Practical course on counting, isolation and culturing of microalgae Part II. Algal Isolation and

45 Pages·2015·0.99 MB·English

Practical course on counting, isolation and culturing of microalgae
Part II. Algal Isolation and Culturing Techniques

Edina Lengyel, Dr. Nóra Kováts, Prof. Dr. Judit Padisák

University of Pannonia
Department of Limnology

TÁMOP-4.1.1.C-12/1/KONV-2012-0015
Higher educational cooperation for the water sector
Contents
1. Sampling for isolation ............................................................................................................ 4
1.1. Sampling of phytobenthos ............................................................................................... 4
1.1.1. Stones ....................................................................................................................... 4
1.1.2. Emerged macrophyta ................................................................................................ 5
1.1.3. Epipelon, episammon ............................................................................................... 5
1.2. Sampling of phytoplankton ............................................................................................. 6
2. Isolation and cultivation ......................................................................................................... 7
2.1. Equipment for the isolation and cultivation .................................................................... 7
2.2. Traditional microalgae isolation technique ................................................................... 11
2.2.1. Enrichment Cultures ............................................................................................... 11
2.2.2. Single-Cell Isolation Technique ............................................................................. 11
2.2.3. Agar ........................................................................................................................ 13
2.2.3. Dilution Technique ................................................................................................. 14
2.2.3. Gravity separation method ..................................................................................... 15
2.3. Cultivation of isolated algae .......................................................................................... 15
2.3.1. Batch cultures ......................................................................................................... 15
2.3.2. Continuous Cultures (chemostat) ........................................................................... 16
2.4. Culture media ................................................................................................................ 17
2.4.1. Chemical composition ............................................................................................ 18
2.4.2. Preparation of media .............................................................................................. 21
2.4.3. Recipes of media .................................................................................................... 22
2.5. Other limiting physical chemical factors ...................................................................... 22
2.5.1. Light intensity ........................................................................................................ 22
2.5.2. Temperature ........................................................................................................... 23
2.5.3. pH ........................................................................................................................... 24
3. Application of cultures ......................................................................................................... 25
3.1. Application in agriculture .............................................................................................. 25
3.2. Application for consumption ......................................................................................... 25
3.3. Industrial utilization ...................................................................................................... 25
3.4. Ecophysiological investigation ..................................................................................... 26
4. Sterilization .......................................................................................................................... 27
4.1. Sterilization methods ..................................................................................................... 27
2
4.1.1. Heat treatment ........................................................................................................ 27
a) "Flame" method ........................................................................................................ 27
b) "Autoclaving" method .............................................................................................. 28
c) "Dry heat" method .................................................................................................... 29
d) "Pasteurization" method ........................................................................................... 29
e) "Tyndallization" method .......................................................................................... 30
4.1.2. Chemical treatment ................................................................................................ 30
a) Ethanol ..................................................................................................................... 30
b) Bleach ...................................................................................................................... 31
4.1.3. Physical treatment .................................................................................................. 31
a) "Filtration" method ................................................................................................... 31
4.1.4. Electromagnetic wave treatment ............................................................................ 31
a) "UV (ultraviolent radiation)" method ...................................................................... 31
4.2. Sterile techniques .......................................................................................................... 32
4.2.1. Laminar flow hood ................................................................................................. 32
4.2.2. Transferring liquid cell cultures ............................................................................. 33
5. Acknowledges ...................................................................................................................... 36
7. Appendix .............................................................................................................................. 41

3
1. Sampling for isolation
The sampling of algae can be carried out from different water bodies: ponds, lakes, channels,
streams, rivers, seas, oceans, wetlands, etc. The sampling techniques depend on those algae
that are required to isolate (phytoplankton or phytobenthos). Materials used for sampling
equipment can include in both cases glass, plastics, tapes, special clothes.
1.1. Sampling of phytobenthos
The sampling must be carried out from the upper region of the water body (10-30 cm). Be
sure that the sampling point is covered by water for 4 weeks (sufficient time is needed for the
benthos to develop). There are several substrate types where the samples can be collected.
1.1.1. Stones
The stones (Fig. 1) should be placed on a tray. The benthos can be removed from the surface
with knife, scalpel or any kind of toothbrush using little amount of water (distilled water, de-
ionized water or from the water body) (Fig. 2). After it, the removed benthos should be
collected into a little vessel. The best vessel is made from glass ensuring the sufficient light
for the microorganisms.

Fig1. The phytobenthos developed on the surface of the stones
4
Fig. 2. The process of the sampling phytobenthos

1.1.2. Emerged macrophytes
The substrate can be any kind of macrophytes, most commonly it is reed (Phragmites
australis) (Fig. 3). The sample should be collected from the 10-20 cm of the stem under the
water surface. Cut this part of the plant, place it on a tray and remove the benthos with knife,
scalpel or any kind of toothbrush.

Fig. 3. The phytobenthos developed on macrophytes

1.1.3. Epipelon, episammon
Make sure that only the benthos will be removed instead of the substrate. The sampling can be
carried out with pipettes (Fig. 4-5), sediment sampling equipment (Fig. 6-7) or spatula and
Petri dishes. In the cases of the latter, the Petri dishes should be placed on the sediment that
should be removed with a spatula after it (Ács and Kiss, 2004).
5
Fig. 4-5. The sampling of phytobenthos with pipettes

Fig. 6-7. The sampling of phytobenthos with sediment sampling equipment

1.2. Sampling of phytoplankton
Samples should be collected from surface water (1-50 cm), from deep water by a bottle
(different form and size are available) or a water column using a special devices. The water
sample must be concentrated using a plankton net (~ 10 µm pore size) (Fig. 8) or by allow it
to precipitate to get more cell for the isolation process.
6
Fig. 8. Plankton net

2. Isolation and cultivation
2.1. Equipment for the isolation and cultivation
Certain equipment is necessary for preparing stock solutions and culture mediums:
- glass wares (etc. Erlenmeyer flasks, reagent bottles, cylinders, Petri dishes, stirring rods,
tubes, beakers, pipettes, etc.) (Fig. 9).

7
Fig. 9. Glass wares for isolation and cultivation

- Plastic wares (e.g. pipettes, micro multiwell plates, Eppendorf tubes)
- Analytical balance
- Magnetic stirrer
- Gauze and cotton wool for the plugs (Fig.10)

Fig. 10. Cotton wool plugs in different sizes

- An autoclave is usually essential for sterilization, as being the most popular sterilization
methods. All of the mentioned materials should always be sterile.
8
- Filtration equipment could also be needed for sterilization, if non-heat-resistant medium
(e.g. f/2 medium) is required. A vacuum source or filter syringe, filter holder, membrane
filters are the minimum materials for doing it.
- The chemical ingredients are essential for preparing stock solution, culture mediums (agar
and liquids). There are several companies with many catalos for having these materials.
Before the order, make sure that the ingredients of the chemical constituents are studied,
because many of them could contain levels of trace metals or others, which may disturb
during the experiments or inhibit some species.
- An equipment to get de-ionized water (Fig. 11) for the mediums.

Fig. 11. Smart Purification Pack for Direct-Q 3

- A refrigerator (maybe with a freezer part) is necessary for maintaining stock solutions and
culture media.
- UV lamps for sterilizing the air and workspace.
- The most popular option for manipulating cultures and working with sterile equipments the
laminar flow hood which is also necessary in the laboratory.
- Microscopy for the isolation (Fig. 12). Dissecting and inverted microscopes are the most
widely used. Make sure that the microscopy has good optical lenses and sufficient
9
magnification. The best magnification is from 40 up to 80. It can be supplemented with a
micromanipulator (Fig. 12) for make the isolation easier.

Fig. 12. Inverted light microscopy with micromanipulator

- Roller shakers may be required for keeping the cultures on the move (Fig. 13), because some
species could need it. Removable rolls are suitable for shaking of larger tubes.

Fig. 13. Shaker

10