Isolation and phenotypic characterization of bacteriashodhganga.inflibnet.ac.in/bitstream/10603/62311/10/10_chapter 3.… · Isolation and phenotypic characterization of bacteria

Chapter – 3

Isolation and phenotypic characterization of bacteria

3. Isolation and phenotypic characterization of bacteria

3.1. Introduction

The CTCRI EPN collection have 67 live EPN isolates collected and

maintained alive by sub culturing them on laboratory reared Galleria mellonela

larvae. It is felt necessary to identify the bacteria in order to characterize them and

take up further studies.

3.2. Materials and methods

Materials

The kits used are listed in appendix (I). The culture media, buffers and

solutions used are listed in appendix (II). Chemicals and indicators used for the study

were listed in appendix (III). Sterile nuclease-free and protease free glass wares and

plastic wares were used for the preparation and storage of reagents and to carry out

experimental procedures.

Methods

Culture of insect and nematode

Greater wax moth (Galleria mellonella) was reared continuously in the

laboratory using artificial diet following Woodring and Kaya (1988). This diet was

made up of wheat flour (200 gm), corn flour (200 gm), milk powder (100 gm), yeast

(75 gm), honey (125 ml) and glycerin (125 ml). EPN isolates were raised

continuously on the laboratory reared Galleria mellonella larvae (Plate I Fig.3.1).

Bacterial strains

The bacteria were isolated from infective third stage dauer juveniles (Plate I

Fig.3.2) of the nematode isolates collected from different parts of India as listed in

Table 3.1.

Table 3 .1. Place of collection of entomopathogenic nematode isolates

Name of the Isolates Place of Collection

B Bangalore, Karnataka N Thiruvananthapuram, Kerala K Kasaragod, Kerala SI Namakkal, Tamil Nadu BEC Bangalore electronic city, Karnataka RRI Jorhat, Assam 532 Vellayani, Kerala NED Nedumangad, Kerala KAV Kavanad, Kollam, Kerala KO Kovvur, Andra Predesh M2 Madurai, Tamil Nadu DH Dhanbad, Jharkand Hy Hyderabad, Andrapradesh

Isolation of Bacteria from infective juveniles

Infective juveniles (IJs) of nematodes (30 nos.) were transferred to 2 ml

distilled water, treated with streptomycin (5000 units/ml) solution for one hour for

surface sterilization. The nematodes were triple rinsed in sterile distilled water

(Akhurst, 1980) and transferred into a micro tube having 2 ml nutrient broth (beef

extract 3 gm and peptone 5 gm in I litre of distilled water). It was then kept in a vortex

shaker for 24 hr. The solution was then streaked on to nutrient agar plates (Woodring

and Kaya, 1988) and kept at room temperature for 24 hr.

Isolation of Primary colonies of bacteria

Nutrient agar (NA) of 100 ml was prepared and 0.0025 gm Bromo Thymol

blue (BTB) was added and autoclaved at 121ºC for 15 min. Just before pouring the

medium Triphenyl Tetrazolium Chloride (TTC) (0.04 gm) was added. Bacteria were

streaked on the plates and incubated the plates at 37ºC. The isolates were examined

for the main phenotypic characteristics of the genus Xenorhabdus, using the methods

of Boemare and Akhurst (1988). The bacterial isolates were maintained on nutrient

agar slants at 5ºC.

Macro morphological tests

To check the possibility of the strains to grow on different media, mannitol

salt agar, pseudomonas agar, kligler iron agar, eosin-methylene blue agar, cetrimide

agar base, and MacConkey's agar plates were used. The cultivation conditions were

37ºC and pH=7.0. Based on these tests, size, colour, form, margin of the colony and

pigmentation were described. Length, width, area and perimeter of colonies were

taken after 24 hr growth on NA.

Micro morphological tests

Cell morphology was observed under a Zeiss Trinocular Research Microscope

(10,00 ×). 24 hr old cultures were used to describe shape of the cells and motility. The

strains were stained to check their response to Gram staining and possibility to form

spores and capsule.

Scanning electron microscopy

Scanning electron micrograph of bacterial isolate N was taken by the

following protocol

1) Bacterial pellets were fixed in 3% glutaralehyde in phosphate buffer (Sorensen

phosphate buffer, pH 7.2-7.4).

2) Washed the pellets with phosphate buffer, 15 minutes

3) Dehydration

a) 30% ethanol - 15 min, 2 changes

b) 50% ethanol - 15 min, 2 changes




4) Pellets were dried using Critical point dryer and covered in filter paper

5) Gold coating was done using Ion sputter unit

6) Viewed under SEM and image was taken

Biochemical and physiological characterization

All tests were conducted at 37°C. The cell size of each bacterium was

measured. Catalse activity was tested by National Standard Method BSOP TP 8.

Lecithinase was tested on egg yolk agar by the method of PML microbiologicals,

Technical data sheet # 320 Rev.2. Urease was tested on urea agar medium by National

Standard Method BSOP TP 36. Oxidase test was performed by using oxidase discs (as

per the directions of Himedia laboratories). Citrate utilization was checked by the

method of Biomerieux Technical data sheet # 695 Rev.3. Indole production test by

National Standard Method BSOP TP 19. MR-VP test by the method of PML

microbiologicals, Technical data sheet # 505 Rev.2. Hydrogen sulphide production

test with lead acetate paper strip, as per the directions of Himedia laboratories. Nitrate

reduction with nitrate reagent discs, twin pack, as per the directions of Himedia

laboratories. Decarboxylase activity with Moller decarboxylase broth with lysine

hydrochloride, Himedia Laboratories, Technical data. Litmus milk reaction was

carried out by PML microbiologicals, Technical data sheet # 455 Rev.2. MIO test

(Motility Indole Ornithine) was done by stabbing the MIO medium as a straight line

(Biomerieux Technical data sheet # 525 Rev.3). Siderophore production test by CAS-

agar universal test. The Cromeazurol (CAS) agar assay was described by Schwyn and

Neilands (1987) and was modified by Silva-Stenico et al. (2005). The phenyl alanine

deaminase activity was also tested on Phenyl alanine agar. Phenyl alanine agar is a

modification of the medium developed by Ewing et al. (1957). Beta-galactosidase

activitiy was tested using ONPG discs (as per the directions of Himedia laboratories).

Proteolysis was performed by the Frazier's method. 12 gm/liter of gelatin was added

to LB agar powder and the plates were streaked. After the prerequisite incubation,

those plates were flooded with a solution of 12 gm HgCl2; 16 ml of 12N HCl and 80

ml distilled water. The method of Boemare and Akhurst (1988) was used to test

lipolysis on Tween 20.

Lipase detection was done by preparation of chromogenic plates (Rajni et al.,

2006). The plates were prepared by using phenol red (0.01%) along with 1% lipidic

substrate (olive oil), 10 mM CaCl2 and 2% agar. The pH was adjusted to 7.3-7.4 by

using 0.1 N NaOH. Starch hydrolysis and casein hydrolysis were also carried out by

the method of Dr. Gary Kaiser (Biol 230, Lab Manual, Lab 8).

Acid and gas production by the bacterial species from various carbohydrates

such as 1% glucose, lactose, sucrose, starch, mannitol, maltose, cetrimide, sorbitol

and inulin were determined by visual observation of colour changes in 1% (w/v)

peptone water containing 0.0025% (w/v) bromo thymol blue (BTB) and 1% (w/v)

carbohydrate as described by Smibert and Krieg (1981). Because of the limitation of

BTB's sensitivity to pH changes, the pH level of the bacterial culture tubes were

measured also using a pH meter to confirm the production of acid.

Measurement of bacterial growth

The bacterial growths of twelve cultures were measured with

Spectrophotometer at a wavelength of 600 nm (A600). Broth culture of 5 ml was

aseptically transferred to 100 ml of nutrient broth. The initial OD at 600 nm was

determined. The inoculated culture flask was then placed in the shaker set at 120 × g

at 37°C for 24 hr. After 24 hr incubation, 5 ml of culture was aseptically transferred to

a cuvette. The optical density of the sample was determined at 600 nm. The steps 4

and 5 were repeated at each 24 hr interval for a period of 216 hr.

Effect of incubation temperature on bacterial growth

Sterile nutrient broth tubes (5 ml) were prepared and inoculated with 0.1 ml

each of twelve bacterial cultures. Incubated the tubes at different temperature viz.

0 ºC, 5ºC, 10ºC, 20ºC, 25ºC, 30ºC, 35ºC, 40ºC,45ºC, 50ºC and 55ºC. After 24 hr, the

optical densities of the inoculated cultures were determined at 600 nm.

Effect of pH on bacterial growth

Tubes containing sterile, buffered nutrient broth (5 ml) of pH 2.0, 2.5, 3.0, 3.5,

4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5 and 12.0

were inoculated by adding 0.1 ml of the culture. Incubated the tubes at 37ºC for 24 hr

and the OD of the inoculated cultures were determined at 600 nm.

Effect of salt concentration

Nutrient agar plates containing sodium chloride concentrations of 2%, 4%,

6%, 8% and 10% were prepared and inoculated with the twelve bacterial cultures by

making a single line loop inoculation. The plates were incubated at 37ºC for 24 hr.

3.3. Results

All bacterial isolates were characterized as Gram-negative (Plate I Fig.3.3),

motile, non-spore forming rods and catalase positive. Colonies formed on NA by most

of the symbiotic bacteria were creamy white, irregular with entire edge and had

smooth surface. Colonies of K and NED were convex, circular, smooth, mucoid and

glistening.

The length, width, area and perimeter of each cell showed variability

depending upon the strains and it is presented on Table 3.2. Pigmentation was not

observed in the case of all strains. The strain KO was dirty yellow in colour with

moist surface. Scanning electron micrograph of bacterial isolate B was shown in Plate

I Fig.3.4.

Table 3.3-3.5 showed the morphological, physiological and biochemical

characteristics of the twelve bacterial strains studied. Oxidase was negative for

isolates K, B, N, NED, BEC and SI. Oxidase delayed positive for RRI, 532 and DH.

Oxidase test was positive for KAV, KO and M2. All strains were negative for indole

and Voges-Proskauer reaction and positive for MR. The strains K, NED, BEC, SI,

DH, KAV, KO and M2 were positive for simmon's citrate test where as the strains B,

N, RRI and 532 were negative for the test. All strains were ONPG negative. Urease

test was positive for all strains. In TSI reaction isolate B, N and DH had shown

alkaline slant and acid butt. Alkaline slant, acid butt and H2S production were

observed for the isolates K, NED, 532, RRI, KAV and M2. Acid slant, acid butt and

H2S productions were observed for the isolates BEC and SI. Isolate KO had shown

the presence of acid slant and acid butt. The results of MIO test indicated that all

isolates were motile, indole negative and ornithine positive. Among the twelve strains

K, NED, BEC and SI were positive for nitrate reduction test. Except four strains (RRI,

532, DH and KO) others showed caseinase activity.

Starch hydrolytic activity was positive for the strains B, N, K, BEC, NED, SI

and KO. Gelatinase activity was shown by strains K, RRI, NED, 532, DH, KAV and

M2. All strains were positive for egg yolk reaction. With the exception of DH, all

other isolates were lipase producers. Litmus milk reaction was positive for all strains.

Siderophore productions were positive for BEC, SI, DH, KAV and M2.

The strains BEC, SI, KAV and M2 were positive for phenyl alanine. Strains B, N, DH

and KO were negative for hydrogen sulphide production test. The strains BEC, SI,

DH, KAV and M2 were capable of decarboxylate L-lysine hydrochloride. All strains

showed colour changes in litmus milk.

All strains grew on mannitol salt agar, pseudomonas agar, kligler iron agar,

eosin-methylene blue agar, cetrimide agar base, and Mac Conkey's agar. The results

of gas and acid production by the 12 bacterial isolates from various carbohydrates are

shown in Table 3.6 and 3.7. The optimum temperature for the isolates SI and BEC

were 25°C (Fig.3.5-3.16). The optimum pH was found to be 7.5 and 8. Results of

optimum temperature and pH are shown in (Fig.3.17-3.28). Bacterial isolates 532 and

KO showed maximum growth at 48 hr, all other isolates showed maximum growth at

24 hr (Fig.3.29-3.40). The study of optimum temperature for the 12 isolates indicated

that except two isolates the optimum temperature was 35°C. Salt concentration for

optimum growth was 2-6%; very slight growth was recorded at 8% and no growth

was observed at 10% salt concentration.

Table 3.2. Measurement of 12 bacterial strains

Bacterial strains Length Width Area Perimeter

B 3.74±0.46 1.05±0.02 8.44±0.34 3.96±0.21

N 3.61±0.16 1.04±0.05 8.04±1.07 3.75±0.58

K 3.49±0.40 1.02±0.02 8.09±0.86 3.83±0.97

SI 3.17±0.28 1.05±0.01 7.77±0.58 3.32±0.32

BEC 3.25±0.30 1.07±0.03 8.07±0.69 3.45±0.39

RRI 4.57±0.60 1.19±0.05 10.34±1.35 4.62±0.72

532 4.70±0.64 1.23±0.09 11.20±1.46 4.74±0.88

NED 3.43±0.39 1.07±0.03 7.98±0.83 3.79±0.32

KAV 4.73±0.55 1.30±0.06 11.17±1.43 5.20±0.90

KO 5.01±0.82 1.02±0.01 11.82 ±1.52 5.42±0.5

M2 4.68±0.44 1.26±0.04 10.96±1.39 5.12±0.83

DH 3.95±0.71 1.21±0.10 9.67±1.65 4.22±0.87

Table 3.3. Colony morphology of bacteria on Nutrient agar

Pigment production

Optical character

Elevation

Edge Form Surface Growth Colony colour Bacterial

strains

Negative Translucent Flat Entire Irregular Smooth Rapid Creamy white B

Negative Translucent Flat Entire Irregular Smooth Rapid Creamy white N

Negative Translucent Convex Entire Circular Smooth, Mucoid,

Glistening

Rapid Creamy white K

Negative Translucent Flat Entire Irregular Smooth Rapid Creamy white SI

Negative Translucent Flat Entire Irregular Smooth Rapid Creamy white BEC

Negative Translucent Flat Entire Irregular Smooth Rapid Creamy white RRI

Negative Translucent Flat Entire Irregular Smooth Rapid Creamy white 532

Negative Translucent Convex Entire Circular Smooth, Mucoid,

Glistening

Rapid Creamy white NED

Negative Translucent Raised Entire Irregular Smooth Moderate Creamy white KAV

Negative Translucent Flat Entire Irregular Moist Rapid Dirty yellow KO

Negative Translucent Raised Entire Irregular Smooth Moderate Creamy white M2

Negative Translucent Flat Entire Irregular Smooth Rapid Creamy white DH

Table 3.4. Colony morphology of bacteria on Nutrient broth

Sediment Clouding Surface growthBacterial strains

Scanty Slight Ring B

Scanty Slight Ring N

Flaky Heavy Ring K

Scanty Slight Pellicle SI

Scanty Slight Pellicle BEC

Scanty Slight Ring RRI

Scanty Slight Ring 532

Flaky Heavy Ring NED

Flaky Heavy Ring KAV

Flaky Heavy Ring KO

Flaky Heavy Ring M2

Flaky Heavy Ring DH

Table 3.5. Biochemical characteristics of 12 bacterial isolates

Characteristics B N K RRI BEC NED SI 532 DH KAV KO M2

Catalase + + + + + + + + + + + + Oxidase - - - D+ - - - D+ D+ + + + Indole - - - - - - - - - - - - MR + + + + + + + + + + + + VP - - - - - - - - - - - -

Citrate - - + - + + + - + + + + ONPG - - - - - - - - - - - -

Motility + + + + + + + + + + + + Indole - - - - - - - - - - - -

Ornithine + + + + + + + + + + + + Urease + + + + + + + + + + + +

Nitrate reduction - - + - + + + - - - - - Casein hydrolysis + + + - + + + - - + - + Starch hydrolysis + + + - + + + - - - + - Gelatin hydrolysis - - + + - + - + + + - +

TSI A A A H2S AH2S Ac H2S A H2S Ac H2S A H2S A A H2S Ac A H2S A – Alakaline slant acid butt A H2S - Alakaline slant acid butt H2S production

Ac H2S – Acid slant acid butt H2S production Ac- Acid slant acid butt D+ - Delayed positive

Table 3.5. Continued

M2 KO KAV DH 532 SI NED BEC RRI K N B Characteristics

+ + + + + + + + + + + + Egg yolk Reaction

+ + + - + + + + + + + + Lipid hydrolysis

Alkaline

reaction at surfac

e

Coagulation and litmus

reduction

Alkaline

reaction at

surface

Acid with

reduction and curd

Coagulation and litmus reduction

Alkaline reaction

Alkaline reaction

Alkaline reaction

Coagulation and litmus reduction

Alkaline reaction

Acid production

curd formation and gas

Acid production

curd formation and gas

Litmus milk reaction

+ - + + - + - + - - - - Siderphore producton

+ - + - - + - + - - - - Phenyl alanine deaminase reaction

+ - + - + + + + + + - - Hydrogen sulphide production

+ - + + - + - + - - - - Decarboxylation of L-Lysine

hydrochloride

Table 3.6. Gas production from carbohydrates

DH M2 KO KAV NED 532RRI BECSI K N B Carbohydrates

+ - + - + - - - - + - - Glucose

- - - - + - - - - + - - Lactose

+ - + - + - - - - + - - Sucrose

- - - - + - - - - + - - Starch

+ - + - + - - - - + - - Mannitol

+ - + - + - - - - + - - Maltose

- - - - - - - - - - - - Cetrimide

+ - - - - - - - - - - - Sorbitol

- - - - - - - - - - - - Inulin

Table 3.7. Acid production from carbohydrates

DHM2KO KAV NED532RRI BECSI K N B Carbohydrates

+ + + + + + + + + + + + Glucose

- - + - + - - + + + + + Lactose

+ + + + + - - + + + - - Sucrose

- + + + + - - - - + - - Starch

+ - + - + - - + + + - - Mannitol

+ + + + + + + + + + + + Maltose

- - - - - - - - - - - - Cetrimide

+ - - - - - - - - - - - Sorbitol

- - - - - - - - - - - - Inulin

Fig.3.5-3.16. Effect of incubation temperature on bacterial growth

Fig.3.5. Isolate B Fig.3.6. Isolate N

Fig.3.7. Isolate K Fig.3.8. Isolate SI

Fig.3.9. Isolate BEC Fig.3.10. Isolate RRI

Fig.3.11. Isolate 532 Fig.3.12. Isolate NED

Fig.3.13. Isolate KAV Fig.3.14. Isolate KO

Fig.3.15. Isolate M2 Fig.3.16. Isolate DH

Fig.3.17-3.28. Effect of pH on bacterial growth







Fig.3.29-3.40. Bacterial growth by turbidity measurements (spectrophotometric

method).







3.4. Discussion

Dutky (1937) and Bovien (1937) were the first to point out the association

between nematode parasite of insects and their specific bacteria. We compared the

phenotypic characteristics of 12 bacterial strains isolated from the infective juveniles

of nematode isolates collected from different parts of India. Evaluation of the present

study showed that all of these bacterial strains were Gram-negative motile rods which

do not produce indole and are catalase positive.

In the present study, none of the strains has produced acid from inulin and

cetrimide. All strains produced acid from glucose and maltose. The strains B, N, RRI,

BEC, SI, 532, KAV and M2 do not produce gas from glucose, lactose, sucrose,

starch, mannitol, maltose, cetrimide, sorbitol and inulin. Moller (1955) was probably

the first to develop a practical amino acid decarboxylase test for the identification of

bacteria. This test has been used primarly in the identifiaton of the

enterobacteriaceae.

In the present study, five strains viz. BEC, SI, DH, KAV and M2 were

capable of decarboxylating L-Lysine hydrochloride. Phenyl alanine deaminase

activity was showed by the isolates BEC, SI, KAV and M2. However, positive

reactions for this test were weak and appeared 15 to 30 sec after the addition of ferric

chloride as a green colour at the periphery of the growth on phenyl alanine agar. This

green colour was best seen by viewing the agar from the side.

Lipase activity was tested with fresh egg yolk emulsion rather than with egg

yolk extract. Another method of lipase detection used i.e. by preparation of

chromogenic plates. It is a very simple, highly efficient, reliable and rapid method for

qualitative detection of lipases on plate. Lipase detection by chromogenic plate

method indicated a colour change from pink to yellow. Phenol red had an end point at

pH 7.3-7.4 where it was pink, and a slight decrease in pH (7.0-7.1) turned it yellow.

The chromogenic substrate was kept at pH 7.3. As soon as hydrolysis initiated, the

dye became yellow, indicating lipolysis. Within 10 min the activity of lipolase on

olive oil could be observed.

Lecithinases are bacterial virulence determinants which have known roles as

toxins or cytolysins, as well as a means of securing supplies of phosphates. In

Xenorhabdus spp. it is not known whether proteins such as lecithinase, which are

subject to phase variation, have a role in the virulence for the insect host, or, play a

role in the association of Xenorhabdus with the symbiotic nematode vector.

Many Xenorhabdus species produce extra cellular enzymes like proteases,

lipases, phospholipases and DNAases are involved in breaking down insect tissues to

provide nutrients for both the nematode and bacterial symbionts (Forst and Nealson,

1996). Schmidt et al. (1988) purified an alkaline metalloprotease from Photorhabdus

luminescens culture broth and inferred that proteases might have a role in insect

toxicity via analogy with proteases produced by other insect pathogens.

The fact that the effect of temperature on bacterial growth occurs sporadically

and is somewhat transient within any given group of cultures does not simplify the

taxonomic problem. The test results of effect of incubation temperature on bacterial

growth have indicated that within the range of 25 to 35°C the bacterial strains showed

maximum growth. Many laboratories employ incubators in this range for routine

bacteriological analysis of milk and other food products and it would appear to be a

logical procedure to carry out any taxonomic study on the organisms at the

temperature at which they are initially isolated.

One of the first published discussions of bacteria associated with

entomopathogenic nematodes appeared in 1959 (Dutky 1959), although the bacteria

were neither fully characterized nor named. Most of the studies of symbiotic bacteria

associated with EPN already reported are about Xenorhabdus and Photorhabdus. The

identification of the bacteria is performed traditionally by isolating the organism and

studying it phenotypically by means of Gram staining, culture and biochemical

methods, which have been the gold standard of bacterial identification. However,

these methods of bacterial identification have drawbacks such as they can not be used

for non-cultivable organisms and are occasionally faced with organisms with

biochemical characteristics that do not fit into patterns of any known genus and

species.

3.5. Conclusion

This study provides the first characterization of symbiotic bacteria from

entomopathogenic nematode Rhabditis sp. Each nematode isolate is associated with a

distinct bacterial isolate belonging to different species or subspecies. This research

results strengthen the hypothesis of a strong specificity in the symbiotic interactions

between the rhabditid EPN and bacteria. Comparison of biochemical tests of the

newly isolated twelve bacterial strains from EPN isolates does not fit the

characteristics of Xenorhabdus and Photorhabdus. Since the discovery of the

polymerase chain reaction (PCR) and DNA sequencing of these bacteria provide an

alternative method in the identification of these new bacterial strains.

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Isolation and phenotypic characterization of bacteriashodhganga.inflibnet.ac.in/bitstream/10603/62311/10/10_chapter 3.… · Isolation and phenotypic characterization of bacteria