V{tÑàxÜ 3
MATERIALS AND METHODS
In the present study, water samples from different stations of Pamba river were
collected and analyzed for water quality status during pre-monsoon, monsoon and
post- monsoon seasons. Two selected fishes Cyprinus carpio (Linnaeus, 1759) and
Puntius sarana (Hamilton-Buchanan, 1822) were maintained in the laboratory in
river water collected during different seasons and exposed to different bacterial
inocula to study the haematological, biochemical and histopathological changes due
to the stress induced by the bacteria.
3.1 Study Area
The river Pamba, the third largest river in Kerala, is originating from the Piramedu
plateau, second segment of forest in the southern Western Ghats, adjacent to the
north of Agasthyamalai range. The main tributaries are Kakki Ar, Azhutha Ar,
Kakkad Ar and Kalli Ar which join with the Pamba river in the high lands. The rivers
Manimala and Achenkovil join with it in the plains. After meandering through
Idukki, Pathanamthitta and Alleppey districts in Kerala over 176 km, the river
ultimately flows into the Vembanad lake. The Sabarigiri Hydroelectric Project, the
second major Hydel Scheme in Kerala, is situated in this river. The famous forest
shrine of Swami Ayyappa (Sabarimala Pilgrim Centre) is in the north western
foothills of Pamba plateau and the mount plateau. Over the years it has become one
of the most popular pilgrim centres in South India and millions of pilgrims visit the
shrine during the months of December and January and also during the first day of
every Malayalam month.
Chapter 3 Materials and Methods
36
3.1.1 Sampling stations
Ten sampling stations (Site-1 to Site-10) were selected for investigation (Fig.1). The
sampling sites were fixed as per the requirements of the study and extending an area
of about 80 km. from Azhutha Ar in the upstream to Thakazhi area in the down
stream.
3.2 Water quality analysis
The water samples for the present investigation were collected at monthly intervals
from ten sites of Pamba river for a period of two years from January 2005-December
2006. Surface water samples were collected in sterilized bottles from the identified
sites of River Pamba, brought to the laboratory taking necessary precautions and
analyzed for the various physico-chemical and bacteriological parameters following
standard methods (APHA, 1998). The study was carried out during three seasons,
Pre-monsoon, Monsoon and Post-monsoon.
3.2.1 Physico-chemical parameters
Temperature (oC)
The temperature of water was recorded using a digital thermometer at the site itself.
Turbidity
Turbidity of the water sample was analyzed by a Nephelometric turbidity meter
(Systronics). When light is passed through a sample having suspended turbidity,
some of the light is scattered by the particles. The scattering of the light is
proportional to the turbidity. The turbidity of a sample is thus measured from the
amount of light scattered by the sample taking a reference with standard turbidity
suspension. Nephelometer measures the scattered light at the right angle of the path
of the incident light. Turbidity is expressed in NTU.
Chapter 3 Materials and Methods
37
Electrical conductivity (EC)
Electrical conductivity is the measure of the ability of an aqueous solution to carry an
electric current. It was measured with the help of a conductivity meter having a
conductance cell containing electrodes of Platinum coated with Pt Black or Carbon.
The unit of conductivity measurement is µ/m Siemens(S)/cm. The conductivity of
most waters is generally low and hence the unit µ mhos /cm is commonly used.
Total Dissolved Solids (TDS)
Total Dissolved Solids was determined using TDS-conductivity meter (Systronics)
that was calibrated using standard KCl solution and expressed in ppm.
Hydrogen ion concentration (pH)
Hydrogen ion concentration (pH) was measured by a digital pH meter (Eutech).
Total Alkalinity
Total alkalinity is the measure of the capacity of the water to neutralize a strong acid.
The alkalinity in the water is generally imparted by the salts of carbonates,
bicarbonates, phosphates, nitrates, borates, silicates etc. together with the hydroxyl
ions in the free state. Alkalinity is measured as carbonates and bicarbonates by the
titration method with a strong acid (HCl or H2SO4) first to pH 8.3 using
Phenolphthalein as an indicator and then further to pH between 4.2 and 5.4 with
Methyl Orange indicator. In the first case, the value is called as Phenolphthalein
Alkalinity (PA) and in the second case it is Total Alkalinity (TA) and expressed as
mg Ca CO3 / l.
Total Hardness
Hardness of water is defined as the presence of significant concentration of salts of
metallic cations mainly Ca2+ and Mg 2+ions dissolved in water. It is expressed in
terms of CaCO3 concentration in mg/l. The hardness may range from zero to
hundreds of milligram per litre, depending on the source and treatment to which the
Chapter 3 Materials and Methods
38
water has been subjected. It is determined by complexometric titrimetric method
using Ethylene Diamine Tetra Acetic acid (EDTA) as titrant and Eriochrome Black-
T (EBT), a dye used as an indicator.
Chloride
Chloride, in the form of chloride ions is one of the major inorganic anions in water
and wastewater. Chloride is estimated using Argentometric method with standard
silver nitrate as titrant and potassium chromate as the indicator solution. The end
point is indicated by the formation of brick red colour.The chloride content is
expressed in mg / l.
Dissolved Oxygen (DO)
The azide modification of Winkler’s method is used for the determination of DO and
expressed in mg O2 / l.
Biochemical Oxygen Demand (BOD)
BOD is the measure of the degradable organic material present in a water sample,
and can be defined as the amount of oxygen required by the microorganisms in
stabilizing the biologically degradable organic matter under aerobic conditions. A
five day BOD test was conducted to measure the BOD of the sample using BOD
incubator. The reagents used are same as that of DO. The difference of the initial and
the final DO is the BOD. BOD mg O2 / l = Initial DO - Final DO.
Nitrate – nitrogen
Nitrate was determined using the Brucine -sulphanilic acid method. An aliquot of
sample was refluxed with 30% NaCl, 4:1 H2SO
4 and Brucine-sulphanilic acid. The
intensity of the yellow colour formed was measured at 410nm using
spectrophotometer (Genesys-Thermospectronic). The reaction is highly dependent
upon the heat generated during the test and was carried out in controlled heating at
Chapter 3 Materials and Methods
39
constant time. The concentration of NO3N was found out from the standard curve
and expressed as mg / l.
Phosphate- phosphorous
Phosphate was determined by the stannous chloride method. An aliquot of sample
was made into alkaline by adding phenolphthalein followed by NaOH. The solution
decolourised with H2SO4. Then the sample was subjected to persulphate digestion for
converting all the forms of phosphates into one form. After 30 minutes of digestion,
the sample was cooled to room temperature and one drop of phenolphthalein
indicator was added. Then neutralized the solution using NaOH or H2SO4.
Ammonium molybdate and stannous chloride was added to the digested solution to
give intensively coloured molybdenum blue and the colour developed was measured
at 690nm using spectrophotometer. The concentration of phosphate was determined
from the standard curve and expressed as mg / l.
3.2.2 Bacteriological parameters
3.2.2.1 Collection of sample
Water samples for bacteriological analysis were collected in sterilized containers and
kept in icebox and carried to the laboratory as early as possible. The samples were
then analyzed for bacteriological parameters such as faecal coliforms (FC) and total
heterotrophic bacterial (THB) load.
3.2.2.2. Faecal coliform
The most probable number (MPN) of coliform bacteria was determined by the three
tube dilution method using EC broth. A 10ml, 1ml and 0.1ml of appropriate diluted
samples were inoculated into respective dilution tubes containing inverted Durham’s
tubes. 10ml samples were inoculated into 10ml double strength EC broth and 1ml
and 0.1ml sample into single strength medium of 10ml each. Inoculated tubes were
incubated at 44.4°C for 24-48 hours and examined for the growth and gas
Chapter 3 Materials and Methods
40
production. The MPN index was determined by checking the number of positive
tubes in each set and comparing the values with standard MPN table (APPENDIX-II).
3.2.2.3 Isolation of total heterotrophic bacteria (THB)
One ml of the water sample from each site was serially diluted with sterile distilled
water. Appropriately diluted water samples were plated on Nutrient Agar (NA)
medium using spread plate and pour plate technique. The inoculated plates were
incubated at 37oC for 48-96 hours. After incubation plates with countable range (30-
300 colonies) were selected for counting using colony counter and the bacterial load
in the sample was expressed as total colony forming units (cfu) per ml of water
sample.
3.2.2.4 Characterization of total heterotrophic bacteria (THB)
Morphologically different colonies were isolated, restreaked to ensure purity and
maintained on Tryptic Soy Agar (TSA) vials for further characterization. The
cultures were then identified as various genera as per the Bergey’s manual of
determinative bacteriology (Buchanan and Gibbons, 1984).
Gram staining
The Gram staining was done to classify bacteria into two groups, viz., Gram-positive
forms and Gram-negative forms. The Gram-positive forms are those which retain the
primary stain when washed with a decolourizing agent and the Gram-negative forms
are those which do not retain the primary stain when a decolourizing agent is used
but then take up the colour of the counter stain. In this method (Aneja, 1996) the air-
dried and heat fixed bacterial smear is subjected to the following staining reagents in
the order of sequence listed below:
Gram's Crystal violet Gram's Iodine Alcohol (decolorizing agent) Gram's
Safranin
Chapter 3 Materials and Methods
41
Spore staining
Spore staining was done to characterize Gram-positive cultures. Smears were
prepared on clean slide, air-dried and heat fixed. The smears were flooded with 5%
aqueous malachite green and subjected to steaming for 3 minutes by passing a
Bunsen burner flame under the slide. Care was taken to keep the slide wet by adding
sufficient amount of malachite green. The slides were washed with running tap water
and counter stained with 0.5% safranin for 30 seconds. The smears were again rinsed
with tap water, blot dried and observed under the microscope, the spores took up the
colour of malachite green were green, and the vegetative cells stained pink colour of
safranin.
Motility test
The motility of bacteria was observed indirectly by using soft agar (0.4% agar
concentration) deeps. A small amount of culture is inoculated aseptically into the
motility agar medium by stabbing. The tubes were then incubated at 37oC for a
certain period to allow the growth of the organism. If the bacteria are motile, the
turbidity radiates outward from the stabbed line. If it is not motile the culture will
grow only along the stabbed line. The procedure works well for those organisms that
do not require more oxygen for growth.
Oxidase test
During aerobic respiration, oxidase enzyme plays a vital role in the electron transport
system. Cytochrome oxidase catalyses the oxidation of a reduced cytochrome by
molecular oxygen, resulting in the formation of water or hydrogen peroxide. This test
depends on the presence of certain oxidases in bacteria that will catalyse the electron
transport between electron donors in the bacteria and a redox dye tetramethyl-p-
phenylene-diamine dihydrochloride. The dye is reduced to an indophenol, which has
got a deep purple colour. The test was carried out by scratching a small amount of
culture using a sterile wooden toothpick on the dry filter paper soaked in freshly
prepared tetramethyl p-phenylene diamine dihydrochloride. A positive reaction
Chapter 3 Materials and Methods
42
indicated by an intense deep purple blue appearing within 5-10 seconds, a delayed
positive reaction by colouration in 10-60 seconds, and a negative reaction by absence
of colouration or colouration later than 60 seconds.
Catalase test
This was done to test the presence of catalase enzyme. Little growth from the
nutrient agar slant was transferred to a clean glass slide and few drops of 3%
hydrogen peroxide were placed over the culture. An evolution of gas bubbles from
the culture is considered positive for catalase test. The evolution of gas bubbles
results from the splitting of hydrogen peroxide with the help of catalase enzyme
leading to the production of water and oxygen.
Oxidation / fermentation test
Sterile oxidation fermentation (O/F) medium tubes were prepared with glucose as
carbohydrate source. A pH indicator is incorporated in the medium used to
differentiate between oxidative and fermentative breakdown of glucose. A small
amount of culture is inoculated into the medium by stabbing the butt and streaking
the slant, and incubated at 37oC for 24 hours. If acid is produced at the surface of the
medium, where the conditions are aerobic, the breakdown of sugar is considered as
oxidative. If acid is found throughout the tube including the lower layer, where
conditions are anaerobic, the breakdown is considered as fermentative.
3.3 Survey of fishes
A representative survey of fishes was carried out to throw light into the fish fauna of
Pamba river. The fishes were collected using gill nets and cast nets and preserved in
7% formalin. The identification of fishes was done with the help of standard manuals
(Day, 1865, Day, 1878, Munro, 1955).
Chapter 3 Materials and Methods
43
3.4 Experimental studies
3.4.1 Experimental fish
The fishes selected for the present study are Cyprinus carpio (Plate–I A) and Puntius
sarana (Plate-I B) which belong to the family Cyprinidae of order Cypriniformes and
class Teleostei.
Cyprinus carpio
It is widely known as common carp, a widespread freshwater fish recognized by its
small eyes, thick lips with two barbels at each corner of the mouth, large scales and
strongly serrated spines in the dorsal and anal fins. The colour is variable, but often
olive green to silvery grey dorsally, fading to silvery yellow on the belly. The carp
lives alone or in small schools in quiet, weedy, mud bottomed ponds, lakes and
rivers. It is omnivorous in diet and a commercially important species. Common carp
is a major cultivated species in freshwater aquaculture.
Puntius sarana
Small size with terminal mouth having maxillary and rostral barbels, commonly seen
in rivers. Fins are dusty brown to orange in colour having a dark caudal blotch
behind anal fin, whereas the young ones with two or three additional spots. It is well
known that they are susceptible to the oxygen content of the water and could be used
as an indicator for dissolved oxygen (DO) content of water.
3.4.2 Selection of bacteria
Three bacteria, Aeromonas hydrophila, Escherichia coli and Pseudomonas isolated
from the water samples of Pamba river were selected as test microorganisms for the
experimental study. Pseudomonas and A. hydrophila were selected considering their
ubiquitous presence in the aquatic environment and their role as an important fish
pathogen in tropical fishes. E. coli was selected because of high load and was
frequently encountered in the water samples from the study area. These
Experimental fishes
A-Cyprinus carpio
B- Puntius sarana
Chapter 3 Materials and Methods
44
microorganisms were selected hypothesizing that in a stressed environment the
different bacterial species could negatively affect the fish fauna.
3.4.3 Preparation of inoculum
Pure cultures of A. hydrophila, Pseudomonas and E. coli were inoculated separately
into 10 ml sterile nutrient broth and incubated at 37oC for 16-18 hours. After
incubation the cells were harvested by centrifugation at 3000 rpm for 15 minutes.
The cell pellets were washed in isotonic saline and re-suspended in 10 ml sterile
isotonic saline. The initial inoculum density was determined by spread plate method
on nutrient agar after desired dilution of inoculum in sterile distilled water or isotonic
saline.
Cumulative percentage mortality of the experimental fish for 96 hours was
graphically represented and from the graph the load for 96 hour LD50 was noted. Sub
lethal dosage was calculated and it was found to be 105 cfu / ml.
3.5 Experimental set up
Cyprinus carpio procured from the Pannivelichira fish seed farm, Pathanamthitta
district and Puntius sarana collected from Pamba river were brought to the
laboratory. The fishes were kept in properly aerated large tanks containing river
water from Site-1 for one month for acclimatization and were fed daily. Water from
Site-1 (upstream) was selected as control medium as the water sample was found to
be pristine with least bacterial concentration. Fishes of about the same weight and
size irrespective of sex were selected for the experiment. Five set of aquarium tanks
(A to E) in triplicate were set up, four tanks (A to D) with water from Site-1 and tank
E with water from Site-2. Thirty experimental fishes were maintained in each tank.
Tank A stands as control. Fishes in tank-B series (B1, B2, B3) were exposed to
E. coli, tank C series (C1, C2, C3) with A.hydrophila, tank D series (D1, D2, D3) with
consortium I (Aeromonas, Pseudomonas and E.coli). Each tank were administered
with an initial inoculum density of 105 cfu / ml. Tank E (E1, E2, E3) was set up with
water sample from Site-2 (post-monsoon) as consortium II. Water sample from Site-
Chapter 3 Materials and Methods
45
2 was selected for the experiment as it recorded the highest load with varied bacterial
species during the pilgrim season (post-monsoon). The experiments were conducted
for a period of one month. For the analysis, the fish was caught individually using a
small hand net from aquarium tanks. The analyses were carried out on days 1, 3, 5,
10, 15, 20, 25 and 30. Physico-chemical parameters such as temperature, turbidity,
pH, dissolved oxygen and bacterial load of the aquarium water were also maintained
by regular monitoring. Five sets of experiments were carried out for each
bacterial exposure and the mean values were taken.
3.6 Haematological analysis
The fish was cleared off the water from the body surface, then the caudal peduncle
was severed and the blood was collected using a disposable syringe from the caudal
artery and transferred into heparinized tubes. Approximately 2 ml blood was drawn
from test as well as control fish at day 1, 3 and 5 and every 5 days interval of
exposure, for the analysis of haematological parameters. The red blood cell (RBC)
count and the white blood cell (WBC) count were done, the haemoglobin (Hb) and
packed cell volume (PCV) or the haematocrit (Hct) concentration were determined
(Dacie and Lewis, 1977).
The RBC count and WBC count were carried out using a Neubauer’s double
haemocytometer. Sahli’s haemoglobinometer was used for estimating haemoglobin.
The packed cell volume (PCV) or haematocrit values were measured by Wintrobe’s
method using haematocrit tube which was centrifuged for 10-15 minutes at 3000-
4000 rpm (Blaxhall and Daisley, 1973b). The derived blood indices of mean
corpuscular haemoglobin (MCH), mean corpuscular volume (MCV), mean
corpuscular haemoglobin concentration (MCHC) and the oxygen carrying capacity
were calculated from the haematological data by the following standard formula.
MCH = Hb in gms/1000ml blood = pg
RBC in millions/mm³
MCV = PCV/1000ml blood = µ3
RBC in millions/mm³
Chapter 3 Materials and Methods
46
MCHC = Hb in gms/1000ml blood x 100 = % (g/100ml)
PCV/100 ml
O2 carrying capacity =Hbx1.25 O2 combining power of Hb/g (ml O2/g Hb)
Blood smears were prepared over grease free clean slides and air dried. Subsequently
the slides were stained with Wright’s stain (Hesser, 1960) and morphological
changes in control and experimental fishes were observed. Photographs were taken
using Nikon H-III microscope with photomicrographic equipment.
3.7 Biochemical analysis
After the fish blood has been withdrawn for haematological studies, the tissues viz.,
gill, muscle and liver were dissected out from the control and treated fish from each
group for the analysis of lipid peroxidation level, total protein, total amino acids and
soluble sugar content using standard methods.
Lipid peroxidation
Membrane lipid peroxidation was measured in terms of malondialdehyde (MDA)
accumulation. MDA level is routinely used as an index of lipid peroxidation. To
estimate MDA content, thiobarbituric acid (TBA) test was performed using the
procedure of Heath and Parker (1968). The absorbance of the extract was read at 532
nm and the values were corrected for non-specific turbidity by subtracting the
absorbance at 600 nm. The concentration of malondialdehyde was calculated using
an extinction coefficient of 155 /mM /cm and expressed as n moles MDA / g wet
weight of tissue.
Total protein
The method developed by Lowry et al. (1951) was followed for the estimation of
total protein content. The colour developed is due to the presence of (i) biuret
reaction of protein with copper ion in alkali and (ii) reduction of phosphotungstic
reagent in Folin-Ciocalteau reagent by tyrosine and tryptophan present in the treated
Chapter 3 Materials and Methods
47
protein. The colour developed was read at 660nm using spectrophotometer and the
protein concentration were calculated and expressed as mg / g wet weight of tissue.
Total amino acid
Total free amino acids in tissues were estimated (Moore and Stein, 1954) using
ninhydrin, a powerful oxidizing agent that carboxylates the alpha amino acids and
yield a bluish purple product. The colour was spectrophotometrically measured at
570nm. The amino acid content was expressed as mg / g wet weight of the tissue.
Soluble sugars
Phenol sulphuric acid method (Dubois et al., 1956) was used for the analysis of total
soluble sugars. In hot acidic medium glucose is dehydrated to hydroxymethyl
furfural. This forms a green coloured product with phenol and the absorbance was
read at 490 nm. The soluble sugar was expressed as mg / g wet weight of tissue.
3.8 Histopathological analysis
Organs like gill, liver and intestine were isolated from control and experimental
fishes. For histopathological examinations the tissues were fixed in 10% phosphate-
buffered formalin solution. The samples were dehydrated and embedded in paraffin
wax. Sections (5µ thick) were taken and stained with Haematoxylin and Eosin
(Roberts, 1978). Photographs were taken using Nikon H-III microscope with
photomicrographic equipment.
3.9 Statistical Analysis
The data (mean values) obtained were tabulated and graphically represented and
were subjected to statistical analysis. ANOVA-Two Factor CRD (Completely
Randomized Design) was employed to test the level of significance of variation
between controls and experimental mean for haematological and biochemical
analysis (Gomez and Gomez, 1984). Pearson’s Correlation coefficient matrix was
applied for the water quality parameters using SPSS 6.1.3 (Norusis, 1996).