Characterization of the morphology, digestion,

osmoregulatory activity of Paramecium sp.

and its reaction to various stimuli

AGRIPO | FORONDA | MATAAC | MONSALVE | PICART

Institute of Biology, College of Science, UP Diliman

 Introduction : Paramecium
● Free-living unicellular eukaryotic organism
● Kingdom: Chromalveolata; Phylum: Ciliophora
( Cerutti & Casas-Mollano, 2006; Kaplunovsky et al., 2011; Tassin, et al., 2016)
1. Cilia: locomotion, feeding, tactile ● Covered with cilia
perception, mating including oral groove
2. Rigid pellicle and a plasma membrane: ● Asexual reproduction :
protection and shape maintenance binary fission
3. Membrane-bound 2 contractile ● Sexual reproduction:
vacuoles: osmoregulation Conjugation
4. Heterokaryons: two types of nuclei ● Motility
a. Micronucelus : sexual reproduction ● Heterotrophic mode of
b. Macronucleus : metabolic control nutrition
● No cell wall
● Animal like
(Wichterman, 986; Tassin et al., 2016; Rangaswami & characteristics
Bagyaraj, 2005) (Reece et al., 2013)
 TYPICAL RESPONSES
MOST STUDIED | Model Organisms
(Sonneborn, 1970; Fokin et al., 2001) TAXIS
Widely distributed
● Positive Taxis
Easy to cultivate
○ Towards the stimulus
Reproduce rapidly
● Negative Taxis
Size and Space Efficient
○ Away from the stimulus
“...organelles are functionally KINESIS
equivalent to the organs evolved by
higher animals” Change in direction ( + or -)
(Pocsidio, 2009)
Due to Sensory Structures | Paramecium movement is sensitive to:

Touch (Thigmotaxis)

Electricity (Galvanotaxis)

Gravity (Geotaxis)

Light (Phototaxis)

Chemicals (Chemotaxis)
 To observe the following in the Paramecium

1. Gross Morphology and Locomotion

2. Responsiveness to various stimuli: touch,
Objectives
chemicals, gravity, light, and electrical
field
3. Digestion and Food Movement
4. Osmoregulatory activity of the contractile
vacuoles
Methodology
 1. 1-3 drops of the Paramecium culture
was mixed with 2% agar solution or
Gross morphology cotton fibers were placed
2. LPO -> HPO
and movement
Structures observed:
● Slipper shape
Paramecium culture prepared prior to ● Cilia across surface
experiment ● Posterior and Anterior end
● Oral Groove
● Cotton thread/2% Agar : prevent the ● Contractile vacuole
movement so physical characteristics ● Food vacuole
can be observed
● 1% Methylene blue : observe the 3. A drop of methylene blue solution
macronucleus was added to a drop of the culture
4. Movement observed under HPO
 Reaction to various stimuli

THIGMOTAXIS

1. Cotton fibers were added to a drop of the culture

2. Observe: movements of the Paramecium when it

comes in contact with the cotton fibers with its
anterior and posterior end
 Reaction to various stimuli

CHEMOTAXIS

1. Solutions prepared:
● 0.1 M HCl | 0.2 % Acetic acid | 0.1 M NaHCO3 | 3 % NaCl | 5% Sucrose

2. Lengths of strings were dipped in one of the solutions and

the string was placed across the middle of a drop of culture

3. Observe: s going towards or away from the string, and if it

dies in the presence of the string with respective solution
 Reaction to various stimuli

GEOTAXIS

1. A small tube was filled to a quarter of its volume with a rich

culture of Paramecium

2. Placed in a rack and under bright illumination

3. Observe: after 15 minutes, location of aggregates, white

cloud, whether positively or negatively geotaxic
 Reaction to various stimuli
PHOTOTAXIS

1. A small tubes was filled to a quarter of its volume with a rich

culture of Paramecium
2. Stopper was placed at the mouth
3. In a lengthwise manner, half of the test tube was covered in
aluminum foil and was placed on its side with the covered part on top
and the uncovered portion below
4. Bottom was illuminated with a very bright light
5. Observe: location of aggregates, white cloud, whether positively or
negatively phototaxic
 Reaction to various stimuli
GALVANOTAXIS

1. A glass slide with paraffin on the sides was prepared

2. 3 to 5 drops of Paramecium culture and 3 to 5 drops of Ringer’s
solution was placed in the glass slide well from solidified wax
3. Wires were placed on each end of the slide, and different voltages
of batteries were added ( incrementsof 1.5 V)
4. Observe: motion of the Paramecium, whether it’s going toward
the positive( cathode of battery, anode solution) or the negative
end ( anode of battery, cathode solution)
 1. A mixture of a yeast solution (0.1 g
Food movement dry yeast/ 40 mL water boiled 8
min)
and digestion
2. Drops of 1% Congo Red was added
Congo Red solution : pH indicator and for a few minutes allowed to
Alkaline solution → red diffuse into the yeast
Acidic solution → blue
3. On a slide, immobilized Paramecium
Observations:
was mixed with Congo red-stained
● Ingestion
yeast with a toothpick
● Food vacuole formation
4. It was then observed where the
● Cyclosis
food droplet will enter the
● Egestion
paramecium, the color changes, and
where it exits
 1. One drop of Paramecium culture
Role of Contractile was placed on a slide with one drop
of corresponding solutions
vacuole
Neutral red : pH indicator 2. Observe: Record rate of pulsations
Solutions of the contractile vacuole per minute
● Distilled water
● NaCl (2.5%, 5%, 7.5%, 10%) 3. Paramecium was stained with
● M/500 KCN/13 mg in 100 mL neutral red to determine if vacuole is
distilled water acidic or basic
● Paramecium culture fluid ( Panicum
sp. grass extract, sterile water and
feed)
Results
Gross Morphology

Macronucleus
Locomotion
Reaction to various stimuli
Table 1. Thigmotaxis response of Paramecium by
anterior and posterior region bumping

Bumps Anterior Posterior

Thigmotaxis Goes backward and changes Goes to opposite direction of

direction the barrier obstructed

Table 2. Chemotaxis response of Paramecium with

various solutions

Chemical Chemotaxis

0.1M HCl Avoided (-)

0.2% CH3COOH Avoided (-)

0.1 M NaHCO3 Attracted (+)

3% NaCl Attracted (+)

5% Sucrose Attracted (+)

Reaction to various stimuli
Table 3. Geotaxis and Phototaxis response of Table 4. Galvanotaxis response of Paramecium
Paramecium

Geotaxis Phototaxis Voltage (V) Movement

Response Go towards Go towards light 1.5 Towards anode (-) solution; cathode (+) battery
direction of (+)
gravity (+) 3 Towards anode (-) solution; cathode (+) battery

4.5 Towards anode (-)solution; cathode (+) battery

6 Towards anode (-)solution; cathode (+) battery

7.5 Skewed towards anode(-)solution; cathode (+)

battery, rotating

9 Skewed towards anode -)solution; cathode (+)

battery, rotating, rotating

10.5 Slow rotation leading to death

12 Dead
Digestion and movement of food
Oral groove → cytopharynx → food vacuole → anal pore

*Food color change was not observed

Role of
contractile
vacuole
Chemical Pulsations per minute

dH2O 32 Contractile vacuole

Paramecium Media 12

2.5 % NaCl erroneous

5% NaCl erroneous

7.5 % NaCl 5

10% NaCl 2

KCN erroneous
DISCUSSION
 Gross morphology

Shape : Slipper shape

● Anterior end: Blunt
● Posterior end: Pointed

Streamlined with swift motion

(101science.com/paramecium.htm)
STRUCTURE FUNCTION

PELLICLE Outermost gelatinous covering with hexagonal structure ( cilia outside and
trichocyst inside); PROVIDE SHAPE AND ELASTICITY

CYTOPLASM ECTOPLASM: outer, thin, trichocysts and base of cilia;

ENDOPLASM : inner, granular, cell organelles and cell inclusions

CILIA Fine hair-like structures; HELP IN LOCOMOTION AND MECHANISM FOR

OBTAINING FOOD

TRICHOCYST Spindle-shaped structure; DEFENSE ORGANS ( discharged out as needle like

structures when stimulated)

PLASMA MEMBRANE Primary border; SEPARATE INTERIOR FROM EXTERIOR ENVIRONMENT

ORAL GROOVE Oblique aperture runs backward and situated ⅔ part of body

VESTIBULE Base of oral groove; conical shaped; CONNECTS AN OPENING,

CYTOSTOME. Opens into GULLET, terminates in FOOD VACUOLE

ANAL PORE Or Cytoproct; UNDIGESTED MATTERS PASSED OUT

STRUCTURE FUNCTION

NUCLEUS MACRONUCLEUS: SOMATIC NUCLEUS FOR METABOLIC PROCESSES;;

larger kidney shaped
MICRONUCLEUS: GERMINAL NUCLEUS FOR SEXUAL REPRODUCTION;;
smaller spherical

CONTRACTILE 2 ; consists of 5-10 radial canals (star like structure)

VACUOLE Radial canal has three parts: ampulla, terminal and injecting canal’
ABSORPTION OF WATER FROM THE BODY AND RELEASE OF EXCESS
WATER - OSMOREGULATION
 Locomotion
● Spiral motion
● Zigzagging while simultaneously rotating its body
counterclockwise along its longitudinal axis.
● Unique movement attributed to the difference in ciliary
beat direction and frequency (Randall et al., 2002)
 Thigmotaxis
Avoidance reaction

- Stimulation at anterior end

- Changes in membrane potential
- Calcium channels

http://www.watpa.org/10706/MSProject/avoidreact.htm
 Thigmotaxis
● Anterior end
○ Depolarization Influx of Ca2+ ions
○ Movement of cilia is reversed
○ Change in direction occurs
● Posterior end
○ Hyperpolarization Efflux of K+ ions
○ Accelerated movement of cilia
 Chemotaxis
- Effect of certain chemicals on movement depend on its ability to
hyperpolarize or depolarize the cell membrane

● Attractants-Weak acids (Acetic acid, HCl), Sucrose- Hyperpolarize

- Hyperpolarization causes faster beating of cilia

● Repellants- Cations (NaCl, NaHCO3) and HCl- Depolarize

- Depolarization causes influx of Ca2+ resulting to a change in direction
of ciliary beating
 Geotaxis and Phototaxis
- Negatively geotactic
- Due to mechanism of buoyancy-oriented upward
swimming
- Body structure of Paramecium exhibits variable
density
- Rotational torque orients the organism upward
- Center of gravity varies
 Geotaxis and Phototaxis
- Indifferent to ordinary light
- Except for the green Paramecium bursaria which is positively
phototactic
- Negative response to:
a. Sudden and sharp increase in light intensity
b. Ultraviolet rays
 Geotaxis and Phototaxis
SET-UP IN DIFFERENT ORIENTATION
● Geotaxis: Vertical movement greater
● Phototaxis: Horizontal general direction of movement is
perpendicular to the direction of stimulus
 Galvanotaxis
Organism is directed towards
the cathode regardless of its
initial position
When voltage becomes very high, the Paramecium will move towards the
anode until death.(Grell, 2013)
 Galvanotaxis
Ludloff Phenomenon:
Electric field causes the cilia on the posterior
end to beat quickly (ciliary augmentation),
and those on the anterior end to beat
quickly as well but in the opposite direction
(ciliary reversal)

(N. Ogawa et al, 2006)

 Digestion and movement of food
● Oral groove→ cytopharynx→
food vacuole→ anal pore
○ Cyclosis carries food vacuole along a
definite course through the
Paramecium
● Digestion starts as acidic
digestion followed by an alkali
digestive process

Verma, P. (2001). Invertibrate Zoology. S. Chand Publishing.

Digestion
Mechanism
○ cilia sweeps water and “food”
○ endocytosis
○ Vacuole formation
○ Smaller vesicles formation for
absorption efficiency
○ anal pore
Change in Food Color
Congo Red solution : pH indicator
Alkaline solution → red
Acidic solution → blue
● changes in acidity caused
by secretions from the
cytoplasm
● change in size was caused
by changes in osmotic
pressure
Size of cell decreases = red to
blue = high acidity
Size of cell increases = blue
to orange = low acidity
(Mast, 1947)
 Role of Contractile Vacuole
Contractile Vacuoles - osmoregulatory organelle which controls intracellular
water balance by accumulating and expelling excess water., allow cells to
survive under hypotonic stress (Du et., al 2008)

○ Temporary: contractile vacuole together with its membrane, and the

vesicles which fuse to form it
○ Permanent: CV pore including its discharging tubule, and feeding canals
Contractile Vacuole Pulsations
1. Hypotonic solution - faster contraction
● Salt content balanced between organism and
external environment ; prevent lysis
2. Hypertonic solution - slower contraction
● Control of water being expelled; prevent shrivelling

KCN - the CV exhibits decrease in activity or becomes

paralyzed (swelling of the cell)
○ reversible
○ vacuolar activity dependent on ATP
- cyanide inhibits cytochrome c which is involved in the
electron transport chain.
 Role of Contractile Vacuole
Anterior and Posterior Contractile Vacuoles
● Posterior:
- functions stronger as it is near the cytopharynx
● Contracts alternatingly

Fluid / contents of the CV has a pH of 6.4

References
Eckert, R. (1972). Bioelectric Control of Ciliary Activity. Science, 176(4034), 473-481.
Fukui, K., & Asai, H. (1985). NEGATIVE GEOTACTIC BEHAVIOR OF PARAMECIUM CAUDATUM IS
COMPLETELY DESCRIBED BY THE MECHANISM OF BUOYANCY-ORIENTED UPWARD
SWIMMING. Biophysical Journal, 47(4), 479-482. doi:10.1016/S0006-3495(85)83940-0
Grell, K. G. (2013).Protozoology. SpringerScience & Business Media.
Mast, S. O. (1947). The Food Vacuole in Paramecium1. 92:1, 31-72. Retrieved from
http://www.journals.uchicago.edu/doi/abs/10.2307/1537967?journalCode=bbl&
Nakatani, I. (1968). Chemotactic Response of Paramecium caudatum. JOURNAL OF THE FACULTY OF
SCIENCE, 16(4), 553-563.
Valentine, M., Yano, J., & Van Houten, J. L. (2008). Chemosensory Transduction in Paramecium. japanese
journal of protozoology, 41(1).
Verma, P. (2001). Invertibrate Zoology. S. Chand Publishing.
Wichterman, R. (2012). The Biology of Paramecium. Springer Science & Business Media.