MAINTENANCE OF PURE CULTURE OF MICROALGAL STRAINS AND

MICROSCOPIC STUDY OF THEIR MORPHOLOGY

SHARDA SCHOOL OF AGRICULTURAL SCIENCE

SHARDA UNIVERSITY, GREATER NOIDA (201310)
 COMPILED AND SUBMITTED BY:

ANUJ RAJ - 2019006826

ASHSIH PATHAK - 2019007275
GURAVNEET SINGH MALWA - 2019650557
JAPHET JOHN - 2019006588
SMUTIRANJAN PANDA - 2019003880
UJJU KUMARI - 2019004012

B.Sc.(Hons.) Agriculture
8th Semester
Batch (2022-23)

UNDER SUPERVISION OF,

Dr. SWATI HAZRA
(ASSISTANT PROFESSOR)
(SSAS)
 CONTENTS

1. INTRODUCTION
2. OBJECTIVES
3. METHODOLOGY
4. RESULTS
5. DISCUSSION
6. FUTURE ASPECTS
7. REFERENCES
 1. INTRODUCTION

• Blue-green algae are eukaryotic phototropic organism requiring

light for photosynthesis.

• They may be unicellular or multicellular, filamentous or colony

forming.

• Soil and water is the most important sources for the isolation of
algae.

• BGA have been used for various purposes, including food,

feed, medicine, and bioremediation.
 2. OBJECTIVES

 To Maintain Pure Cultures of different BGA Strains in

suitable growth media

 To study the morphological features of different BGA

strains.

Fig.-Cyanobacteria
 3. METHODOLOGY
Material Reqired
EQUIPMENTS
 Mother Culture of BGA • Autoclave
 Double-distilled water • Laminar Air Flow
 Micro pipette • Bunsen Burner
 Micro tip (1ml) • Volumetric Shaker
 Cotton • Microscope
• Weighing Machine
 Stirring rod
 1000 ml conical flask CHEMICALS REQUIRED
 Spectula  BG 11 Broth Powder
 Measuring Cyclinder  NaCl Tablets
 Hydrochloric Acid(HCl)
 Rubber
 Aluminium Foil
 How to Inoculate Mother Culture with BG 11 solution in +N media

Composition of BG 11 BROTH
S.No. Ingredients gm/L.

1 K HPO 0.0314g
2 4

2 MgSO .7H O 0.0360g

4 2

3 CaCl .7H O 0.0367g

2 2

4 Citric Acid 0.0056g

5 (FeNH ) 0.0060g
4

6 EDTA 0.0010g
7 Na CO 0.0200g
2 3

8 NaNO 1.5000g
3
STEPS:-

1. Add 1.62gm of BG-11 Broth (Blue-Green Medium)

Powder to a 1000-ml conical flask and add double-
distilled or deionized water to it.
Fig.- Weighing Machine

2. Bring the volume to 1.0 liters and mix thoroughly.

3. Check the pH level and adjust it to 7.1–7.2.

4. If the pH is high, lower it by adding 1 mM NaCl, and if it

is low, make it higher by adding a few drops of HCl.

5. Cover mouth of conical flask with cotton plug and

aluminum foil by tight rubber. Fig.- Sample Covering in Laminar flow
6. Autoclave solution at 15 psi pressure at 121 °C for 15 minutes.

7. Switch on laminar flow and turn on UV light for 15 minutes. Then turn off the UV light
and turn on the light and fan. After this, put the conical flask in laminar flow to cool it.

8. After the solution gets cool, ring the mother culture in and start the burner to get the
sample sterilized and contaminant-free.

9. Use a small measuring cylinder to pour 50 ml of solution into each mother culture with
full precautions.

10. Put the sample in a cool, dry place with white light.
 How to make (–N) Media Sample from BG 11 Solution

Materials for (-N) stock solution

S.No. Ingredients In 100 ml In 2.5 ml Trace Metal
Ingredients gm/L
1 K HPO 0.4g 2.5 ml
2 4

2 MgSO .7H O 0.75g 2.5 ml

4 2
H BO 2.86g/L
3 CaCl .7H O 0.36g 2.5 ml 3 4 In 1L
2 2
MnCl 1.81g/L Double
4 Citric Acid 0.6g 2.5 ml 2 Distilled
ZnSO .7H O 0.22g/L water
5 FeNH 0.6g 2.5 ml 4 2
4
Na MnO .2H O 0.39g/L
6 EDTA 0.01g 2.5 ml 2 4 2

CuSO .H O 0.07g/L
7 Na CO 0.2g 2.5 ml 4 2
2 3
Co(NO ) .6H O 0.049g/L
8 Trace Metal 1 ml in 1 L 2.5 ml 3 2 2
Steps:-

1.Take 7 piece 150 ml Conical flask.

2.Weight above 7 sample and add in conical flask with double distilled

water and adjust it to 100ml respectively.

3.Now add trace material in 1000 ml conical flask with double distilled

water and adjust it to 1000 ml. Fig.- Volumetric Shaker

4.Put it on Volumetric shaker to mix well.

5.Now add 2.5ml solution from each conical flask to 2.5 L Double

distilled water. Pour it in laminar flow with full precautions.

6.Autoclave Solution at 15 psi pressure at 121 °C for 15

minutes.

7.After that cool it in laminar flow.

8.Pour 200 ml Sample in 150-200 ml conical flask each.

9.Now put 1ml BGA species in conical flask of –N media in

laminar flow.
Fig.- Media Prepaed
10. Cover each conical flask with cotton plug and put it in

white light.
4. Results
a. Maintained culture media
 b. MICROSCOPIC STUDY OF BGA STRAINS
I) Anabaena
II) Tolypothrix Tenuis
III) Scenedesmus Dimorphus
IV) Phorimidium
V) Aulosira
VI) Oscillatoria
VII) Chlorella ulgaris
VIII) Lyngbya
IX) Nostoc Muscorum
X) Westiellopsis Prolifica
I) ANABAENA
Phylum : Cynobacteria

Class : Cyanophyceae

Order : Nostocales

Family : Nostocaceae

Morphology:-

 It is filamentous blue green algae. They are both planktonic or make coating on aquatic organism.

 The thallus is unbranched filament cells are joined end to end.

 Vegetative reproduction and fragmentation. Sexual reproduction is absent.

 Found in a variety of aquatic environments including freshwater, marine, and even terrestrial habitats.
 The filaments can range in length from a few millimeters to several
centimeters and are typically visible to the naked eye. The individual cells of
Anabaena are typically cylindrical in shape and have a diameter of around few
micrometers.
II) TOLYPOTHRIX TENIUS
Phylum : Cynobacteria

Class : Cynophyceae

Order : Nostocale

Family : Tolypothrichaceae

Morphology:-

 Tolypothrix is a genus of cyanobacteria that occurs in small tufts either floating in

torpid water or attached to plants and rocks.

 These are photosynthetic prokaryotes and ubiquitous in nature and are found
commonly in lakes, ponds, springs, wetlands, streams, and rivers.

 It is a filamentous cyanobacterium that typically forms thin, unbranched filaments.

 The filaments can grow up to several millimeters in length and are usually
visible to the naked eye.

 The individual cells of Tolypothrix tenius are cylindrical in shape and measure
around few micrometers in diameter.

 They are typically arranged end-to-end within the filament, with no visible
separation between them.
III) SCENEDESMUS DIMORPHUS
Phylum : Cynobacteria

Class : Chlorophyceae

Order : Sphaeropleales

Family : Scenedesmaceae

Morphology:-

 Scenedesmus dimorphus is a freshwater, unicellular green alga that typically forms colonies or aggregates.

 The individual cells of Scenedesmus dimorphus are oval-shaped and measure about few micrometers in length
and few micrometers in width.

 Each cell has two flagella, whip-like appendages that it uses for movement. The flagella are typically of unequal
length, giving the cell a characteristic curved or crescent shape.
 The cell is surrounded by a cell wall made of cellulose and pectin, which helps to
protect it from environmental stresses.

 Inside the cell, there is a nucleus and other organelles such as chloroplasts, which
are responsible for photosynthesis.
IV) PHORIMIDIUM
Phylum : Cynobacteria

Class : Cyanophyceae

Order : Oscillatoriales

Family : Oscillatoriaceae

Morphology:-

 Phormidium is a genus of filamentous cyanobacteria that are commonly

found in marine and freshwater environments.

 The filaments of Phormidium are typically unbranched and can be quite

long, reaching lengths of several millimeters or more.
 Filaments without any branching; slightly waved to loosely, irregularly
coiled.

 The individual cells of Phormidium are cylindrical in shape and measure

around few micrometers in diameter.
V) AULOSIRA

Class : Cyanophyceae

Phylum : Cynobacteria

Order : Nostocales

Family : Fortieaceae

Morphology:-

 Aulosira is a genus of filamentous, heterocystous cyanobacteria

that are found in freshwater and soil environments.

 The filaments of Aulosira are unbranched and can range from a

few millimeters to several centimeters in length.
 The individual cells are cylindrical in shape, measuring around few
micrometers in diameter, and are separated by thin septa.
VI) OSCILLATORIA
Phylum : Cynobacteria
Class : Cyanophyceae
Order : Oscillatoriales
Family : Oscillatoriaceae
Morphology:-

 Oscillatoria is a genus of filamentous cyanobacteria that is commonly found in

freshwater and marine environments.

 The filaments of Oscillatoria are unbranched and can range in length from a few
millimeters to several centimeters.

 The individual cells of Oscillatoria are cylindrical in shape, measuring around few
micrometers in diameter, and are arranged end-to-end within the filament.
 Oscillatoria is characterized by its ability to move via oscillatory
motion, which is caused by the coordinated movement of its
individual cells.

 This movement is facilitated by short, motile filaments that break

off from the main filament and move independently
VII) CHLORELLA VULGARIS
Phylum : Chlorophyta

Class : Trebouxiophyceae

Order : Chlorellales

Family: Chlorellaceae

Morphology:-

 Chlorella vulgaris is a single-celled green algae that is commonly found in freshwater

environments.

 The cell of Chlorella vulgaris is roughly spherical, measuring around few micrometers in diameter.

 The cell is surrounded by a thin, flexible cell wall made of cellulose and pectin, which allows the
cell to change shape and squeeze through narrow spaces.
 Inside the cell, there is a nucleus and other organelles such as chloroplasts, which
are responsible for photosynthesis.

 Under certain conditions, Chlorella vulgaris can form dormant structures called
cysts, which allow it to survive in adverse environments until conditions improve.
VIII) LYNGBYA
Class : Cyanophyceae

Order : Oscillatorials

Phylum: Cynobacteria

Family : Oscillatoriaceae

Morphology:-

 Lyngbya is a genus of filamentous cyanobacteria that is commonly found in marine and freshwater
environments.

 The filaments of Lyngbya are unbranched and can range in length from a few millimeters to several
centimeters.

 The individual cells of Lyngbya are cylindrical in shape, measuring around few micrometers in
diameter, and are arranged end-to-end within the filament.
 The cells are surrounded by a mucilaginous sheath that helps to protect
them from environmental stresses.

 Under certain conditions, Lyngbya can also produce specialized cells

called heterocysts, which are involved in nitrogen fixation.
IX) NOSTOC MUSCORUM
Phylum : Cynobacteria

Class : Cyanophyceae

Order : Nostocales

Family : Nostocaceae

Morphology:-

 Nostoc muscorum is a filamentous cyanobacterium that is commonly found in soil and aquatic environments.

 The filaments of Nostoc muscorum are unbranched and can range in length from a few millimeters to several
centimeters.

 The individual cells of Nostoc muscorum are cylindrical in shape, measuring around few micrometers in
diameter, and are surrounded by a mucilaginous sheath that helps to protect them from environmental stresses.
 Nostoc muscorum is characterized by the presence of specialized cells called
heterocysts, which are involved in nitrogen fixation.

 Heterocysts are typically spaced at regular intervals along the filament, with
several vegetative cells between them.
X) WESTIELLOPSIS PROLIFICA
Phylum : Cynobacteria

Class : Cyanophyceae

Order : Nostocales

Family : Hapalosiphonaceae

Morphology:-

 Main filaments consisting of short barrel-shaped cells, with broad and as long as broad or slightly
longer; branch-filaments thinner and elongate, not constricted at the crosswalls, with elongate cylindrical
cells, and broad.

 Heterocysts oblong cylindrical, broad and long. Gonidia formed singly from each cell of the
pseudohormocy.
 Filaments intensely true-branched, composed of primary thicker, more or less
creeping, generally thinner and growing in erect T-type of branching without
visible sheaths.

 Trichomes intensely constricted at crosswalls, only young branches

sometimes consist in some segments of long, cylindrical cells, without
constrictions at crosswalls; branches not attenuated towards the ends.
 5. DISCUSSION
 Microscopic study of BGA can help us to have a detailed study of morphological features
of different strains.

 Maintenance of pure culture can play a crucial role in the characterization, conservation
and sustainable use of microorganisms in agriculture and allied sectors.

 Researchers require reliable cultures for their research, therefor, there is a strong desire for
the long term preservation of microbial cultures for present and future use.
 6. FUTURE ASPECTS

Microorganisms(BGA) represent a Black Box that has potential solutions to various

problems pertaining to food, health, environment and poverty in both developed and
developing countries.
7. REFERENCES

1. Belton, T. J., Nielsen, K. J., Mandalam, R. K., Sundaram, S., Bolch, C. J., & Blackburn, S. I. (2019). Mass cultivation of the cyanobacterium Arthrospira
platensis (Spirulina) and microalgae for biotechnology. Journal of Applied Phycology, 31(2), 1233-1263.

2. Bhattacharya, A., & Ghosh, A. (2019). Microscopic study of morphology of the cyanobacterium Spirulina platensis (Nordstedt) Geitler. In Microscopy
Techniques for Materials Science (pp. 155-169). Springer, Singapore.

3. Singh, R. P., & Sharma, N. K. (2018). Blue-green algae: In vitro culture and its biotechnological applications. Journal of Genetic Engineering and
Biotechnology, 16(1), 1-10.

4. Soni, R., & Prasanna, R. (2019). Isolation and characterization of blue-green algae: a practical approach. Springer Protocols Handbooks, 23, 69-84.

5. Stanier, R. Y., Kunisawa, R., Mandel, M., & Cohen-Bazire, G. (1971). Purification and properties of unicellular blue-green algae (order Chroococcales).
Bacteriological Reviews, 35(2), 171-205.

6. Thajuddin, N., & Subramanian, G. (2005). Cyanobacterial biodiversity and potential applications in biotechnology. Current Science, 89(1), 47-57.