SUMMER INTERNSHIP REPORT

Title: Effect of sinusoidally modulated light on the growth of

Chlamydomonas reinhardtii(CC-125)

Organization:Faculty of Science,Maharaja Sayajirao University,Vadodara

Duration: 6 weeks

Principal Investigator:Dr Debjani Bagchi,Department of Physics,Faculty of

Science,MSU,Vadodara

Internship Done By:

Somilya Mishra
SEM III

Introduction
Chlamydomonas species have been distinguished by differences in overall size and body
shape,shape and position of the chloroplast and pyrenoids,flagellar length,number and position of
contractile vacuoles and more subtle structural features visible at the light microscopic level.Ettl
recognized 459 species of Chlamydomonas and 9 major morphological groups.

So in 459 species, Chlamydomonas reinhardtii has emerged as the predominant laboratory

species because of its ability to grow non-photosynthetically with acetate as its sole Carbon
source.

Currently one of the most active research topics is with Chlamydomonas eugametes and
Chlamydomonas moewusii is phospholipid mediated signal transduction.Sequence of the nuclear
genes including 18S ribosomal RNA,the nuclear ribosomal DNA spacer ITS2 and chloroplast
ribosomal RNAs place Chlamydomonas moewusii and Chlamydomonas eugametos in a group
more closely allied to Haematococcus and Chlorogonium than to Chlamydomonas reinhardtii
cluster.

Volvox Carteri,Pandorina morum,and some other colonial Volvocales used as research

organisms appear to be closely allied with Chlamydomonas reinhardtii.Although analysis of
transposon insertion sites and chloroplast DNA restriction digests strongly suggest that all these
strains do have a common origin,especially when compared with the interfertile isolates from
other localities.
 We basically do this project to know the conditions which are suitable for the Chlamydomonas to
produce Triacylglycerol efficiently and feasible to use for human population,which is commonly
known as Biodiesel.

The project basically constitutes of two aspects, first is the designing of the light intensity
modulator with help of ARDUINO Uno board and second is the culturing of the
Chlamydomonas reinhardtii and microscopic studies.

Workflow
1) Programming the microprocessor with the help of Arduino UNO IDE
window,according to the photoperiod required such as 12hrs light and 12hrs dark
2) Design of the circuit using following components
 Mosfet IRZ447N
 Resistors
 PCB Board
 Single Stranded Wire
 Solder and flux
 LED Light
 ARDUINO UNO BOARD

3) Preparation of the Tris-Acetate-Phosphate (TAP) Media for Chlamydomonas (100 ml pH=7)

 1M Tris base 2ml

 Phosphate buffer II 0.1ml
 Solution A 1ml
 Hutner’s trace elements 0.1ml
 Glacial Acetic Acid 0.1ml
 Make up the volume upto 100ml
 Autoclave the media at 121ºC,15psi for 20 minutes
 Add 12ug/ul of Ampicillin to avoid any bacterial infection.We should use an
antibiotic whose mode of action should not hinder the metabolic pathways of the
Chlamydomonas

4) Culturing of the Chlamydomonas reinhardtii by the following techinques

 Plate to Broth:This technique was used to make the primary culture of the
Chlamydomonas.The strain CC-125 of Chlamydomonas reinhardtii was grown
on plate and then transferred to test tubes containing the media
 Broth to Broth:This technique was used to make the secondary culture for the
Chlamydomonas which can be used for test.The primary culture was centrifuged
for 5 times for 3mins at 5000rpm (MCT Volume-2ml) and then it was washed
 with sterile distilled water,the cells isolated were used as the inoculum for the
secondary culture or daughter culture.

5) Measurement of the growth of the culture by Optical density measurement with help of
Spectrophotometer

 Optical Density: It is also referred as OD,it’s a measurement of a refractive

medium or optical component’s ability to slow or delay the transmission of
light.The slower the light that is able to travel through a given medium,the higher
is the optical density of the medium

 OD of the primary culture at Day 0

1. MCT 1:0.432abs
2. MCT 2:0.458abs
3. MCT 3:0.448abs
4. MCT 4:0.514abs
o Test tube no 4 was having the maximum growth

6) Imaging of the specimens under Bright field Microscope

 Eyespot was visible at periphery of the cell membrane

 Live movement of Chlamydomonas and its flagella(whiplash type) was observed
 Some lipid bodies were also observed

Techniques Learned
1. Subculturing: This is a technique in which we harvest the cells from primary culture or
the mother culture with help of centrifuge and inoculate it in a new fresh medium.The
culture obtained is known to be daughter culture or secondary culture
2. Spectrophotometery:It is a method to measure how much a chemical substance absorbs
light by measuring the intensity of light as a beam of light passes through sample
solution.The basic principle is that each compound absorbs or transmits light over a
certain range of wavelength.
3. Bright Field Microscopy:It is the most elementary form of microscope illumination
techniques and is generally used with compound microscopes.The name “brightfield ” is
derived from the fact that the specimen is dark and the background is bright.

Note: The project is still under progress