ISOLATION OF ALGAE FROM SOIL

& WATERGuide:DR.S T GIRISHADepartment of Microbiology & Biotechnology,Bangalore University

Presented By:SHRIHITH A1st MSc Microbiology,Department of Microbiology & Biotechnology,Bangalore University

CONTENTSINTRODUCTIONHABITATSGeneral Methods• Micromanipulation• Streak plating• sprayingMATERIALSISOLATION OF ALGAE FROM SOILISOLATION OF ALGAE FROM WATERCONCLUSIONREFERENCE

INTRODUCTIONThe 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.

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.

HABITATSAlgae 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

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

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.

•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.

MICROMANIPULATION PROCESS

• 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.

STREAK PLATINGMETHOD:-

•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.

•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.

•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 .

STREAK PLATE METHOD

SPRAYINGMETHOD:-

• 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.

•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.

•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.

• 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.

SPRAYING

ISOLATION OF ALGAE FROM

SOIL & WATER

MATERIAL REQUIREMENTSNatural samples.Culture media. -Beneck’s media -Chu’s mediaSterile conical flasks.Sterile test tubes.Sterile pipettes.Distilled water.

Bunsen burner.Growth chamber.Light of suitable intensity.

BENECK’S MEDIA

COMPOSITION OF CHU’S MEDIA

CHU’S MEDIA

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

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.

• 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.

•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.

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.

•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.

•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.

(Water)

ISOLATION OF ALGAE FROM SOIL BY SERIAL DILUTION METHOD

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.

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

• 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.

reference

ANY QUESTIONS