.81 6.48 6.66

Vanella sp., Acanthamoeba sp., Vanella sp., Amoeba Type B, Vanella sp., Vahlkamphia sp., Sappinia sp., Vanella sp., Sappinia sp., Thecamoeba sp., Vanella sp., Platyamoeba sp., Amoeba Type B, Vanella sp., Acanthomaeba sp., Vanella sp., Rosculus sp., Vanella sp., Platyamoeba sp. Vanella sp., Thecamoeba sp., Platyamoeba sp., Vanella sp., Sappinia sp.,

W3a
W3b W4a W4b W5a W5b

6.39
7.11 6.43 6.36 6.45 6.7

24.52
24.57 24.56 24.63 24.63 24.62

6.72
6.81 6.84 7.03 6.80 6.67

0.024
0.025 0.025 0.026 0.021 0.024

0.016
0.016 0.016 0.017 0.015 0.015

Amoeba species in soil

POINT S1a S1b S2a S2b S3a S3b S4a S4b Vanella sp. Vanella sp. Vanella sp., Thecamoeba sp., Sappinia sp., Amoeba Type A Vanella sp., Sappinia sp, Thecamoeba sp. Vanella sp., Platyamoeba sp. Vanella sp. Vanella sp. Vanella sp. Species

S5a S5b

Vanella sp., Platyamoeba sp., Thecamoeba sp.

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 Vanella sp. Thecamoeba sp. Sappinia sp. Platyamoeba sp. Water Soils

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

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 Pages descriptions.

POINT

pH

Temperat ure (C)

Dissolved oxygen (mg/L) 6.5 6.30 6.92 6.52 6.72 6.81

Water conductivi ty (ms/cm) 0.024 0.031 0.025 0.023 0.024 0.025

Turbidity

W1a W1b W2a W2b W3a W3b

7.2 6.81 6.48 6.66 6.39 7.11

24.65 24.66 24.54 24.52 24.52 24.57

0.017 0.018 0.015 0.015 0.016 0.016

W4a
W4b W5a W5b

6.43
6.36 6.45 6.7

24.56
24.63 24.63 24.62

6.84
7.03 6.80 6.67

0.025
0.026 0.021 0.024

0.016
0.017 0.015 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

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.