PRACTICAL 2 : Microalgal identification & isolation

INTRODUCTION

Algae can be found in fresh water and marine. They can tolerance for a broad range of pH, temperature, turbidity, oxygen and carbon dioxide. Structures of algae are no roots, no stem, no leaves, and no well-defined vascular tissue. Algae also do not form embryos and their cells are potentially fertile. Algae classifications have four kingdoms. There are bacteria, plantae, chromista and protozoa. Algae can be differentiating by three types which are planktonic algae, periphytic algae and benthic algae.

Isolation of microalgae has three techniques. There are single-cell isolation by micropipette, streak plating and serial dilution. 

MICROALGAL IDENTIFICATION

Procedure 

1. The microalgal samples given known as "culture x" was indentified by the aid of a compound microscope.
2. It was then described and differentiated.

Results

	Tetraselmis sp.	Chlorella sp.	Isochrysis galbana	Chaetoceros sp.
Class of microalgae	chlorophyta	chlorophyta	prymnesiophyta	bacillariophyta
Cell shape	Cordiform and almost spherical	Spherical and has a cup-shaped chloroplast.	Spherical shape	Elliptic cylinder
Live form & its distinctive feature	4 equal flagella in 2 opposite pairs, motile, green coloration	Without flagella, contains the green photosynthetic pigments, non-motile,	Unequal flagella,yellow-brown, motile, unicellular phytoflagellate	Yellow brown coloration, non-flagellate, simple colonies or chain of cells,
Reproduction type	Asexual division in the non-motile stage	Asexual by non-motile reproductive cells (autospores).	Sexual	Asexually and sexually
Distribution	marine	Freshwater/terrestrial species.	marine environment	Freshwater and marine, cosmopolitan

ISOLATION OF MICROALGAE

1)      Technique 1: Micro pipetting

Micro pipetting technique is usually perform with a Pasteur pipette or a glass capillary. With a minimal practice this technique become quick and easy but the beginner must practice some time before reliable production of micropipette is achieved. The goal of micropipette is to pick up a cell from the sample, deposited the cell without damage into a sterile droplet, pick up the cell again and transfer it to a second sterile droplet. This process is repeated until a single algal cell, free of other protist, can be confidently placed into culture medium

Material

1)Inverted microscope or stereo microscope with magnification up to 200 x. 2) capillary tubes or haematocrit tubes.3) Bunsen burner or small flame. 4)silicone tubing to fit over end of capillary tube. 5) hot plate with beaker containing distilled water. 6)Glass slides. 7) agar plates. 8) pasteur pipettes. 9)sterile media 10)sterile tissue culture multi-well plates.

Preparation of a micropipette from a Pasteur pipette.

a)      The Pasteur pipette is held in the hottest region of the flame, supported on the left by a hand and on the right by forceps. The pipette should be rotate as the glass is heated to soft pliable condition.

b)      When the glass is soft, the pipette is quickly removed from the flame with a gentle pull to produce a thin tube.

c)      The forceps is then relocated to the appropriate region of the thin tube.

d)     The forceps is used to gently bend the thin area so that it breaks, forming a micropipette.

e)      An enlarged tip of a micropipette, showing a jagged break; this tip is not suitable for use.

f)       An enlarged tip with a very smooth break, this tip is suitable for use.

Procedure

1. With a fine flames from a bunsen burner heat and draw out (holding at both ends) the capillary tube to form two micropipettes. The narrow end should be about twice the diameter of the cell to be micromanipulated.

2. Heat distilled water to simmering point on hot plate.

3. Place drops of sterile medium onto 1.5% agar plates with a sterile pasteur pipette. Alternatively place three drops on a glass slide.

4. With silicone tubing attached to micropipette suck up and blow out with mouth a small amount of hot distilled water.

5. Locate algal cell to be isolated in drop of enrichment sample. While observing the cell, suck up into the micropipette.

6. Transfer the cell to a drop of sterile medium on agar plate or glass slide.

7. Sterilize the micropipette.

8. Repeat this process to “wash” the cell. The more times a cell is washed the less likely is bacterial contamination. However, the risk of cell damage increases with the number of times a cell is handled. The optimum number of washes will depend on the type of algae.

9. Transfer the cell to dilute medium in a tissue culture plate, petri dish or culture tube.

10.  Place culture vessel under low light at appropriate constant temperature. Check microscopically for growth.

2)      Technique 2: Streak plating

Isolation of cell on algal plate is most common and it is most preferred isolation method for many coccoid algae. It is easy method and also axenic culture which can be directly established without further treatment. For successful isolation onto agar, the algae must grow on agar. The algae such as Tetraselmis sp., Pavlova and Clamydomonas grow very well on agar. Isolation is accomplished by streaking the natural sample across the agar surface which is identical to the technique used for isolating bacteria. A bacterial loop is loaded with a small amount of sample then the sample is spread with the loop across the agar. The origin of the streak typically has too many cells that are not separated but as the distance from the origin increase single cell begin to separate. After streaking, the agar plate is incubated until colonies of cell appear.  

Materials

Petri dish, Media, Agar, Wire loops, Bunsen burner or small flame, Parafilm

Procedure

1) Prepare petri dishes containing growth medium solidified with 1-1.5% agar medium.

2) Place 1-2 drops of mixed phytoplankton sample near the periphery of the agar. Flame sterilizes a wire loop. Using aseptic technique use the sterile loop to make parallel streaks of the suspension on the agar.

3) Cover and seal plate with parafilm. Invert and incubate under low light at constant temperature.

4) Select colonies that are free of other organisms for further isolation.  Remove a sample using a sterilized wire loop and place in a drop of sterile culture medium on a glass slide. Check microscopically that the desired species has been isolated and is unialgal.

3)      Technique 3: Serial dilution

Dilution technique is very effective for organisms that are abundant in the sample. A serial dilution is the step-wise dilution of a substance in solution. Usually the dilution factor at each step is constant, resulting in a geometric progression of the concentration in a logarithmic fashion. A ten-fold serial dilution could be 1 M, 0.1 M, 0.01 M, 0.001 M... Serial dilutions are used to accurately create highly-diluted solutions as well. For a ten-fold dilution on a 1 mL scale, vials are filled with 900 microliters of water or media, and 100 microliters of the stock algae solution are serially transferred, with thorough mixing after every dilution step. The dilution of algae is very important to get to algae diluted enough to count on a spread plate.

Materials

Culture tubes, Test-tube racks, Media, Automatic dispenser, Glass pipettes 1 ml or Pasteur.

Procedure

1) Using aseptic technique, dispense 9 ml of media into each of ten test tubes with sterile automatic dispenser or sterile 10 ml pipettes. Label tubes 10-1 to 10-10 indicating dilution factor.

2)  Aseptically add 1 ml of enrichment sample to the first tube (10-1) and mix gently.

3) Take 1 ml of this dilution and add to the next tube (10-2), mix gently.

4)  Repeat this procedure for the remaining tubes (1O-3 to 10-10).

5) Examine cultures microscopically after 2-4 weeks by withdrawing a small sample aseptically from each dilution tube. A unialgal culture may grow in one of the higher dilution tubes e.g. 10-6 to 10-10. 

RESULTS
ISOLATION OF MICROALGAE
1)      Technique 1: Micro pipetting
        Our group didnt manage to get the single cell as this procedure needs to practice a lot.

2)      Technique 2: Streak plating

        

3)      Technique 3: Serial dilution
       
       

The higher the dilution factor,the lower the microalgae number.