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).