# IV. Hemolymph Microflora

IV. 1. Introduction

IV.2. Materials and Methods

IV.3. Results

IV.3.1. Hetrotrophic Bacterial genera in the three snail species

IV.3.2. Coliform Bacterial genera in the three snail species.

IV.4. Discussion

IV. 1. Introduction

Mollusc - bacteria interactions occur in natural conditions since bacteria

constitute an important part of the micro flora of the environment (Oubella etal., 1994).

It is not known whether the presence of bacteria in the hemolymph is normal or an

indication of disease (Brinkley et al., 1976). Many studies refer to microflora in the

hemolymph as causing pathogenic infections rather than as being part of the normal

flora (Stewart and Robin, 1970; Rosenmark and Conklin, 1983).

Molluscs can act as vectors for the spread of certain diseases that are pathogenic

for humans such as Vihrio cholerae, Vibrio vu1nfIcus and Vibrioparahemolyticus (Colwell,

1984; Tamplin et al., 1982; Tamplin and Capers, 1992). Bacteria may constitute a

substantial proportion of the diet of marine filter feeders (Zo Bell and Feitham, 1938)

and colonize their integument and gut (Mc Henery and Birkbeck, 1985). Thus marine

bivalves accumulate large numbers of gram-positive and gram-negative microorganisms

(Colwell and Liston, 1962; Murchelano and Brown, 1968).

Considering the above studies, there is a dearth of work on the microflora of the

hemolymph of T. vittata, P. globosa and I. exustus. This study has been undertaken to

identify and quantify bacteria in the body fluid of these snails.

IV. 2. Material and methods

IV. 2. 1. Sampling procedure

The three species of gastropods snails were brought to the laboratory and the

external surfaces of the shells were cleaned and wiped with alcohol before collecting the

hemolymph. Hemnolymph from all the three snails were collected as described in the

earlier chapter (1.2.1) and transferred to separate sterile test tubes, sealed tightly with

sterile cotton. These hemolymph samples were utilized immediately for bactciological

analysis.

IV. 2. 2. Media and preparation

Dehydrated media (Hish media, Bombay) were used throughout the study.

Standard tests used to determine the bacteriological quality in water systems in terms of

presumptive, confirmed and completed tests (Boyd et al., 1984) were done on the

hernolymph of the three snail species and carried Out according to Bordner and Winter

(1978).

(A) Presumptive test medium

Lauryl tryptose broth was used for the detection and enumeration of coliform

bacteria in the hemolymph of the gastropods.

Composition

Tryptose

Lactose

Sodium chloride

Dipotassium phosphate

Monopotassium phosphate

Sodium lauryl sulphate

Distilled water

Final pH (at 27°C)

20.0 g

5.0 g

5.0 g

2.75 g

2.75 g

0.1 g

1000 ml

6.8 ± 0.2

The medium (10 ml each) was distributed into test tubes with inverted Durham's

tubes and sterilized by autoclaving at 15 lbs sq. inch pressure (121°C) for 15 minutes

(Salle, 1979).

(B) Confirniative test medium

Brilliant green 2% bile broth (Total coliform) and EC broth (Faecal coliform)

were used for confirmative tests.

Brilliant green 2.0% Bile B rot!,

Peptone 10.0 g

Lactose 10.0-0

Oxgall 20.0-

Brilliant green 0.0133-

Distilled water 1000 ml

Final pH (at 27°C) 7.2 ± 02

EC Broth

Tryptone 20.08 g

Lactose 5.08 g

Bile Salt mixture 1.5 g

Dipotassium phosphate 4.08 g

Monopotassium phosphate 1.5 g

Sodium chloride 4.08 g

Distilled water 1000 ml

Final pH (at 27°C) 6.9 ± 0.2

The medium (10 ml each) was distributed with inverted Durham's tubes and

sterilized by autoclaving at 15 lbs / (121°C) for 15 minutes.

(C) Completed test mediumLevine's eosin methylene blue agar (LEMBA) was used for the isolation of

coliform organisms.

69

Composition of Lt lB/I

Peptone

Dipostassium phosphate

Lactose

Agar

Eosin - Y

Methylene blue

Distilled water

Final pH (at 27°C)

10.0 g

2.0 g

10.0-

15.0 g

0.4 g

0.065 g

1000 ml

7.1 ± 0.2

The medium was boiled to dissolve completely and sterilized at 15 lbs/ pressure

(121°C) for 15 minutes. It was cooled to 50°C and shaked in order to oxidize the

methylene blue (ie., to restore its blue colour), and to suspend the flocculant precipitate

which is an essential part of the medium.

IV. 2. 3. Bacteriological procedures employed

For the enumeration of Escherichia colt in the hemolymph of the three snail

species, procedures to find Out most probable number (MPN) were followed as in

Bordner and Winter (1978). This method has three stages: the presumptive, the

confirmed and the completed tests. In the presumptive test, a series of lauryl tryptose

broth fermentation tubes were inoculated with decimal dilutions of samples (1 ml, 0.1

ml). The formation of gas at 35 °C within 48 hours constitutes a positive presumptive

test for the members of the total coliform and facal coliform groups.

However, the MPN must be carried through the confirmed test for valid results.

In this test, inocu]a from positive presumptive tubes were transferred to tubes of

brilliant green lactose bile broth (BGLB) and EC broth for total coliform and faecal

I'll

coliorni respectively. Gas production after incubation for 24 hours to 48 hours at 35°C

constitutes a positive confirmed test for total coliform and gas production after

incubation for 24 hours at 44°C incubation constitutes a positive confirmed test for

faecal coliform.

In the completed test, inoculum is streaked from the positive BGLB and EC

tubes on to sterile, air dried EIvIB agar plates and incubated for 24 hours at 35°C.

Typical and atypical colonies are transferred into nutrient agar slants for pure culture.

IV. 2. 4. Tests employed in bacterial identification

The isolated bacterial colonies were identified by using various biochemical tests.

Biochemical tests have always been performed along with positive and negative

controls.

Characterization of heterotrophic bacteria

Morphologically different colonies were streaked on sterile, air dried nutrient

agar plate for purity and stored in nutrient agar slants.

For characterization studies, the fresh overnight nutrient broth cultures of

bacterial isolates, isolated from the hemolymph samples of the three chosen gastropods

were subjected to the following tests. For differentiation and characterization, the

method of Simudo and Also (1962) was adopted.

(A) Gram's staining

This is the widely used differential staining technique in microbiology. In this

process, the fixed bacterial smear is subjected to the following staining order: Gram's

crystal violet, Gram's iodine solution, decolourising agent (acetone-alcohol) and Gram's

Gram staining

Positive Negative

Cocci

Micrococcus sp

Small rods Rods

]Spore

PositiveBacillus sp.

Non - fermentative

Pseuclomonas sp.

Sensitive toPencillin

(2.5 1U/disc)

Negativeresistance

Hush &Leifson

Fermentative

Acid & No gas Acid &gas

Non - luminesentVilirio sp.

Kovacs oxidase test

Positive Negativesp IEnterobacter sp.

Scheme of Identification of bacterial isolates from hemolymph samples of threechosen gastropods (Simudu and Aiso, 1962)

S

1tiT1

(a) (c)Control

(b) NegativPositive reactionreacgfl

(c)(a)NegativeControl Positivereaction

W -

A

B

:.-

(a) (b) (c)Positive

Control Negativereaction

(a)(b) (c)

Positive NegativeControl reaction reaction

B

, -

t

Plate. 11 Shows the characterization of heterotrophic bacteria, adoptingdifferent standard methods (A, B, C and D)

A. Indole test B. Methyl Red test C. Voges - Proskauer test D. Citrate test

safranin. The stain has its greatest use in characterizing bacteria into Gram positive

(purple) and Gram negative (pink) on the basis of their cell wall chemistry.

(B) Indole test

Indole production was tested in a peptone water culture after 48 hours

incubation at 37°C. This test demonstrates the production of indole from tryptophan.

After incubation, 0.5 ml Kovac's reagent was added and shaken gently. A red colour

indicates positive and yellow colour indicates negative reaction.

(C) Methyl red test

This test is employed to detect the production of acid during fermentation of

glucose and maintenance of a pH below 4.5 in an old culture. Five drops of 0.04%

solution of methyl red are added to the culture in glucose phosphate medium, incubated

at 35 C C for five days, mixed well and read at once. Red colour is considered positive

while yellow signifies a negative test.

(D) Voges - Proskauer test

This procedure detects the production of acetyl methyl carbinol (acetoin) which

in the presence of alphanaphthol and potassium hydroxide develops a reddish (crimson

red) colour. The test was performed by adding 0.6 ml of 5.0% solution of naphthol in

ethanol (Barrit's reagent A) and 0.3 ml of 40% KoH to 0.1 ml of glucose phosphate

(Barrit's reagent B) to the culture of the organism and incubated at 30°C for 48 hours.

In a positive reaction, a pink colour appears in 2-5 minutes' from magenta in

half an hour. Traces of pink colour is neglected.

(E) Citrate test

Simmons citrate medium has citrate as the sole source of carbon. The

inocu.atecl iar medium is incubated at 35'C for 48 hours. The ability to use this

substance is seen b y a change of colour from grecIl to blue on the basis of shift in

media pH.

IV. 3. ResultsThe following table shows the total viable heterotrophic bacteria (CFU/ml) and

coliform bacteria MPN index/ 100 ml) in the hernolymph of 7: vittata, P. globosa and

I. exustus.

Snail Heterotrophic Coliform bacterial density

species bacterial density (MPN index / 100 ml)

(CFU / ml) Total coliform Faecal coliform

7: vittata 29.17 x 10 < 1100

< 1100

P. globosa 17.17 x 10 < 1100

< 1100

I. exustus < 1100

iL.I.]13.76 x 106

IV. 3. 1. Heterotrophic bacterial genera

IV. 3. 1. 1. Trachia vittata

In the hemolymph of 7:. vittata, six genera of heterotrophic bacteria was

identified viz., Bacillus, Micrococcus, Vibrio, Pseudom onas, A eromonas and Enterobacter.

Among these six genera, Vibrio showed the highest percentage of occurrence (25%) in

which five strains were isolated, followed by Pseudornonas (20%; 4 strains), Bacillus,

Aeromonas and Enterobacter (each 15%; 3 strains each) and Micrococcus (10%; 2 strains)

(Fig. IV. 1).

IV. 3. 1. 2. Pilaglobosa

In the henolymph of P. globosa five genera of heterotrophic bacteria were

identified viz., Bacillus, Vi brie, Pseitdomonas, Aeromonas and Enterohactor. Among these five

Percentage distribution of heterotrophic bacteriain the hemolymph of T vittata, P. g1obosa and 1. exustus

Fig. IV. 1.

Enterobacter sp.20%

Bacillus sp.20%

Trachia vittataEnterobacter sp. Bacillus sp.

15% 15%

Pila globosa

Pseudomo25%

Indoplanorbis exusrus

Aeromonas sp.5%

sp.'jtJ%

rseuaomonassp.

20%

Bacillus sp.15%Enterob acter sp.

20%

Aeromonas sp10%

Micrococcus sp.5%

to sp.

ococcus sp.10%

Aeromonas15%

Pseuciomonassp.

20%

V.25%

genera, Vibilo genus showed the highest percentage of occurrence (30%) in which 6

strains were isolated followed by Ps'udomonas (25%; 5 strains), Bacillus and Enterohactor

(each 20%, each 4 strains) and Aeromonas (5%; 1 strain) (Fig. IV. 1).

IV. 3. 1. 3. Indoplanorbis exustus

In the hemolvmph of I. exustus, six genera of heterotroph bacteria were

identified viz., Bacillus, Micro coccus, Vi&rio, Pseuc/ornonas, Aeromonas and Enterobacter.

Among these six genera, Vihrio genus showed the highest percentage of occurrence

(30%) in which 6 strains were isolated followed by Pseua'omonas and Erzterobacter (each

20%; each 4 strains) and Bacillus (15%; 3 strains), Aeromonas (10%; 2 strains) and

Micrococcus (5%; 1 strain) (Fig.

IV. 3. 2. Coliform bacterial genera

From the hemol ), mph of three gastropod molluscs, 5 coliform bacterial genera

were recorded viz., Escberichth co/l, Klebsiella, Enterobactor, Proteus and Citrobacter.

III. 3. 2. 1. Trachia vittata

In the hemolymph of T vittata, among these five coliform bacterial genera.

Eschericbia colt showed the highest percentage of occurrence (30%) in which 6 strains

were isolated followed by Klebsie/la (25%; 5 strains), Proteus (20%; 4 strains), Enterobacter

(15%; 3 strains) and Citrobacter (10%; 2 strains) (Fig. IV.2).

IV. 3.2.2. Pilaglobosa

In the hemolymph of P. globosa, the coliform bacteria genera of Eschericbia co/l'

and Klebsiella showed the highest percentage of occurrence (each 30%; each 6 strains)

followed by Proteus (20%; 4 strains), Citrol2acter (15%; 3 strains) and Enterobacter (5%, 1

strain) (Fig. IV.2).

Enteroba_.15%

Proteus sp.20%

Kiebsiella sp.25%

Fig. IV. 2. Percentage distribution of coliform bacteriain the hernolymph of T. villa/a, P. globosa and I. exwlz's

Trachia vittataCitrobacter sp.

10% E. co/i

Proteus sp.20%

Proteus sp25%

Pila globosaCitrobacter sp

15% E. co/i

Kiebsiella sp.30%

Indoplanorbis exusrusCitrobacter sp.

10%

Kiebsiella sp.20%

Enteroba.5%

EnteruL,aur, p.10%

co/i5%

IV. 3. 2. 3. Indoplanorbis

Among the 5 coliform bacterial genera identified in the hemolyrnph of!. exustus,

Escherichia coil recorded the maximum percentage of occurrence (35%; 7 strains)

followed by Proteus (25%; 5 strains), KIebsiella (20%; 4 strains) and Enterobacter and

Citrobacter (each 10%; 2 strains) (Fig. IV.2).

IV. 4. Discussion

Unlike mammals, healthy invertebrates might have bacteria in their body fluid

and tissues (Fancy, 1977; Stein etal., 1987). Disease may result from massive increases

in numbers of otherwise harmless bacteria, saprophytes or gut and surface-associated

non-pathogens, that proliferate in a compromised host or may result from changes in

environmental factors (Lauckner, 1983). The current report also revealed the

occurrence of different types of heterotrophic and coliform bacteria in the hemolymph

samples of three active snails T vittata, P. globosa and I. exustus from three different

natural habitats. Similarly Ueda etal. (1993) have detected heterotrophic bacteria in the

hemolymph of five crustacean species. Tubiash et al. (1975) isolated a large number of

bacteria from the hemolymph of healthy specimens of blue crab Callinectus sapidus.

Bacteria found in the hemolymph of the three gastropod species namely T vittata,

P. globosa and I. exustus were assumed to gain access through diet (Zo Bell and Felthum,

1938), integument and gut (Mc Henery and Birkbeck, 1985). Oubella et al., (1994)

suggested that mollusc - bacteria interactions occur in natural conditions since bacteria

constitute an important part of the microflora of the environments.

Marine bivalves -contain large number of microorganisms including Gram

negative and Gram positive bacteria and only minor variation in this flora were found

arnoii bivalves from different locations. In the present study the heterotrophic

bacterial density was high in the aquatic snail I. exustus and lesser in the terrestrial snail

T vittzta and less in the amphibious snail P. globosa. It may be because I. exustus are non-

operculate snails, inhabiting freshwater ponds Satyamurti, 1960) and feeds entirely

upon aquatic vegetation which may result in the increased bacterial density. Zo Bell and

Feltham (1938) supported this view and suggested that bacteria constitute a substantial

portion of the diet of marine filter feeders. Filter feeding may result in the concentration

of potential pathogens and, bacteria in large numbers may persist in bivalves without

causing diseases (Olafsen etal., 1993).

Vibrio and Pseua'ornonas spp. are normal constituents of the microflora of healthy

Oysters (Murchelano and Brown, 1968) even from different habitats. The current study

also revealed that the predominant species of bacteria found in the hemolymph of

T vittata, P. globosa and I. exustus were Vibrio spp. and Pseucloinonas spp. This findings are

in agreement with the results of Sizemore etal., (1975), Ortigosa et al., (1989) and Ueda

etal., (1993). The tendency to harbour indigenous Vibrio spp. is evident from the fact

that Vibrio spp. persist in shell fish hemolymph even after depuration in UV - treated

seawater (Eyles and IDavery, 1984; Greenberg etal., 1982), whereas Escherichia colt counts

are rapidly reduced under similar conditions (Olafsen et al., 1993). Molluscs can act as

vectors for the spread of Vibrios that are pathogenic for human (Colwell, 1984; Tamplin

et al., 1982; Tamplin and Capers, 1992) and this association could result from the

I existence of host lectins with the ability to bind vibrios (Fisher, 1992; Hardy etaL, 1977;

Olifsen et a/., 1992; Tamplin and Fisher, 1989). Apart from Escherichia coli, other

coliform bacteria K!eb5ie!1a, Enterobactor, Proteus and Citrobacter observed in the

hemolymph of the three snail species of the present study have not yet been observed in

gastropod molluscs, thus forming a pioneering record.

Healthy adult bivalves, apparently possess efficient defense mechanisms against

bacterial disease (Grimes et al., 1984). Invertebrate hemolymph and body parts contain

lectins that agglutinate various species of bacteria and may act in their defense by

facilitating opsonization and phagocytosis (Cheng etai., 1984; Olafsen, 1986; Olafsen,

1988; Renwrantz, 1986). The hemolh of Crassostrea gigas agglutinates marine Vibrio

and other bacteria (Hardy et al., 1977; Olafsen et A, 1992). Olafsen et al. (1992) found

that the hemolymph agglutinin activity of oysters increased after challenge with Vibrio

anguillarum in seawater. The defense processes could also complement the

hemolymphatic system and may act to limit bacterial proliferation (Paillard etal., 1994).

The present results demonstrate that bacteria are normally present in the body fluids of

healthy gastropods of T vittata, P. globosa and I. e.xustus. Since gastropods concentrate

bacteria, they thus could serve as a reservoir for pathogens (Malek and Cheng, 1974;

Suresh and Mohandas, 1990). The above characteristic feature indicates the possibility

of these gastropod snails acting as bioindicators of pathogenic organisms. Further, it is

suggested that the gastropod feed supplements for human and other consumption may

be an important source of food borne infections and endotoxin intoxications.