This document provides instructions for isolating algae from soil and water samples. It describes several general methods for isolation, including micromanipulation of single cells, streak plating of mixed samples, and spraying of cell suspensions. Specific protocols are given for isolating algae from soil and water using serial dilution methods. A variety of culture media and materials are listed for growing isolated algae cultures. The aim is to obtain pure, uni-algal cultures free of bacterial contamination through careful manipulation and transfer of algal cells from natural samples onto solid and in liquid growth media.

1. ISOLATION OF ALGAE FROM
SOIL & WATER
Guide:
DR.S T GIRISHA
Department of Microbiology &
Biotechnology,
Bangalore University
Presented By:
SHRIHITH A
1st MSc Microbiology,
Department of Microbiology &
Biotechnology,
Bangalore University

2. CONTENTS
INTRODUCTION
HABITATS
General Methods
• Micromanipulation
• Streak plating
• spraying
MATERIALS
ISOLATION OF ALGAE FROM SOIL
ISOLATION OF ALGAE FROM WATER
CONCLUSION
REFERENCE

3. INTRODUCTION
The algae belongs to the kingdom “Protista” and domain
“Eukarya”.
They may be microscopic or macroscopic ranging from
unicellular to multicellular forms.
There are at least 20,000 identified species of algae.
Soil & Water is the most important sources for the
isolation of algae.

4. Due to there high capacity for morphological &
physiological adaptations to different environments , both
algae often act as pioneer microorganisms in terrestrial
ecosystems & aquatic ecosystems.
Several methods & basic culture media are developed in
the late 1800 & early 1900.
Over a century ago algae were first studied in culture by
“Bejerinck” in 1890 & he was the first person to work
with ‘axenic cultures’.
“Pringsheim” in 1946 suggested that the selection of
suitable culture media for isolation of algae.

5. HABITATS
Algae are found in a wide range of habitats.
A good general description of the habitats is given by
“Round” in 1981. In most cases the species composition in
each habitat is quite unique , so it is advisable to isolate
microalgae from as many as possible to enhance the
chances of finding species with useful characteristics.
POND WATER WASTE WATER

6. Algae are probably best known as the dominant plants in
most aquatic habitats, both fresh water & marine water.
While one generally thinks of algae as aquatic organisms,
many are in fact adapted to living in dry terrestrial
habitats, including soil, the surfaces of rocks, even in
desert sand.
Most soil types contain rich communities of algal species
Surface of rocks Fresh water

7. GENERAL METHODS
MICROMANIPULATION (single-cell)
METHOD:-
• With a fine flame from a Bunsen burner heat & draw out the
capillary tube to form two micropipettes. The narrow end should
be about twice the diameter of the cell to be micromanipulated.
• Heat distilled water to simmering point on hot plate . This
is used for sterilizing the micropipette between each
transfer.

8. • Place drops of sterile medium onto 1.5% agar plates with
sterile Pasteur pipette. Alternatively place three drops on
a glass slide.
• With silicone tubing attached to micropipette suck up &
blow out with mouth a small amount of hot distilled water.
This sterilizes the micropipette.
• Locate algal cell to be isolated in drop of enrichment
sample . while observing the cell, suck up into the
micropipette.
• Transfer the cell to a drop of sterile medium on agar
plate or glass slide.

9. MICROMANIPULATION
PROCESS

10. • Sterilize the microtips.
• 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
handled. The optimum number of washes will depend on type
of algae.
• Transfer the cell to dilute medium in a tissue culture plate,
Petri dish or culture tube.
• Place culture vessel under low light at appropriate constant
temperature. Check microscopically for growth or wait until
microscopic growth can be detected(3-4 weeks after
transfer).
• A clonal uni-algal culture should result from this method.

11. STREAK PLATING
METHOD:-
•Prepare petri dishes containing growth medium
solidified with 1-1.5% agar medium .The agar
should be ½-2/3 the depth of the dish.
•Place 1-2drops of mixed phytoplankton sample near
the periphery of the agar . Flame sterilize a wire
loop . Using aseptic technique use the sterile loop
to make parallel streaks of the suspension on the
agar.

12. • Cover & seal plate with parafilm . Invert & incubate
under low light at constant temperature.
• Select colonies that are free of other organisms for
further isolation. Remove a sample using a sterilized
wire loop & place in a drop of sterile culture medium on
a glass slide . Check microscopically that the desired
species has been isolated & is uni-algal.

13. • Transfer selected colonies to liquid or agar medium.
• Repeat the streaking procedure with the algal cells
from a single colony & again allow colonies to develop.
This second streaking reduces the possibility of
bacterial contamination & of colonies containing more
than one algal species .

14. STREAK PLATE METHOD

15. SPRAYING
METHOD:-
•In this technique, a stream of compressed air is
used to disperse algal cells from a mixture onto the
surface of a Petri plate containing growth medium
solidified with agar.
•Hold a petri plate about 18 inches from touching
tips of two Pasteur pipettes.

16. • One of this is attached to an airline via a hose, &
mounted onto a ring stand. The other pipette is
suspended tip-up into a continer holding the algal
mixture.
• The airflow also sprays the suspended algae through the air
, where they can be intercepted by the agar plate.
• The airflow from the first pipette creates a vacuum that
draws a stream of algae – containing lipid up from the
container through the second pipette.

17. • Prepare in advance glass ~15 ml centrifuge/glass
tubes, sealed with a micropipette wrapped in non-
absorbent cotton-wool and then autoclaved.
• Aseptically transfer the algal cell suspension into the
tube and then reclose the tube by placing the cotton
bung/micropipette into the tube.

18. • The plate should be sealed and incubated for a
few days.
• Any discrete algal Colonies observed can be picked
off to initiate potentially bacteria free clonal algal
cultures.
• Current should induce the capillary flow of a fine
mist of liquid and algal cells onto the agar.
• Hold a tube with a flow of compressed air in front
of the top of the micropipette and the air plate.

19. SPRAYING

20. ISOLATION OF ALGAE FROM
SOIL & WATER

21. MATERIAL REQUIREMENTS
Natural samples.
Culture media.
-Beneck’s media
-Chu’s media
Sterile conical flasks.
Sterile test tubes.
Sterile pipettes.
Distilled water.
Bunsen burner.
Growth chamber.
Light of suitable intensity.

22. BENECK’S MEDIA

23. COMPOSITION OF CHU’S MEDIA
CHU’S MEDIA

24. ISOLATION OF ALGAE FROM SOIL BY SERIAL
DILUTION METHOD
METHOD:-
• Collect a natural sample “soil” & weigh it for 1gram .
• All 10 sterilized test tubes are named (labelled) as
10-1, 10 –2 ……… 10 -8 .
COLLECTION OF SOIL SAMPLES

25. 10-1 10-2 10-3 10-4 10-5 10-6 10-7 10-8
SOIL
(1g)
ISOLATION OF ALGAE FROM SOIL BY SERIAL DILUTION
METHOD
• Now aseptically add 1g of soil sample to first tube 10-1 & mix
gently.
• Then using aseptic technique 9ml of distilled water into each of
ten test tube , with steriled 10ml pipettes for different
dilutions.

26. • Repeat this procedure, for the remaining tubes.
• Take 1ml of 10 -1 dilution & add to next test tube 10-2
mix gently.
• Beneck’s broth medium or chu’s media is
prepared(different dilutions) in separate conical flasks
& sterilized at 15 lbs, 121ºC For 15-20 minutes.

27. • After sterilization , cool the medium & add 1ml of serially
diluted soil sample to different conical flasks.
• The conical flasks were incubated in a growth illuminated
chamber under suitable temperature for 15-20 days after
incubation period , observe the flasks for growth of algae.

28. ISOLATION OF ALGAE FROM WATER BY SERIAL
DILUTION METHOD
METHOD:-
• Collect a natural sample “water (1ml)” from fresh
water or pond water.
• All 10 steriled test tubes are named as 10 -1 ,10 -2,………10-10
• Then using aseptic technique 9ml of distilled water
into each of ten test tube, with steriled 10 ml
pipettes for different dilutions.

29. • Now aseptically add 1ml of water sample to first test
tube 10 -1 & mix gently.
• Take 1ml of 10 -1 dilution & add to the next test tube 10
-2 & mix gently.
• Repeat this procedure , for the remaining tubes.
• Chu’s media or Beneck’s broth medium is prepared
(different dilutions) in separate conical flasks &
sterilized at 15lbs , 121˚C for 15-20 minutes.

30. • After sterilization , cool the medium & add 1ml of
serially diluted water sample to different conical
flasks.
• The conical flasks were incubated in a growth
illuminated chamber under suitable temperature for
15-20 days after incubation period , observe the
flasks for growth of algae.

31. (Water)
ISOLATION OF ALGAE FROM SOIL BY SERIAL
DILUTION METHOD

32. Incubate under controlled temperature &
light conditions.
(a) temperature & photoperiod as close to the natural
environment as possible.
(b) Light intensity- slightly lower than natural
environment examine culture microscopically after
2-4 weeks by withdrawing a small sample
aseptically from each dilution from conical flask.

33. CONCLUSION
• Isolation of algae from soil & water is very important
for environmental and industrial purposes, because of
the elimination of the eutrophication in the ponds, heavy
metal removal and also for the production of Biofuels,
pigments and single cell proteins…..
• The algae found in water sources like pond water ,
waste water , fresh water , marine water…etc
• The algae found in soil sources like , terrestrial soil ,
rock soil , surface soil , desert soil…etc

34. • For the isolation of algae from soil and fresh
water, temperature and oxygen is a selective factor
of major importance.
• Algae encompass several groups of relatively
simple living aquatic organisms that capture light
energy through photosynthesis, using it to convert
inorganic substances into organic matter.
• Soil algae excrete growth promoting substances
such as hormones, vitamins, amino acids, and
organic acids that affect other organisms in their
survival and multiplication in many ways.

35. reference

36. ANY QUESTIONS