MATERIAL AND

METHODS

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3.1. Culture media, microbial strains and cell lines used in the study:

All the chemicals and reagents used in the present study, were of analytical

grade and procured from Amersham Biosciences, Glaxo India Ltd., Himedia, Merck,

Qualigens Fine Chemicals, Ranbaxy Labs Ltd., SD Fine Chemicals, Sigma Aldrich,

Sisco Research Labs. Pvt. Ltd., Unisons Pharma, Baddi etc.

3.1.1. Preparation of culture media:

3.1.1.1. MRS medium: Medium is based on the formulations of de Man, Rogosa and

Sharpe (MRS) (Table 3.1). This medium supports luxuriant growth of lactobacilli

from oral, fecal, dairy, and other sources (de Man et al., 1960).

Table 3.1: Composition of MRS Medium

Ingredients Quantity (g/l) Dextrose 20 Proteose peptone 10 Yeast extract 5 Sodium acetate 5 2-Phenylethyl alcohol 3 Ammonium citrate 2 Dipotassium phosphate 2 Magnesium sulphate 0.10 Manganese sulphate 0.05 Bromocresol green 0.040 Cycloheximide 0.004 pH 6.5± 0.2

4.72 g MRS media was reconstituted in 100 ml deionized water. 100 µl Tween

80 was added and pH of the solution was adjusted at 6.5 with help of 1N NaOH

solution. Media was sterilized at 15 lbs for 20 min.

3.1.1.2. Casman Medium: Casman Broth with blood is used for isolation of

fastidious microorganisms from clinical specimens under reduced oxygen tension

(Casman, 1947). Composition of Casman Medium used for the cultivation of G.

vaginalis ATCC 14018 is given in table 3.2a.

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Table 3.2a: Composition of Casman’s Medium

Ingredients Quantity (g/l) Proteose peptone 10.00 Tryptose 10.00 Sodium chloride 5.00 Beef extract 3.00 Dextrose 0.500 Corn starch 1.00 Nicotinamide 0.05 p-amino benzoic acid(PABA) 0.05 Gardnerella vaginalis selective supplement 1ml Defibrinated blood 5% v/v

2.96 g Casman Media was reconstituted in 100 ml deionized water and was

heated to boiling temperature to dissolve the medium completely. Media was

sterilized by autoclaving at 15 lbs pressure (121°C) for 15 minutes. It was cooled to

50°C and 5% defibrinated blood of 5% sterile blood was added aseptically.

G. vaginalis ATCC 14018 Selective Supplement: An antibiotic supplement is

recommended for the selective isolation of Gardnerella vaginalis which consists of

gentamycin sulphate, nalidixic acid and amphotericin B (Table 3.2b).

Table 3.2b: Antibiotic selective supplement

Antibiotic selective supplement Quantity (mg/l)

Gentamycin sulphate 2.0 Nalidixic acid 15.0 Amphotericin B 1.0

Contents of 1 vial were rehydrated aseptically with 2 ml of sterile distilled

water. 500 ml of sterile, molten, cooled (45-50°C) Casman’s Media with 5%

defibrinated blood was aseptically added it to.

3.1.1.3. Nutrient Agar: This is an undefined medium used for the general cultivation

and maintenance of bacteria kept in laboratory culture collection. Nutrient media

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contain all the elements that most bacteria need for growth and are non-selective.

Composition of Nutrient Agar is given in table 3.3.

Table 3.3: Composition of Nutrient Agar

Ingredients Quantity (g/l) Peptic digest of animal tissue 5.0 Sodium chloride 5.0 Beef extract 1.5 Yeast extract 1.5 Agar 15 pH 7.0 ± 0.2

1.3 g of Nutrient agar was added to 100ml of distilled water. pH was adjusted

to 7.0 with 1N NaOH solution. Media was sterilized by autoclaving at 121ºC at 15 psi

for 20 min.

3.1.1.4. Brain Heart Infusion Medium: Brain Heart Infusion Broth is employed for

the propagation of fastidious pathogenic cocci and other organisms associated with

blood culture work and allied pathological investigations (Dowell et al., 1974).

Composition of Brain Heart Infusion Medium is given in table 3.4.

Table 3.4: Composition of Brain Heart Infusion Medium

Ingredients Quantity (g/l) Calf Brains, Infusion from 200 Beef Heart, Infusion from 250 Proteose Peptone 10.0 Dextrose 2.0 Sodium Chloride 5.0 Disodium Phosphate 2.5 pH 7.4 ± 0.2

3.76 g of media was added to 100ml of distilled water and pH was adjusted to

7.0 with 1N NaOH solution. Media was sterilized by autoclaving at 121ºC at 15 psi

pressure for 20 min. For best results, the medium should be used on the day it was

prepared, otherwise, it should be boiled or steamed for a few minutes and then cooled

before use.

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3.1.1.5. Thayer Martin Media: Thayer Martin Agar is a selective and enriched

medium for the isolation and cultivation of Neisseria sp. from mixed flora (Thayer et

al., 1964). Composition of Thayer Martin Media is given in table 3.5.

Table 3.5: Composition of Thayer Martin Media

Ingredients Quantity (g/l) Peptic digest of animal tissue 23.0 Hemoglobin, Bovine 10.0 Starch 10.0 Sodium Chloride 5.0 Dextrose 2.0 Agar 20.000 FD021 (GC ANTIBIOTICS SUPPLEMENT)

1 vial

Final pH 7.4±0.2

5.15 g of Thayer Martin Media was reconstituted in 90 ml distilled water.

Media was sterilized by autoclaving at 121ºC at 15 psi pressure for 20 min. Media

was cooled to 45°C. 200 µl of Antibiotics GC supplement FD021-5VL containing

vancomycin, colistin and nystatin was aseptically added to the media. GC supplement

FD021-5VL is recommended for the selective isolation and cultivation of pathogenic

Neisseria.

3.1.1.6. Cooked Meat Media: Cooked Meat Medium is used for cultivation of

aerobes and anaerobes, especially pathogenic Clostridium (MacFaddin et al., 1985).

Composition of Cooked Meat Media is given in table 3.6.

Table 3.6: Composition of Cooked Meat Media

Ingredients Quantity (g/l)

Beef Heart 98.0

Proteose Peptone 20.0

Sodium Chloride 5.0

Dextrose 2.0 Final pH ( at 25°C) 7.2±0.2

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12.5 g of media was added to 100ml of distilled water and mixed thoroughly.

It was allowed to stand for 15 min until all the particles were thoroughly wetted.

Media was sterilized by autoclaving at 121ºC at 15 psi pressure for 20 min.

3.1.1.7. Yeast Extract Peptone Dextrose Media: Yeast Extract Peptone Dextrose

Media is used for cultivation of fungal sp. especially Candida (Shermann, 1991).

Composition of Yeast Extract Peptone Dextrose Media is given in table 3.7.

Table 3.7: Composition of Yeast Extract Peptone Dextrose Media

Ingredients Quantity (g/l) Bactopeptone 20 Dextrose 20 Yeast extract 10 Uracil 0.1 Adenine 0.1 Tryptophan 0.1

Bacto-agar 20

5.5 g of media was added to 100ml of distilled water and mixed thoroughly. It

was allowed to stand for 15 minutes until all the particles were thoroughly hydrated.

Media was sterilized by autoclaving at 121ºC at 15 psi pressure for 20 min.

3.1.1.8. Mac-Conkey Broth: Mac-Conkey Broth is recommended for the selective

enrichment and enumeration of coliforms (McCrady, 1939). It is a differential

medium recommended for the selective isolation and differentiation of lactose

fermenting and lactose non-fermenting enteric bacilli. Composition of Mac-Conkey

Broth is given in table 3.8.

Table 3.8: Composition of Mac-Conkey Broth

Ingredients Quantity (g/l) Peptone 17 Lactose 10 Bile salts 1.5 Sodium chloride 0.03 Neutral red 0.001 pH 7.4± 0.2

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5.15 g of Mac-Conkey medium was added in 100 ml distilled water and pH

was adjusted. Media was sterilized by autoclaving at 121ºC at 15 psi pressure for 20

min.

3.1.1.9. Reinforced Clostridial Broth: It is used to initiate growth from small inocula

and to obtain the highest viable count of Clostridium. Hirsch and others used the broth

medium for diluting an inoculum of vegetative cells of Clostridium perfringens and

other spore forming anaerobes from cheese (Hirsch et al., 1954). This is a

nonselective enrichment media. Composition of Reinforced Clostridial Broth is given

in table 3.9.

Table 3.9: Composition of Reinforced Clostridial Broth

Ingredients Quantity (g/l)

Pancreatic Digest of Casein 5

Proteose Peptone No. 3 5 Beef Extract 10 Yeast Extract 3

Dextrose 5

Sodium chloride 5 Soluble Starch 1 Cysteine Hydrochloride 0.5

Sodium Acetate 3.0

Agar 0.5 pH 6.5±0.2

3.8 g of Mac-Conkey medium was added in 100 ml distilled water. Media was

sterilized by autoclaving at 121ºC at 15 psi pressure for 20 min.

3.1.1.10. Tryptone Glucose Yeast Extract Agar: Tryptone Glucose Yeast Extract

Agar is recommended for the isolation and enumeration of bacteria (Kaur et al.,

2012). Composition of Tryptone Glucose Yeast Extract Agar is given in table 3.10.

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Table 3.10: Composition of Tryptone Glucose Yeast Extract Agar

Ingredients Quantity (g/l) Tryptone 10 Glucose 10 Yeast Extract 10 MgSO4 0.05 MnSO4 0.05 Agar 0.75 pH 6.5+0.2

4.1g of Mac-Conkey medium was added in 100 ml distilled water pH was

adjusted to 6.5. Media was sterilized by autoclaving at 121ºC at 15 psi pressure for 20

min.

3.1.2. Procurement and maintenance of culture: Microbial cultures were procured

from various culture collection centers such as American Type Culture Collection

(ATCC); National Collection of Industrial Microorganisms (NCIM), Pune; National

Collection of Type Cultures (NCTC) England (UK) and Microbial Type Culture

Collection (MTCC), IMTECH, Chandigarh and some cultures were gifted by Orbit

Biotech, Mohali. Indicator microorganisms were revived and maintained in

recommended media at growth conditions specified by various culture banks given in

table 3.11.

Table 3.11: Growth media and conditions of indicator microorganisms

Microorganism Gram Nature

Growth Medium

Nature Temp. (oC)/pH

Incubation time

BV Associated Pathogens Bacteriodes fragilis MTCC 1045 -ve RCB Anaerobe 37/6.8 5 days B. ovatus MTCC 3298 -ve RCB Anaerobe 37/6.8 48h B. vulgates MTCC 1350 -ve CMM Anaerobe 37/7.2 72h Candida albicans ATCC 10231 Yeast YEPD Aerobe 30/7.2 48h C. albicans MTCC 183 Yeast YEPD Aerobe 30/7.2 48h Gardnerella vaginalis ATCC 14018 +ve CB Anaerobe 37/7.2 48h Micrococcus flavus ATCC 10240 +ve NB Aerobe 30/7.4 48h Neisseria gonorrhoeae ATCC 19424 -ve NB Aerobe 37/7.4 24h N. mucosa MTCC 1772 -ve NB Aerobe 37/7.4 24h Proteus mirabilis NCIM 2387 -ve NB F. anaerobe* 37/7.2 24h Staphylococcus albus ATCC 11631 +ve BHI Anaerobe 25/7.4 5 days S. aureus MTCC 737 +ve BHI Anaerobe 37/7.4 24h

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S. aureus NCTC 7447 +ve BHI Anaerobe 37/7.4 24h Streptococcus agalactiae NCIM 2401 +ve MRS Aerobe 37/6.5 48h S. faecalis MTCC 459 +ve MRS Aerobe 37/6.5 48h S. pyogenes NCTC 10869 +ve BHI Aerobe 37/7.4 48h S. thermophilus MTCC 1928 +ve MRS Aerobe 40/6.5 28h General Human Pathogens Bacillus subtilis ATCC 6633 +ve NB Aerobe 37/7.4 24h Clostridium perfringens MTCC 450 +ve RCB Anaerobe 37/6.8 48h Escherichia coli BL21 DE3 MTCC 1679

-ve NB F. anaerobe 37/7.2 24h

E. coli DH5α MTCC 1652 -ve NB F. anaerobe 37/7.2 24h E. coli KL16 MTCC 1650 -ve NB F. anaerobe 37/7.2 24h Enterococcus faecalis (Lab isolate) +ve MRS F. anaerobe 37/6.5 24h E. faecalis (NDRI isolate) +ve MRS F. anaerobe 37/6.5 24h E. faecalis ATCC 29212 +ve MRS F. anaerobe 37/6.5 24h Klebsiella pneumoniae NCIM 2883 -ve NB F. anaerobe 30/7.2 24h K. pneumoniae NCIM 2401 -ve NB F. anaerobe 30/7.2 24h Leuconostoc mesenteroides MTCC 107

+ve MRS Aerobe 25/6.5 48h

Listeria monocytogenes MTCC 657 +ve BHI Aerobe 37/7.4 24h Pseudomonas aeruginosa ATCC 10662

+ve NB Aerobe 30/7.4 24h

Salmonella typhi NCTC 5760 -ve BHI F. anaerobe 37/7.4 24h Vibrio cholera ATCC 14104 -ve NB Aerobe 37/7.4 24h Yersinia enterocolitica MTCC 861 -ve BHI F.anaerobe 30/7.2 12h Non-Pathogenic Microorganisms Lactobacillus brevis MTCC 1750 +ve MRS F. anaerobe 30/7.4 24h L. bulgaricus NCDC 253 +ve MRS F. anaerobe 37/6.5 24h L. casei NCIM 2651 +ve MRS F.anaerobe 37/6.5 24h L. helveticus NCIM 2126 +ve MRS F.anaerobe 37/6.5 24h L. leichmanni NCIM 2027 +ve MRS F. anaerobe 37/6.5 24h L. pentosus NCIM 2669 +ve MRS F. anaerobe 37/6.5 24h L. plantarum NCIM 2912 +ve MRS F. anaerobe 37/6.5 24h Lactococcus lactis subsp. cremoris MTCC 1484

+ve MRS Aerobe 20/6.5 24h

Pediococcus acidilactici LB 42 +ve MRS Aerobe 30/6.5 24h F. anaerobe*- Facultative anaerobe

3.1.3. Procurement and maintenance of cancerous cell lines: Cell lines were

procured from NCIM, Pune and few of them were gifted by Dr. Sanyog Jain, NIPER,

Mohali. Cell lines were revived and maintained in recommended media at growth

conditions specified by various culture banks given in table 3.12.

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Table 3.12: Growth media for cell lines

S. No.

Cell Line Growth media Atmosphere Temperature

1. HEP G2 CRL-10741

1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 medium with 2.5 mM L-glutamine, 15 mM HEPES, 0.5 mM sodium pyruvate and 1200 mg/l sodium bicarbonate and supplemented with 0.4 mg/ml G418, 90%; fetal bovine serum, 10%

5% carbon dioxide (CO2)

37.0°C

2. HEK 293 CRL-1573

The base medium (RPMI 1640); 4mM L-glutamine

5% carbon dioxide (CO2)

37.0°C

3. MCF7 (HTB-22)

Eagle's Minimum Essential Medium, 0.01 mg/ml bovine insulin; fetal bovine serum to a final concentration of 10%

5% carbon dioxide (CO2)

37.0°C

4. Sp2/0-Ag14 (CRL-1581)

Dulbecco's Modified Eagle's Medium, fetal bovine serum to a final concentration of 10%

5% carbon dioxide (CO2)

37.0°C

5. HeLa (CCL-2) Eagle's Minimum Essential Medium, fetal bovine serum to a final concentration of 10%

5% carbon dioxide (CO2)

37.0°C

3.2. Isolation of bacteriocin producing endogenous species of vaginal LAB strain

which inhibits the growth of Gardnerella vaginalis.

3.2.1. Collection of vaginal swabs:

Samples used in the present study includes vaginal swabs from healthy and

fecund females of reproductive age group (20 to 45 years) which were collected with

informed consent (annexure I). 100 samples were collected from Darshanjot Private

Nursing Home, Phase X, Mohali and Gynecology Department, Government Medical

College and Hospital, Sector 32, Chandigarh. Samples were collected by the

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Gynecologist using sterilized ear buds and transferred immediately to sterilized

normal saline (NaCl). Guidelines of Institutional Ethical Committee (IEC) vide

reference no. IEC/IRB N: 005 were strictly followed during sample collection.

3.2.2. Cultivation of LAB microflora of the samples:

LAB microflora present in vaginal samples were cultivated in MRS medium

pH 6.5 at 37ºC for 24 hours. Appearance of yellow color in broth indicates presence

of acid producing bacterial strains. Samples were subcultured thrice before

proceeding with bacterial isolation and bacteriocin activity assays. LAB strains were

isolated on MRS agar plates using pure culture techniques (Old et al., 1970).

3.2.3. Isolation of the bacteriocin producing LAB:

Pure cultures were isolated from the above isolates by quadrant streaking on

MRS agar plates (Old et al., 1970). Isolates were purified by repeated streaking and

were used for screening of bacteriocin producing LAB strains.

3.2.4. Bacteriocin activity assays:

Bacteriocinogenic activity of vaginal LAB isolates was assayed using

following methods:

3.2.4.1. Agar-Spot Assay: Agar-spot assay was carried out by with little

modifications was followed to assay the Bac+ activity of the isolated cultures (Sarkar

and Banerjee, 1996). Plates of MRS agar (1% w/v agar) were allowed to set

overnight, spotted with 18h old producing cultures (3-5 spots per plate) and incubated

at 37°C for 24h. TGE supplemented with 0.75% (w/v) agar termed TGE soft agar,

was tempered to 45°C and seeded with 18h old 105-106 cfu/ml indicator strain. The

number of cells was determined, using haemocytometer and microscope under 45X

magnification. The spotted plates were heat treated and overlaid with 4-6 ml of the

seeded TGE soft agar, incubated at 30°C for 24h and observed for inhibition zones.

Inhibition was scored positive, if the width of the clear zone around the colonies of

the producing strain was 0.5 mm or larger with indicator bacteria used in study.

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3.2.4.2. Agar Well Diffusion Assay: Bacteriocin activity assay associated with the

isolated cultures was done in accordance to the standard protocol with little

modifications (Toba et al., 1991). Isolated cultures, with positive results in agar-spot

test against the respective indicator bacteria, were tested for the antibacterial activity

in neutralized cell free supernatants (CFS) by well diffusion assay. CFS of 24h old

culture was neutralized to pH 6.5 with 1N NaOH and heat treated in boiling water

bath for 5 min. MRS bottom agar plates were overlaid with TGE soft agar seeded with

18h old culture with 105-106 cfu/ml indicator bacteria. Wells of 6mm diameter cut

with sterile cork borer were filled with 50µl of the CFS. Plates were kept at 4°C for

1h to allow diffusion of CFS out of the wells. Plates were then incubated at 37°C for

24h and the antimicrobial activity of supernatant was detected and quantitated by

measuring the width of the clear zones around the wells in the resultant lawn culture.

3.2.4.3. Spot-on-Lawn Assay: Spot-on lawn assay was carried out by spotting 5µl of

CFS and dilutions prepared on MRS 1% (w/v) bottom agar and overlaid with TGE

0.75% (w/v) soft agar pre-seeded with approximately 106 cfu/ml of P. acidilactici

LB42 indicator strain. Assay plates were incubated at 37°C for 24h. The titre was

expressed as the reciprocal of the highest dilution showing a definite zone of

inhibition/lysis in the resultant lawn culture (Pucci et al., 1988). Titre multiplied with

dilution factor i.e. 200, so as to get total activity units per ml of the sample.

3.2.4.4. Disc diffusion assay: 1% selective media agar plates were prepared and

mixed with 30µl of 24 hour old indicator culture in 0.75% soft agar. Discs of

approximately 6mm diameter were placed on agar plates and spotted with 5µl of CFS

or purified bacteriocin or antibiotic suspension in various assays. Plates were

incubated at 37°C for 24 h and the inhibition zones around the discs were observed

(Jorgensen et al., 2007).

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3.3. Study of antibiotic susceptibility of the vaginal LAB isolates:

Antibiotic susceptibility of human vaginal LAB isolates was studied at

different concentrations of some commonly prescribed antibiotics (2 to 8 µg/ml) to

BV patients. Ampicillin, amoxicillin, amoxicillin and clavalanic acid, azithromycin,

ciprofloxacin, co-trimoxazole, erythromycin, gentamycin, metronidazole, nalidixic

acid, ofloxacin, penicillin, rimphicin, tetracycline, tinidazole and vancomycin

susceptibility of LAB isolates was evaluated by agar well diffusion (Toba et al., 1991)

and disc diffusion methods (Jorgensen et al., 2007; Kaur et al., 2012).

MRS bottom agar plates were pre-seeded with 18h old 105-106 cfu/ml

indicator bacteria. Wells of 6mm diameter cut with sterile cork borer were filled with

100µl of antibiotic suspension made in the range from 2 to 8 µg/ml. Plates were kept

at 4°C for 1h to allow diffusion of antibiotics out of the wells and were then incubated

at 30°C for 24h. The antibiotic sensitivity activity of isolates was detected and

quantitated by measuring the width of the clear zones around the wells in the resultant

lawn culture. Similarly, disc diffusion assay was performed with different

concentrations of antibiotics.

3.4. Study of Antimicrobial range of bacteriocin produced by LAB isolates:

Antimicrobial activity of bacteriocins produced by LAB isolates was

characterized using spot-on-lawn and well diffusion method (Pucci et al., 1988; Toba

et al., 1991). Microorganisms were divided into two categories i.e general human

pathogens and non-pathogens (table 3.11). General human pathogens used in the

study in the were Bacillus subtilis ATCC6633, Clostridium perfringens MTCC450,

Escherichia coli BL21 (DE3) MTCC1679, MTCC1652 and MTCC1650,

Enterococcus faecalis (Lab isolate), ATCC29212, Klebsiella pneumoniae NCIM2883,

NCIM2401, Leuconostoc mesenteroides MTCC107, Listeria monocytogenes

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MTCC657, Pseudomonas aeruginosa ATCC10662, Salmonella typhi NCTC5760,

Vibrio cholera ATCC14104 and Yersinia enterocolitica MTCC861. Non-pathogenic

microorganisms assayed in the study included Lactobacillus brevis MTCC1750, L.

bulgaricus NCDC253, L. casei NCIM2651, L. helveticus NCIM2126, L. leichmanni

NCIM2027, L. pentosus NCIM2669, L. plantarum NCIM2912, Lactococcus lactis

subsp. cremoris MTCC1484 and Pediococcus acidilactici LB-42.

3.5. Identification of the bacteriocin producing anti-Gardnerella vaginalis LAB

isolate:

Characterization of the bacteriocin producing LAB isolates was carried out in

two steps:

3.5.1. Identification of microorganism at Generic Level:

Bacteriocin producing strains isolated from healthy human vaginal was

identified and characterized biochemically as per “Bergey’s Manual of Determinative

Bacteriology” – 9th Edition. As the isolation of LAB strains was carried out by

enrichment culture technique using MRS modified broth (supplemented with 3%

NaCl and 0.01% cycloheximide), only those strains were allowed to grow, that

belongs to genera viz. Lactococcus, Pediococcus, Lactobacillus etc. Different

morphological and biochemical properties studied include:

1. Gram-Staining: The Gram staining was carried out as per instructions given

with the Gram Staining Kit (Hi-media).

2. Morphological Examination: Gram stained slides of the producer strains

were viewed at 100X magnification under oil immersion and the most

common appearance of the cells and their predominant arrangements was

observed using binocular microscope.

3. Micro-Aerophilic Nature: 10ml MRS broth (pH 6.5) in the test tubes and

100 ml MRS broth in 250 ml flasks were inoculated with producer strains at

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1% (v/v) inoculums and incubated at 37°C for 24h. Growth was observed by

taking OD of 1ml aliquot of the culture at 600nm after appropriate dilutions of

the pelleted cells with normal saline, using UV-VIS spectrophotometer (E.

Merck).

4. Motility: A drop of culture was poured on a sterile and clean glass slide.

Cover slip was placed on the culture drop and viewed under the microscope at

45X magnification and observed for wriggling movements of cells.

5. Growth at Different Temperatures: Growth pattern of LAB isolates at

different temperatures reflected a good picture of the putative bacterial species

present in the culture. MRS broth were inoculated at 1% (v/v) inoculums from

bacteriocin produced LAB isolate. Initial pH of the media was set 6.5 using

1N NaOH or diluted HCl. Samples were incubated at three different

temperatures viz. 10°, 20°, 30°, 37°, 40° and 60° C for 24h. OD600 was

measured using a UV-VIS spectrophotometer after 24h culturing and diluting

the pelleted cells with 1N saline.

6. Growth at different pH: MRS broths were adjusted to pH 4.2, 7.5, 8.5 and

9.6, 11 autoclaved and inoculated at 1% (v/v) inoculums of the producing

strain. After 24h of the incubation at 37°C, growth was measured by taking

OD of 500µl pelleted cells at 600 nm using a UV-VIS spectrophotometer as

described earlier.

7. Growth in the presence of 6.5% NaCl: MRS broth (pH 6.5) was prepared

and NaCl was added so as to get a final concentration of 6.5%. It was

inoculated with 1% (v/v) inoculums of the producer strain and incubated at

37°C for 24h. After incubation was over, growth was compared with controls

lacking inoculums, by measuring OD600 after making appropriate dilutions of

cultured cells of control and test samples with 1N saline.

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8. Growth with 40% Bile : To MRS broth (pH 6.5), bile salt mixture was added

at final concentration of 40% (w/v) and inoculated using 1% (v/v) inoculums

of the producer isolate. It was inoculated along with a control lacking

inoculums at 37°C for 24h and growth was compared with that of control by

measuring OD600 using UV-VIS spectrophotometer, after diluting cultures

with 1N saline according to the requirement.

9. Catalase Reaction: The reaction was carried out using methodology as

described by Smibert and Krieg (1994). To 0.5 ml culture broth of each

producer strain, an equal volume of 3% H2O2 was added and observed for

bubbling immediately after the addition of H2O2 and after 5 min of H2O2

addition. Bubbling in the reaction indicates a positive catalase activity.

10. Oxidase test: Oxidase test was performed by adding tetramethyl paraphenyl

diamino dihydrochloride to a test tube containing 24h old culture (Kaur et al.,

2012).

11. Sugar fermentation profile: The sugar fermentation profile of isolate was

checked for glucose (1%), lactose (5%), mannitol (1%), sucrose (1%), dulcitol

(1%), rhamnose (1%), sorbitoland (1%), xylose (1%) and trehalose (1%) by

adding 1 ml of Andrades indicator in MRS broth containing inverted

Durham’s tube (Kaur et al., 2012).

12. Indole test: Indole test was performed using peptone water. Peptone water

was inoculated and incubated overnight. After 24h Kovac’s reagent was added

and color was observed (Kaur et al., 2012).

13. Methyl Red test: Methyl-Red test was carried out to study the ability of strain

to perform mixed-acid fermentation. Organisms that perform mixed-acid

fermentation produce enough acid to overcome the buffering capacity of the

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broth, so a decrease in pH results. After incubation, the pH indicator methyl

red is added to the broth. Methyl red is red at pH below 4.4 (this would be a

positive result) and yellow at pH above 6.0. An orange color indicates an

intermediate pH and would be considered a negative result (Kaur et al., 2012).

14. Voges-Proskaeur test: It was performed by inoculating in glucose phosphate

medium. Production of ammonia from arginine was assessed in arginine broth.

Arginine deamination was detected using Thornley’s semi solid medium

(Kaur et al., 2012).

15. Lactic acid production by HPLC: Lactic acid production was determined by

HPLC on C18 reverse phase column by maintaining flow rate of 0.8ml/min

using phosphate buffer (pH 2.4) in PDA 214 nm detector and compared with

C-18 reverse phase chromatogram of pure lactic acid (Kaur et al., 2012).

16. Lactic acid production by GC: Lactic acid production was also analyzed by

gas chromatography using polyethylene glycol (PEG) column and flame

ionization detector. Detector temperature 250oC, injector 150oC, oven

temperature 150oC (Kaur et al., 2012).

3.5.2. Characterization of LAB Isolates at Species Level:

The 16SrRNA gene sequencing of the selected HV6b isolate was carried out

by Bangalore Genei Private Limited, Bangalore, India. 16SrRNA gene sequence was

uploaded to Genbank database and the strain was deposited with MTCC culture bank,

India.

3.6. Study of localization of genes responsible for bacteriocin production in Anti-

Gardnerella LAB isolate:

3.6.1 Isolation of Plasmid DNA:

3.6.1.1 Principle: Bacterial plasmids exist as ccc molecules having –ve super helical

twists. In plasmid isolation most of procedures make use of the closed circular form

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of bacterial plasmids and their small size in reaction to bacterial chromosome. EDTA

is included in plasmid isolation procedures as chelate metal ions and thus inhibit

cation dependent nucleases. In isolation procedure, first step is to lyse the cells by

treating them with EDTA and lysozyme which breakdown the cell wall. The outer

membrane is disrupted by EDTA and thus allows lysozyme to degrade the

mucopeptide layer of the cell wall so that sphaeroblasts are formed. Sphaeroblasts in

an isoosmotic solution are then lyzed by adding a lysis reagent. Alkaline conditions

(pH 12-12.5) which denature linear DNA molecule and do not denature ccc

molecules. DNA precipitates, when the cell extract is neutralized at high salt

concentration due to reassociation of long ss DNA molecules occur at multiple sites to

form an insoluble mass. Cellular RNA and most of the proteins also precipitates

because of presence of SDS during reaction (Birnboin and Doly, 1979; Menon et al.,

2009).

3.6.1.2. Principle of Alkaline extraction method:

Preparation of Solutions for Plasmid Extraction:

(i) Alkaline lysis Solution I:

50mM Glucose - 0.99g

10mM EDTA - 0.37g

25mM Tris - 0.30g

Contents were mixed in 90ml deionised water and pH was adjusted to 8.0

using diluted HCl. Final volume was made 100ml with deionised water and stored at

4°C.

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(ii) Lysozyme Stock Solutions:

100mg of lysozyme was added to 1ml of solution I to prepare lysozyme stock

I and its 10µl aliquot (containing 1mg lysozyme) was further diluted 10 times in

solution I, to prepare lysozyme stock II. Lysozyme stock II was freshly prepared at

the time of use.

(iii) Alkaline lysis Solution II:

0.2M NaOH - 0.8g

1%(w/v) SDS - 1.0g

Contents were dissolved in 100ml deionised water and stored at room

temperature. A fresh solution was prepared every week for isolating plasmid DNA.

(iv)Alkaline lysis Solution III:

24.6g anhydrous sodium acetate was dissolved in 50ml deionised water and

the pH was adjusted to 4.8 using glacial acetic acid. Deionised water was then added

to make the final volume 100ml.

(v) Alkaline lysis Solution IV:

0.1M sodium acetate (anhydrous) - 0.82g

50Mm Tris - 0.605g

Contents were dissolved in 90ml deionised water, pH was adjusted to 8.0 with

dil. HCl and final volume was made 100ml with deionised water.

(vi) TE Buffer:

10mM Tris - 0.12g

1mM EDTA - 0.037g

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Contents were dissolved in 90ml of deionised water and pH was adjusted to

7.5 with dil. HCl. Final volume was raised to 100ml.

3.6.1.3. Procedure:

L. fermentum HV6b was grown in MRS broth for 16-18h. Cells were

harvested by centrifugation of 3ml culture broths in eppendrofs for 5min at 5000rpm

in a microfuge centrifuge (Tarsons). Pellets were resuspended in 100µl of solution I

and 20µl of lysozyme stock II with a final lysozyme concentration of 2mg/ml.

Eppendrofs were kept in dry ice for 15 min after a gentle vortexing. 200µl of solution

II was added and the contents were mixed by inverting and again kept in dry ice for

another 5 min.150µl of solution III was added and the contents were gently mixed by

inverting and incubated the eppendrofs in dry ice for a time period of 30 min. After

the incubation was over, cell debris and fragments of bacterial chromosome attached

to cell envelop were removed by centrifugation at 4°C for 5 min at a full speed, “A

Clearing Spin”. Clear supernatant was separated in a fresh tube and 1ml of chilled

ethanol was added to the samples, which were kept in dry ice for 5-10 min and then

centrifuged at full speed for 10 min at 4°C. Supernatant was removed carefully and

pellet was resuspended in 100µl of solution IV. 1ml chilled ethanol was added to each

eppendrofs. Samples were vortexed, kept in dry ice for 5 min and then centrifuged for

5 min at 4°C. Supernatants were discarded and the pellet were washed twice with

400µl of chilled ethanol. The eppendrofs were centrifuged at 4°C for 5 min, the

supernatants were discarded. Pellets were allowed to get dry in a desiccator and the

resuspended in 30µl of TE buffer. All the plasmid preparations were stored at -20°C.

3.6.2. Curing of Plasmid DNA:

Curing of plasmid DNA was accomplished through the growth in the presence

of ethidium bromide. 1% (v/v) inoculums of L. fermentum HV6b (18 hour old

83

culture) was added to sterile MRS broth containing 20µg of ethidium bromide per ml.

After incubation at 37°C for 24h, sub culturing was carried out using 1% (v/v)

inoculums in MRS broth lacking ethidium bromide and it was incubated for another

24h at 37°C. Then the culture was serially diluted and plated onto MRS agar plates,

which were incubated at 37°C for 24h. Colonies were selected at random and tested

for their Bac- characterstics (Batish and Gupta, 1992). Cured HV6b derivatives were

then grown in MRS broth and plasmid isolation was carried out as described above.

Analysis of Bac+ L. fermentum HV6b and cured HV6b derivatives was carried out by

agarose gel electrophoreses along with that of a standard plasmid MW marker

(Novagen).

3.6.3. Agarose Gel Electrophoresis:

3.6.3.1. Principle: Agarose gel electrophoresis method is used to measure the

molecular size of DNA, RNA and plasmid molecules. Negatively charged plasmid

DNA molecules are separated on agarose gel matrix according to their molecular

weight upon electrophoresis. The position of plasmid DNA in the agarose gel is

visualized by staining the gel with low concentration of a fluorescent intercalating dye

like ethidium bromide. Smaller molecules move faster and migrate farther than larger

ones because the migrate ion rates of plasmid DNA molecules are inversely

proportional to the logarithms of the molecular weights. This method is frequently

performed to determine the size of an unknown Plasmid DNA fragment by comparing

it with plasmid DNA ladders of known size. A standard curve can be obtained by

plotting the molecular size of the fragments of the marker against the reciprocal of

their respective mobility. The relative mobility (Rf) of the plasmid DNA ladder

depends upon the log of its relative molecular weight. The Rf value can be determined

after dividing the distance traveled by the DNA by distance traveled by tracking dye.

The molecular size of the test sample can be obtained from the standard curve

(Sambrook and Russel, 2001).

84

Agarose gel is transparent and is the material of choice for electroplating

nucleic acids. Ethidium bromide was either added to gel or to buffer after the run was

over. It gets intercalated between the stacked bases and emits orange fluorescence

when UV light falls on it. Thus, nucleic acid bands can be easily viewed. Molecular

weight determination of unknown DNA fragment/plasmid can be carried out easily

against a ladder of known marker standard plasmid MW marker.

3.6.3.2. Preparation of Solutions:

(i) Tris Borate EDTA (TBE) Buffer (10X):

0.89 M Tris base - 21.6 g

0.89M Boric acid - 11.0 g

0.02M Na2-EDTA.2H2O - 1.86 g

pH - 8.3

Contents were dissolved in 150ml deionised water and the final volume was

raised to 200ml. The 10X buffer was diluted 10 times deionized water to prepare

working 1X TBE buffer.

(ii) 0.6% (w/v) Agarose:

0.6g Agarose was dissolved by heating in 100ml 1X TBE buffer.

(iii) 10mg/ml (w/v) Ethidium Bromide:

10mg ethidium bromide was dissolved in 1ml deionised water and stored in dark and

cool place.

(iv) Sample Loading Buffer with Tracking Dye:

40% (w/v) Sucrose - 4g

0.25% (w/v) Bromophenol Blue - 25mg

Dissolved in 10 ml 1X TBE buffer.

85

3.6.3.3. Procedure:

The gel casting tray was sealed with cello tape. 0.6% agarose gel was prepared

by heating. Ethidium bromide was added to it after cooling down the gel to 45°C, at a

concentration of 5µg/ml of the gel. The gel was poured in casting tray, comb was

inserted carefully, air bubbles were removed using micropipette and it was then

allowed to set for 30min. Cello tape sealing and the comb were removed with care

and the gel was placed in tank filled with 1x the tank buffer. Apparatus was connected

to the power supply. Samples were loaded after mixing them with an equal proportion

of sample loading buffer and electrophoresis was carried out at 50V for 1.5-2h. The

gel was then viewed and photographed by placing it on a UV transilluminator.

3.7. Mode of action of the purified bacteriocin:

3.7.1. Growth inhibition assay: To study mode of action of bacteriocin produced by

the L. fermentum HV6b, the most sensitive indicator bacteria i.e. G. vaginalis was

selected. Strain G. vaginalis was inoculated at 1% (v/v) in Casman media along with

selective media and incubated for 4-5 hours at 37°C. In the meanwhile, 1ml aliquots

of freshly grown cultures were prepared in the sterile eppendrofs. Whole experiment

was carried out in duplicate sets containing control, 100µg/ml to 500µg/ml of purified

bacteriocin treated cultures. After, incubation at 37°C for 24h, log cfu/ml and OD600,

were analyzed for 1 ml of culture broth using normal saline as a blank using UV-VIS

spectrophotometer for colony counting, appropriately diluted samples in (0.85 %

NaCl) were spread plated onto Casman agar (1.0% w/v) plates and incubated at 37°C

for 24h. After 24h colonies were enumerated and data was recorded as log cfu/ml and

OD600 were plotted against concentration of bacteriocin used (Kim et al., 2000).

3.7.2. Scanning Electron Microscope (SEM) studies: The control and treated cells

of the BV pathogen G. vaginalis were examined by SEM to visualize any

86

morphological change that occurred in the cells following exposure to bacteriocin

50µg/ml produced by human vaginal isolate HV6b. Method used for SEM was

slightly different from that described in a previous publication (Parhusip and

Sitanggang ., 2011). G. vaginalis cell suspension was prepared in distilled water and it

was treated with pure bacteriocin at a concentration of 50µg/ml. Suspension was kept

for 90 min at 37ºC in a hydrated chamber and then centrifuged at 10,000 rpm for 2

min at 4ºC. The bacterial samples were washed gently and treated successively with

2% glutaraldehyde solution, 2% tannic acid solution, buffer solution (0.1M sodium

cacodylate containing 10 mM MgSO4; pH 6.7) and 1% osmium tetraoxide solution.

Each specimen was dehydrated using sequential exposure per ethanol concentrations

ranging from 50 - 100%. Cell pellet was dissolved in minimum quantity of ethanol

and a drop of it was transferred to poly-L-lysine-treated silicon wafer chips, which

were kept for 30 min in a hydrated chamber for the cells to adhere. Chips were

viewed at 10kV accelerating voltage in a Hitachi S-4300 field emission scanning

electron microscope and images of the bacteriocin treated cells for topography

contrast were collected at different magnifications.

3.7.3. Atomic Absorption Spectrophotometer (AAS) Analysis: Leakage of

potassium ions (K+) and calcium ions (Ca2+) from bacteriocin treated G. vaginalis

cells was quantified with AAS (Parhusip and Sitanggang, 2011).

3.7.3.1. Principle: AAS is a spectroanalytical procedure for the quantitative

determination of chemical elements employing the absorption of optical radiation

(light) by free atoms in the gaseous state. This technique is used for determining the

concentration of a particular element (the analyte) in a given sample. Analyte content

is established from the relation between the measured absorbance and the analyze

concentration and therefore relies on the Beer-Lambert Law (Lambert, 1760; Beer,

1852; Walsh, 1955, 1973).

87

3.7.3.2. Preparation of calibration curve for K+ and Ca2+ ions: For the preparation

of calibration curve of potassium standard solution of 2% HNO3 was used for AAS of

1000 µg/ml strength and for calcium standard solution was used of 1000 mg/ml

strength and were dissolved in de-ionized water and different dilutions 25 µg/ml to

120 µg/ml were prepared for both the ions. Wavelength of light used in this

experiment was 766.5 nm for K+ and 422.7 nm for Ca2+.

3.7.3.3. Methodology: G. vaginalis was activated in Casman media containing

selective media and incubated for 12 h at 37°C. Freshly activated bacteria were

collected by centrifuging the broth at 4, 032 g force (rotating at 6 000 rpm; radius of

rotor 100 mm) for 15 min. Cells were then washed for several times using deionized

water and finally resuspended in 10 ml deionized water in a reaction tube for each

bacterial strain. Into the tube, a known concentration of bacteriocin was added and

then incubated for 24 h. The quantification method of those cations followed the

procedure of Prashar (2003), where the suspension was then analyzed to determined

K+ at 766.5 nm using AAS instrument (AAS type flame photometer GBC Avanta

PM), and quantified using a standard calibration curve of potassium ion. Similarily,

Ca2+ efflux at 422.7 nm.

3.8. Production and purification of the bacteriocin produced by L. fermentum

HV6b:

3.8.1. Bacteriocin production studies: To study the production of bacteriocin

produced by L. fermentum HV6b in 1l MRS medium. The media having an initial pH

6.5, were inoculated at 1% (v/v) using an overnight grown producer culture and

incubated at 37oC for 24h. 1ml sample were drawn from both the culture broths after

every 4h and the changes in growth, pH of medium and bacteriocin activity were

studied:-

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3.8.1.1 Preparation of Growth Curve: In order to find out the phase of the culture

associated with bacteriocin production, growth curve of the human vaginal LAB

isolate was prepared. Above drawn sample was centrifuged at 7,000 rpm for 5 min,

the pellet was resuspended in 1ml normal saline and OD was measured at 600nm

using a UV-VIS spectrophotometer. At the end of incubation, OD600 was plotted

against incubation time (h) to construct growth curve of the human vaginal LAB.

3.8.1.2 Variation in pH: Fall in pH due to acid production is an important factor that

starts and regulates bacteriocin secretion in the culture broth. Variation in pH was also

studied each sampling using a pH meter (E. Merck).

3.8.1.3 Calculation of bacteriocin activity (AU/ml): CFS was subjected to a heat

treatment in boiling water bath for 5 min in order to kill the cells left in the

supernatant and then spot-on-lawn assay was performed to calculate AU/ml. Serial

dilutions of CFS up to 1:200 dilution were prepared in sterile deionised water and

spotted on to a freshly overlaid lawn of the indicator P. acidilactici LB42 (Coventry

et al., 1995) and incubated at 37°C for 18-200h. Titre of the bacteriocin was then

calculated as the reciprocal of the highest dilution giving positive inhibition and the

titre was multiplied by dilution factor i.e. 200 to calculate AU/ml.

3.8.2. Production and Purification of the bacteriocin produced by L. fermentum:

Methodology given by Yang et al. (1992) was followed to purify bacteriocin

produced by L. fermentum HV6b by Adsorption-Desorption method. Fermenticin

HV6b was purified by following the original methodology of pH-dependent

adsorption and desorption on to the producer cells that involved three important steps:

(i) Preparation of cells for bacteriocin adsorption.

(ii) Bacteriocin adsorption by the producer cells of L. fermentum.

(iii) Extraction of adsorbed bacteriocin from producer cells.

89

A schematic representation of the Adsorption-Desorption methodology is

shown in figure 3.1.

Figure 3.1: Schematic representation of bacteriocin purification by Adsorption Desorption method (Yang et al., 1992)

3.8.2.1. Preparation of Cells for Bacteriocin Adsorption: Cultures of L. fermentum,

inoculated initially at 1.0% (v/v), were grown to late log phase (18h) in 1000 ml MRS

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broth supplemented with 0.2% (v/v) Tween 80 (pH6.5), at 37°C. After the incubation

was over, culture broth was heated for 20 min in a boiling water bath, to inactivate

proteases and to kill cells. Culture broth after heat treatment, were allowed to cool

down to room temperature and pH was adjusted to 6.5 with 4M NaOH. An aliquot of

the sample was taken, centrifuged and serially diluted. 5µl of CFS and each dilution

was spotted on to a lawn of indicator bacteria i.e. G. vaginalis ATCC 14018 and

activity units per ml were calculated. OD of the sample was also measured at 280 nm

to quantify the amount of protein present in it.

3.8.2.2. Bacteriocin Adsorption by the Producer Cells: After adjusting the pH of

the cell suspension, it was kept overnight on magnetic stirrer at 4°C with slow stirring

in order to facilitate adsorption of the bacteriocin molecules that were secreted out in

the broth, back on to the producer cells. Cells were harvested by centrifugation at

9000 rpm for 20 min at 4°C, so as to remove the bacteriocin adsorbed on to the dead

cells from the culture liquor. CFS was kept for the estimation of the bacteriocin

AU/ml not adsorbed on to the cells. Cell pellet was washed twice in 5mM sodium

phosphate buffer (pH 6.5).

3.8.2.3. Extraction of Adsorbed Bacteriocin from Producer Cells: Pellet was

resuspended in 10ml 100mM NaCl adjusted to pH 1.5 with 5% (v/v) phosphoric acid.

The suspension was stirred overnight at 4°C and the cells were harvested by

centrifugation at 9000rpm for 30 min. Supernatant was filtered sterilized by passing it

through 0.45 µm filters and was stored at -20°C for further experimentation. An

aliquot of the purified bacteriocin preparation was taken. It was adjusted to pH 6.5

and OD280 was measured in order to calculate increase in specific activity after

purification following the adsorption- desorption method. Total AU/ml were

calculated after serially diluting the sample up to 1:6000 times dilution.

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3.8.2.4. Standard Curve of Protein: 200mg BSA was dissolved in 100ml normal

saline. BSA dilutions ranging from 100-1000 µg/ml were prepared in normal saline

and OD was measured at 280 nm, using UV-VIS spectrophotometer (E. Merck).

Absorbance values were plotted against different BSA concentrations and thus BSA

standard curve was prepared (Lowry et al., 1951). To calculate protein concentration

in an unknown sample viz. CFS, crude protein and purified protein, samples were

diluted with normal saline according to the requirement and OD was measured.

Protein concentration was then calculated from the standard curve.

Calculation of specific activity of bacteriocin: It was calculated by using formula

3.8.2.5. SDS-PAGE Analysis of the Purified Bacteriocin Preparation: Purified

bacteriocin preparation was obtained after adsorption and desorption from producer

cells of the culture broth. 15% non-denaturing polyacrylamide gels were prepared and

the bacteriocin preparation, obtained from adsorption-desorption method, were

loaded. Gel was allowed to electrophorese at 150 V for 4-5 hours. After the run was

over, gels were transferred immediately to a box containing fixative. After fixing the

proteins for overnight, gels were silver stained for details see section 3.8.4.4.

3.8.3. Reverse Phase Chromatography:

3.8.3.1. Principle: Reverse phase chromatography is a specialized form of HPLC. As

opposed to the unusual polar stationary phase and a less polar or non-polar mobile

phase, the stationary phase in reverse phase chromatography is hydrophobic

(hydrophobic bonded phase usually possessing C18 or C8 functional groups) and the

mobile phase is polar (fully or partially aqueous), In this case, polar substances will

interact more with polar mobile phase and elute first. As the non-polarity of the solute

92

component increases, their retention time will also increase, since they will interact

more with the non-polar stationary phase. The reverse phase is therefore, more useful

for separation of non-polar solutes. Water, an extremely polar solvent becomes the

weakest elute here. Methanol and acetonitrile are stronger elute than water. Solvents

of intermediate eluting strength can be obtained by mixing one of these solvents with

water. The driving force for the retention of a component is not its interaction with the

stationary phase but the effect of the mobile phase in forcing the component on to the

hydrocarbon bonded stationary phase. Moreover, this squeezing out is proportional to

the non-polar surface area of the solute, thus homologs show a good resolution by

RPC. When the sample has many components covering the wide range of polarity, a

gradient elution has to be carried for optimum resolution. These gradients are

prepared by continuously decreasing the polarity of the eluting solvent. This can be

achieved by gradually increasing the content of organic solvent in water/ organic

solvent mixture (Molnár et al., 1976).

3.8.3.2. Methodology: Freshly prepared purified bacteriocin preparation as obtained

by adsorption-desorption method was analyzed by reverse phase chromatography,

using C-18 Waters column and a 4.6*250mm C18 column. After equilibration of the

column with solvent (70% .1 % trifluoro acetic acid and Acetonitrile), at a flow rate

of .8ml/min, peptides were eluted using the same solvent. Peptides were monitored

spectrophotometrically at 280 nm. Fraction peak was collected manually in vials and

assayed foe bacteriocin activity. Active fractions were separated by carrying out spot-

on-lawn assay with 5µl aliquots of column effluent spotted on to MRS 1%(w/v)

bottom agar plates overlaid with Casman soft agar seeded with 106 cfu/ml indicator

bacteria G. vaginalis 14018. Total AU/ml was calculated after serially diluting the

sample to 1:6000 times with sterile deionized water.

93

3.8.4. Molecular Weight Estimation Using SDS-PAGE:

3.8.4.1. Principle: Polyacrylamide gel is prepared by polymerizing acrylamide

(H2C=CH-CO-NH2) and a small quantity of cross-linking agent,

methylenebisacrylamide (H2C=CH-CO-NH)2-CH2 (bis), in the presence of catalyst,

ammonium persulphate. TEMED is also added to initiate and control polymerization.

A direct contact of air with the gel should be avoided, because oxygen is a potent

inhibitor of polymerization.

SDS is a detergent that readily binds to protein. At pH 7.0 in the presence of

1% (w/v) SDS and 2-mercaptoethanol, proteins dissociate into subunits and bind large

quantities of the detergent. At pH 7.0, most proteins bind at about 1.4g of SDS per

gram of protein which completely masks the natural charge of protein giving a

constant charge to mass ratio and hence the relative mobility depends on its size. A

plot of log10 molecular weight versus relative mobility gives a straight line and thus

the molecular weight of unknown protein is determined by comparing its mobility

with a series of reference protein standards (Shapiro et al., 1967).

Molecular sieving effect of PAG can easily be amended just by altering the

electrophoretic mobility of the glycine buffer system, at pH 8.9 is greater than that of

the protein, so that the buffer boundary always runs ahead of the molecules being

separated. Bromophenol blue is incorporated in to the gels as a marker that marks the

boundary between glycinate and chloride ions. Acrylamide being toxic should be

handled with care, particularly avoiding its contact with the skin. Vertical slab gel

PAGE apparatus (BIORAD, INDIA) was used for the molecular weight analysis of

bacteriocin (Kaur et al., 2003).

3.8.4.2. Preparation of reagents: All the solutions were prepared in deionized water

and stored in dark bottles at 4°C.

94

(a) Tris-Glycine Buffer: It is a concentrated solution of Tris-Glycine-SDS in

distilled/deionized water, especially intended for use as the running buffer in SDS-

PAGE of proteins.

Tris - 6g/l

Glycine - 28.8g/l

pH - 8.9

Diluted up to 1 liter with deionised water filtered and stored at 4°C. Working

Tris-Glycine buffer was prepared by diluting the stock solution to 2 liters with

deionised water.

(b) 30% Acrylamide Stock Solution: Polyacrylamide gels are prepared by the free

radical polymerization of acrylamide and the cross linking agent N N’ methylene bis

acrylamide. Auto-polymerization of acrylamide takes place when it is dissolved in

water due to joining molecules together by head on tail fashion to form long single-

chain polymers. Gel formation requires linking various chains together.

Bisacrylamide is the most frequently used cross linking agent for polyacrylamide gels.

Chemically it can be thought of as two acrylamide molecules coupled head to head at

their non-reactive ends. Bisacrylamide can crosslink two polyacrylamide chains to

one another, thereby resulting in a gel.

Acrylamide - 30g

Bis-acrylamide - 0.4g

Water - 50ml

95

The contents were dissolved by stirring in 50ml deionised water and final

volume was made up to 100ml using deionized water. The solution was stored at 4°C

in the dark.

(c) 1M Tris-HCl Buffer: The Cl- ions from Tris-HCl, move much more quickly in

the electric field and they form an ion front that migrates ahead of the glycine. The

separation of Cl- from the Tris counter-ion (which is now moving towards the

cathode) creates a narrow zone with a steep voltage gradient that pulls the glycine

along behind it, resulting in two narrowly separated fronts of migrating ions; the

highly mobile Cl- front, followed by the slower, mostly neutral glycine front.

Tris - 12.1g

Water - 80ml

pH was adjusted to 8.9 with with dil. HCl and deionized water added to make

final volume 100ml and stored at 4°C.

(d) Sample Loading Buffer:

Sucrose - 5g

Bromophenol Blue - 0.01g

Working Tris-Glycine Buffer - 10ml

Contents were mixed properly by vortexing and then stored at 4°C.

(e) Fixative: Fixation prevents diffusion of proteins, thus keeping the protein bands

sharp and resolved during the staining process. In addition, fixation removes gel

buffer components, most importantly SDS, which may interfere in the staining

process. In some cases, fixatives are used which modify the proteins to enhance the

staining reaction. Fixative was prepared in deionised water.

96

Methanol - 50%

Glacial Acetic Acid - 12%

(f) 10% (w/v) Ammonium Persulphate Solution (APS): APS is a source of free

radicals and is often used as an initiator for gel formation. One hundred mg

ammonium persulphate was added to 1ml deionized water. Use of freshly prepared

ammonium persulphate solution is recommended.

(g)TEMED: After adding TEMED and APS your gel will polymerize fairly quickly,

so do not add these until you are sure you are ready to pour.

(h) 10% (w/v) Sodium Dodecyl Sulphate Solution (SDS): SDS is an anionic

detergent applied to protein sample to linearize proteins and to impart a negative

charge to linearized proteins. In most proteins, the binding of SDS to the polypeptide

chain imparts an even distribution of charge per unit mass, thereby resulting in a

fractionation by approximate size during electrophoresis. One gram of SDS added to

10ml deionised water was allowed to dissolve.

(i) 50% ethanol: It was used for storage of gels.

Table 3.13: Composition of 15% gel for 5ml solution

COMPONENTS RESOLVING GEL(15%) STACKING GEL(5%) 30 % Acrylamide 350 µl 850 µl 1.5M Tris (pH8.8) 1820 µl - 1M Tris (pH6.8) - 630 µl Distilled water 1537 µl 3400 µl 10% SDS 70 µl 50 µl 10% APS 70 µl 50 µl TEMED 2.8 µl 5 µl

3.8.4.3. Methodology used:

Glass plates were placed one on the other with spacers in between them. Two

slides and bottom of plates were sealed with cello tape and allowed to bake at 80°C

97

for 30min. Plates were then allowed to cool down to room temperature before pouring

PAG mixture. Separating gel (15%) was prepared according to composition given in

table 3.13. All the reagents were mixed properly except TEMED and deaerated under

vacuum for15 min. APS and TEMED were added to PAG mixture and swirled gently

to mix for initiating polymerization. Solution was poured immediately within stacked

plates with care and overlaid it with deionized water and allowed it to polymerize for

30 min. Overlay solution was rinsed off the after the polymerization was over.

Similarly, stacking gel (5%), over the separating gel. Comb was inserted, immediately

being careful not to allow air bubbles to become trapped under the teeth, overlaid it

with deionized water and allowed it to polymerize for another 30 min. Then, cello

tape seal was removed from bottom of the plates and excess water was rinsed out so

as to connect upper and lower buffer reservoirs through gel. Comb was removed

carefully and the PAGE apparatus was assembled. Tank buffer was poured and the

electrodes were connected to power supply. Samples (crude bacteriocin preparation as

obtained by adsorption-desorption method and MW marker) were mixed with sample

loading buffer in the ratio of 1:1 and loaded on to the wells. Then, the electrophoresis

was carried out at 150V for 4-5h. After the run was over, gel was detached from

plates and transferred to a box containing fixative. Gel was allowed to fix overnight.

3.8.4.4. Ionic Silver Staining of the Gel:

Ionic Silver Staining of the Gel:

Gels were stained by ionic silver staining method for visualization of protein

pattern bands. Gels were kept on a roacker during staining (Gromova et al., 2006).

After an overnight fixation, gel was given three consecutive washings with deionised

water for 20 min each. Then the gel placed in the oxidizing solution and kept for 5

min, the oxidizing solution was removed from staining box and two more washings

98

were given with deionized water for 2 min duration. Silver nitrate solution was added

to the box and was allowed to react with protein for 20 min. Thereafter, excess silver

nitrate solution was removed and gave quick wash deionised water to the gel. Thus,

AgNO3 left unbound on the gel was washed off with the water. Developer was added

and protein bands were allowed to develop for 15-30 min. Developer was changed 3-4

times during developing. After the development of protein bands was complete, gel

was washed with deionised water for at least one hour to stop the reaction and then

photographed using a normal camera. A sketch of the separations i.e. electrophoretic

mobility versus log 10 molecular weight was prepared, in order to find out the MW of

the unknown sample. Gels were stored in 50% alcohol.

3.9. Molecular Weight Estimation of pure protein using MALDI-TOF analysis:

3.9.1. Theory: MALDI is a soft ionization technique used in mass spectrometry,

allowing the analysis of biomolecules (biopolymers such as DNA, proteins, peptides

and sugars) and large organic molecules (such as polymers, dendrimers and other

macromolecules), which tend to be fragile and fragment when ionized by more

conventional ionization methods. It is similar in character to electrospray ionization

(ESI) both in relative softness and the ions produced (although it causes many fewer

multiply charged ions).

The MALDI is a two step process. First, desorption is triggered by a UV laser

beam. Matrix material heavily absorbs UV laser light, leading to the ablation of upper

layer (~micron) of the matrix material. A hot plume produced during the ablation

contains many species: neutral and ionized matrix molecules, protonated and

deprotonated matrix molecules, matrix clusters and nano droplets. The second step is

ionization (more accurately protonation or deprotonation). Protonation

(deprotonation) of analyte molecules takes place in the hot plume. Some of the

ablated species participate in protonation (deprotonation) of analyte molecules

(Fitzgerald et al., 1993).

99

3.9.2. Procedure: MALDI-TOF mass spectrometry was carried-out for MW analysis

of purified fermentin HV6b. Mass spectra were obtained in the positive ion mode,

with an accelerating voltage of 25 KV. One microliter of the pure protein (without

trypsin digestion) was mixed with 1µl of matrix (10mg of sinapinic acid and 0.1% 4-

hydroxy-α-cyanocinnamic acid in 1 ml of distilled water) and 0.3 µl of this mixture

was applied to the Teflon-coated plate (Fitzgerald et al., 1993). BSA was used for

calibration.

3.10. Characterization of the bacteriocin produced:

Fermenticin HV6b produced by human vaginal isolate L. fermentum HV6b

MTCC No. 10770 was characterized and its inhibitory properties were studied against

the most sensitive indicator bacteria, i.e G. vaginalis ATCC 14018. Agar well

diffusion Assay (Pucci et al, 1988) and Spot-on-lawn Assay (Toba et al., 1991) were

followed to characterized fermenticin HV6b for its physiochemical properties:

3.10.1. Sensitivity of bacteriocin to various enzymes: Purified bacteriocin

preparation (as obtained after Adsorption desorption method) from L. fermentum, BV

was treated with various enzyme including proteinase K, trypsin, α-chymotrypsin

(Alpay-Karaoglu et al., 2003). Enzymes stocks were prepared in 50mM phosphate

buffer (pH 7.0) and were added to the reaction mixture to obtain a final concentration

of 3mg/ml. The mixture was incubated at 37°C for 3h. At the end of the incubation,

all the samples were subjected to heat treatment in a boiling water bath for 5 min in

order to inactivate the enzymes and the residual activity of the bacteriocins, was

assayed using spot-on-lawn method.

3.10.2. Thermostability of the bacteriocin produced: Crude bacteriocin preparation

of the strain of L. fermentum HV6b was heated at 100°C for 5, 10, 15, 20 min and at

121°C for 20min. Heat treatment at 100°C was given in a boiling water bath and that

of 121°C, was given in an autoclave at 15psi. Residual bacteriocinogenic activity was

then assayed using spot-on-lawn assay.

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3.10.3. Sensitivity of the bacteriocin to pH changes: Crude bacteriocin preparation

of L. fermentum HV6b was adjusted to different pH values ranging from 1.0 to 10,

using 5% phosphoric acid, 1N NaOH and dil. HCl. Controls were prepared using

above bacteriocin preparations. After adjusting pH samples were kept at room

temperature for 1h and assayed by spot on lawn method.

3.10.4. Stability of the bacteriocin during refrigerated storage at -20C: For a

bacteriocin to be used as biopreservative, it is essential to have a considerable stability

towards storage conditions. In order to check the stability of the fermenticin HV6b

bacteriocin produced by L. fermentum towards refrigeration conditions, the crude

bacteriocin preparation (prepared by adsorption-desorption method, Yang et al., 1992)

was stored at -20°C for about 3 months. Serial dilutions of the crude bacteriocin

preparation was prepared in sterile deionised water up to a dilution of 1:1000 and the

bacteriocin activity were assayed using spot-on-lawn and agar well diffusion methods

after every 15 days interval.

3.10.4.1. C18 reverse phase HPLC: HPLC of the freshly purified bacteriocin

preparation was carried out. To study the stability of the bacteriocin during storage at

-20°C, RPC was again carried out after 2 and 3 months of refrigerated storage.

Degradation pattern were observed on RP- HPLC for fermenticin HV6b after storing

bacteriocin at different time intervals till 8 months to one year. For a detailed

methodology see section no. 3.7.4.

3.10.5. Circular dichroism spectroscopy: Circular dichroism (CD) spectroscopy

measures differences in the absorption of left-handed polarized light versus right-

handed polarized light which arise due to structural asymmetry. The capacity of CD to

give a representative structural signature makes it a powerful tool in modern

biochemistry with applications. Circular dichroism is exhibited by biological

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molecules, because of their dextrorotary and levorotary components. Even more

important is that a secondary structure will also impart a distinct CD to its respective

molecules. Therefore, the alpha helix of proteins and the double helix have CD

spectral signatures representative of their structures. It determines whether a protein is

folded, and if so characterizing its secondary structure, tertiary structure, and the

structural family to which it belongs Structural elements are more clearly

distinguished since their recorded bands do not overlap extensively at particular

wavelengths as they do. The far CD spectrum of proteins can reveal important

characteristics of their secondary structure. CD spectra can be readily used to estimate

the fraction of a molecule that is in the alpha-helix conformation, the beta-sheet

conformation, the beta-turn conformation, or some other (e.g. random coil)

conformation (Kawai et al., 2004).

3.10.5.1. Methodology used: Circular dichroism was carried out to understand

conformation of fermenticin HV6b. CD model no. J-815, with a band width 0.5 nm

and response period of 1 sec at standard sensitivity was used. Measurement range

included 350 - 200 nanometres, data pitch of 1nm, with a scanning speed of 50

nm/min at room temperature. Elga water was used as solvent in CD studies of

fermenticin HV6b.

3.10.6. Peptide mass fingerprinting of fermenticin HV6b by MALDI-TOF:

In peptide sequencing, selected ion components are either positive (M+H)+ or

negative (M-H)- ions. The selected ions will pass into the collision cell, there are three

different types of bonds that can fragment along the amino acid backbone: NH-CH,

CH-CO, and CO-NH. For each of these fragments, the charge can be maintained by

either the N or C-terminal fragment. There are actually six different types of

fragments that can be formed. The mass difference between two fragments of the

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same type is indicative of a particular amino acid. Peptide mass fingerprinting was

carried out with help of two techniques after trypsinsation or trypsin digestion of pure

protein sample. After confirming purity of protein on single band on SDS-PAGE

and/as a on single peak on HPLC chromatogram of C-18 column reverse phase

chromatography

3.10.6.1. Preparation of reagents:

a) 50 mM ammonium bicarbonate: 0.1 g of ammonium bicarbonate was dissolved

in 20 ml of 30% Acetonitrile.

b) 30% v/v Acetonitrile: 30ml of acetonitrile was dissolved in 70 ml of distilled

water.

c) 1% trifluoroacetic acid: 1ml TFA was dissolved in 9ml of distilled water.

3.10.6.2. Trypsin digestion: 1mg trypsin was dissolved in 1ml of 50 mM of

ammonium bicarbonate in 30% acetonitrile was mixed with added in 2 µl of 1%

trifluoroacetic acid in trypsin vial and incubated at 37oC for 18 h. Sample was

centrifuged with trypsin solution for digestion at 9500 rpm for 20 sec and supernatant

was collected.

3.10.6.3. MALDI-TOF analysis of Trypsin digested protein: MALDI-TOF mass

spectrometry was done Brukers Daltonics Maldi TOF. Mass spectra were obtained in

the positive ion mode, with an accelerating voltage of 25 KV. One microliter of the

trypsin digested protein was mixed with 1µl of matrix (10mg of sinapinic acid and

0.1% 4-hydroxy-α-cyanocinnamic acid in 1 ml of distilled water) and 0.3 µl of this

mixture was applied to the Teflon-coated plate. BSA was used for calibration

(Sommerer et al., 2007). Initially as a precursor and product ion, a mass fingerprinting

can occur where through further analysis and investigation the fragments of ions can

be pieced together to determine a peptide sequence. Since it is known that amino acid

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backbone fragments among the NH-CH, CH-CO, and CO-NH bonds, each type of

bond can give rise to six different type of species, and these can then be pieced

together to provide peptide fragmentation (Sommerer et al., 2007).

3.11. Study of therapeutic potential of the isolate in vitro against opportunistic

pathogens of vaginal ecosystem:

3.11.1. In vitro inhibition of vaginal pathogens by fermentcin HV6b:

Antimicrobial activity of bacteriocins produced by LAB isolates was characterized

using spot-on-lawn and well diffusion method (Pucci et al., 1988; Toba et al., 1991).

BV associated pathogens included Bacteriodes fragilis MTCC1045, MTCC3298 and

MTCC1350, Candida albicans ATCC10231 and MTCC183, Gardnerella vaginalis

ATCC14018, Micrococcus flavus ATCC10240, Neisseria gonorrhoeae ATCC19424,

N. mucosa MTCC1772, Proteus mirabilis NCIM2387, Staphylococcus albus

ATCC11631, S. aureus MTCC737 and NCTC7447, Streptococcus agalactiae

NCIM2401, S. faecalis MTCC459, S. pyogenes NCTC10869 and S. thermophilus

MTCC1928 used to study inhibition of these vaginal pathogens by fermenticin HV6b.

3.11.2. Biomedical application on cancer cell lines:

3.11.2.2. Tissue models for testing anticancerous activity of fermenticin HV6b:

Protocol: Tissues were seeded in culture flasks containing DMEM and RPMI-1640

medium with 10% fetal bovine serum and 100 µg/ml penicillin and streptomycin, and

cultured in a humidified 5% CO2 incubator at 37oC. After reaching 80% confluence,

cells were passaged and cultured. Spent culture medium was discarded. The cell layer

was rinsed with 0.25% (w/v) trypsin- 0.53 mM EDTA solution to remove all traces of

serum which may contain trypsin inhibitor. 6.0 to 8.0 ml of growth medium was

added and cells were aspirated by gently pipetting. Tissues were exposed to different

concentration of fermenticin HV6b ranging from 0.1µg/ml,1µg/ml and 10µg/ml for

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24 and 48 hours. For exposure time over 24 hours, the tissues were fed with fresh

assay media. After the required exposure time, MTT assay was used to determine

overall cell viability. Cell counts of tissue models were checked using

haemocytometer (Kumar et al., 2012).

3.11.2.2.1 Properties of Cell lines used in the study:

1. HEP G2 CRL-10741

Growth properties: Adherent

Organism: Homo sapiens

Deposited as human morphology: Epithelial

Source Organ: Liver

Disease: Hepatocellular carcinoma

Growth medium for culturing: A 1:1 mixture of Dulbecco's modified Eagle's

medium and Ham's F12 g/l medium with 2.5 mM L-glutamine, 15 mM HEPES, 0.5

mM sodium pyruvate and 1200 sodium bicarbonate and supplemented with 0.4 mg/ml

G418, 90%; fetal bovine serum, 10%

Atmosphere: air, 95%; carbon dioxide (CO2), 5%

Temperature: 37 °C

Growth Conditions: These cells are slow to attach after subculture. Allow 4 to 5

days for reattachment.

Subcultivation ratio: A subcultivation ratio of 1:4 to 1:6 is recommended

Medium Renewal: Every 2 to 3 days

Freeze medium: Complete growth medium supplemented with 5% (v/v) DMSO

Storage temperature: liquid nitrogen vapor phase.

2. HeLa (CCL-2)

Adherent organism: Homo sapiens

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Morphology: Epithelial

Organ: Cervix

Disease: Adenocarcinoma

Cell Type: Epithelial

Growth medium: The base medium for this cell line is ATCC-formulated Eagle's

Minimum Essential Medium, Catalog No. 30-2003. To make the complete growth

medium, add the following components to the base medium: fetal bovine serum to a

final concentration of 10%.

Atmosphere: air, 95%; carbon dioxide (CO2), 5%

Temperature: 37 °C

Subcultivation ratio: A subcultivation ratio of 1:2 to 1:6 is recommended .

Medium renewal: 2 to 3 times per week Preservation

Freeze medium: Complete growth medium supplemented with 5% (v/v) DMSO

Storage temperature: liquid nitrogen vapor phase.

3. MCF7 (HTB-22)

Adherent Organism: Homo sapiens

Morphology: epithelial

Organ: mammary gland; breast

Disease: adenocarcinoma

Derived from metastatic site: pleural effusion

Cell Type: epithelial

Growth medium: The base medium for this cell line is ATCC-formulated Eagle's

Minimum Essential Medium, Catalog No. 30-2003. To make the complete growth

medium, add the following components to the base medium: 0.01 mg/ml bovine

insulin; fetal bovine serum to a final concentration of 10%.

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Atmosphere: air, 95%; carbon dioxide (CO2), 5%

Temperature: 37 °C

Subcultivation Ratio: A subcultivation ratio of 1:2 to 1:6 is recommended

4. Sp2/0-Ag14 (CRL-1581)

Organism: Mus musculus (B cell); Mus musculus (myeloma) deposited as mouse (B

cell); mouse (myeloma)

Morphology: Lymphoblast

Source organ: Spleen

Strain: BALB/c

Cell type: Hybridoma, B lymphocyte;

Growth medium: The base medium for this cell line is ATCC-formulated Dulbecco's

Modified Eagle's Medium, Catalog No. 30-2002. To make the complete growth

medium, add the following components to the base medium: fetal bovine serum to a

final concentration of 10%.

Temperature: 37 °C Subculturing:

Medium Renewal: Add fresh medium every 2 to 4 days (depending on cell density)

Freeze medium: Complete growth medium 95%; DMSO, 5%

Storage temperature: liquid nitrogen vapor phase

Subcultivation Ratio: A subcultivation ratio of 1:2 to 1:6 is recommended

5. HEK 293 (CRL-1573.3)

Organism: Human Embryonic Kidney

Morphology: rounded

Source: Organ: Kidney

Age: fetus

Cell Type: hybridoma: endothelial, epithelial, or fibroblasts.

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Growth medium: ells should be grown in a complete SFMII growth medium

supplemented with 4mM L-glutamine. To make the complete growth medium, add

the following components to the base medium: fetal bovine serum to a final

concentration of 10%.

Temperature: 37 °C Subculturing:

Medium Renewal: Add fresh medium every 2 to 4 days (depending on cell density)

Freeze medium: Complete growth medium 95%; DMSO, 5%

Storage temperature: liquid nitrogen vapor phase

Subcultivation Ratio: A subcultivation ratio of 1:2 to 1:6 is recommended

3.11.2.3. MTT viability assay:

The MTT assay was carried out according to the protocol given by Mosmann

(1983). The viability of the cells exposed to bacteriocin, was measured as a direct

proportion of the breakdown of yellow compound tetrazolium to dark blue water

insoluble formazan. Only the metabolically active cells can show this reaction which

can be solubilized with DMSO and then quantified. The absorbance of formazon

directly correlates with the number of viable cells. The liquid in the plate wells was

combined with the liquid from the tissue. Mixture is then assayed

spectrophotometrically at 540nm using 96 wells plate ELISA reader to determine

level of tetrazolium degradation.

3.11.2.4. DNA fragmentation:

Cells (1 X 105) were cultured for 24 h, treated with 1·0 mg/ml fermenticin

HV6b for 48 h, and then lysed with 250 µl lysis buffer. After incubation at 37oC for

90 mins, 200 µg/ml proteinase K was added and incubated again for 60 mins at 50oC.

Lithium chloride (0.2%w/v) was added to assist cell lysis and as an inhibitor of

nucleases. After incubation, suspension was centrifuged at 13,000 rpm for 3 mins,

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aqueous phase was transferred to fresh tube containing deproteinizing mixture of

phenol, chloroform and isoamyl alcohol (25:24:1) and again centrifuged at 3,000 rpm

for 3 min. DNA was precipitated from the aqueous phase with 3 volumes of chilled

ethanol containing 0.3 M sodium acetate at 4oC. Samples were subjected to

electrophoresis in 1% w/v agarose gel using TBE buffer at 50V and visualized on a

UV transilluminator (Kumar et al., 2013).

3.12. Effect of fermenticin HV6b on G. vaginalis adhesion to HeLa cell lines:

HeLa, a cervical cancer cell line was cultured in MEM supplemented with

10% fetal calf serum (FCS) at 37ºC in 5% CO2 Cells were washed by differential

centrifugation three times in 20ml of MEM at 800rpm for 10 min. The cells were

resuspended in 10ml of MEM and filtered through a membrane filter with a pore size

of 8 µm (Millipore). Cells were resuspended in essential medium and adjusted to final

concentration of 2×104 washed epithelial cells per ml, as counted by

haemocytometer counting chamber.

G. vaginalis grown on Casman agar was suspended in phosphate-buffered

saline; pH 7.2 and washed three times in 20ml of phosphate-buffered saline by

centrifugation at 2500 rpm. After resuspension of bacteria in phosphate buffer saline,

the cell counts were adjusted to 108 cfu/ml using haemocytometer. For positive

control, one ml of bacterial suspension was mixed with 1ml of HeLa cell suspension

so that a ratio of 5×103 bacteria per HeLa cell can be achieved. Suspension was

incubated aerobically on a rocker at 37oC for 30 min (pH 7.2) and diluted 1:2 in

phosphate buffer saline. Now, in test samples three different concentrations 100µg/ml,

500 µg/ml of fermenticin HV6b were added to G. vaginalis infected HeLa cells. HeLa

cells were then washed to remove nonadherent bacteria by differential centrifugation

at 800 rpm and filtered it through 8 µm (Millipore) membrane filter. For transferring

the cells to slides, membranes were gently pressed against the glass slides.

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Cells attached with slides were fixed with 95% ethanol for 15 min and air

dried and Gram stained. The number of bacteria adherent to HeLa cells were counted

under a light microscope (Olympus Stereozoom Microscope 1,000X). In each

experiment, 50 HeLa cells were studied after 60 and 90 minutes of bacteriocin

exposure, and triplicates determinations were performed ( Mårdh and Westtöm,

1976).

3.13. Semen sample collection and analysis:

Partially purified fermenticin HV6b preparation was used as spermicide to test

its effect on motility and immobilization of human spermatozoa. Two semen samples

were collected from healthy volunteers in sterile wide-mouth polypropylene

containers with a screw cap by self-masturbation on the day of experimentation.

Within 1h of collection, samples were dispensed. By mixing a drop of diluted

spermicide with a drop of semen and examining under microscope, an approximation

is obtained to the highest bacteriocin concentration that does immobilize spermatozoa

in 2 min, and the series of dilutions to be used in the test are made in a range below

this point. Total sperm count was calculated using a compound microscope (Olympus,

100X) after dilution (1:50) of the semen in normal saline. The sperm suspensions

were made in small glass tubes, one tube being required for each concentration of the

spermicide. The suspensions were made by adding 0.5 ml of semen to each tube of

saline or spermicide solution. Samples were placed in an incubator at 37°C for 15 to

30 min to reach that temperature. The percent sperm motility was determined by the

progressive (forward) and non-progressive (vibrating and zig-zag) movement of

sperm observed in a compound microscope. The sperm count was calculated using

Newbauer haemocytometer from a count of between 100-200 sperms using randomly

selected (100X) (Sutyak et al., 2008).

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3.13.1. Treatment of spermatozoa with fermenticin HV6b:

Sutyak test (2008) was used to determine the effect of purified bacteriocin

fermenticin HV6b on the motility of human spermatozoa with little modifications

(Sutyak et al., 2008). To measure the effect of fermenticin HV6b on sperm mobility

and aggregation after 30 sec exposure time to different concentrations of bacteriocin

ranging from 50 to 200µg/ml of diluted semen sample. The motilities of human

spermatozoa cells from random high magnification fields (100X) of the sample were

determined in duplicate using atomic force microscope. Results were evaluated

according to WHO grade system and motilities of sperms are divided into four

different grades (Wilson, 2005). Grade a sperms have progressive motility. These are

the strongest and swim fast in a straight line. Grade b sperms also exhibit forward

movement but tend to travel in a curved or crooked motion. Grade c sperms show

non-progressive motility because they do not move forward despite the fact that they

move their tails and Grade d sperms are immotile and fail to move at all.

3.14. Sensitivity analysis of G. vaginalis: fermenticin HV6b vs antibiotics:

Antibiotic susceptibility G. vaginalis was studied against different

concentrations (20 to 80 µg/ml) of some commonly prescribed antibiotics to BV

patients including ampicillin, amoxicillin, amoxicillin and clavalanic acid,

azithromycin, ciprofloxacin, co-trimoxazole, erythromycin, gentamycin,

metronidazole, nalidixic acid, ofloxacin, penicillin, rimphicin, tetracycline, tinidazole

and vancomycin by agar well diffusion method and disc diffusion method.

1. Agar well diffusion method: The original methodology was modified a little and

was then used to assay antibiotic sensitivity of G. vaginalis (Sarkar and Banerjee,

1996). The drugs commonly used for the treatment of bacterial vaginiosis. These

drugs have been procured from Unisons Pharma Baddi. Five dilutions of those drugs

were made and filled in the wells on the culture plates of G. vaginalis in Casman

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medium seeded with 18h old 105-106 cfu/ml cells. Wells of 6mm diameter cut with

sterile cork borer were filled with 100µl of the antibiotic solution. Plates were kept at

4°C for 1h to allow diffusion of antibiotics out of the wells and incubated at 30°C for

24h. The antibiotic sensitivity activity of isolates was detected and quantitated by

measuring the width of the clear zones around the wells in the resultant lawn culture.

2. Disc diffusion method: Disc diffusion method 1% Casman media agar plates were

prepared and then mix 30µl of 24 hour old G. vaginalis in 0.75% soft agar and mixed

well and spread gently on selective agar plate. Then discs containing above enlisted

antibiotics were placed on petri plates and 5 µl antibiotic dilution was spotted on

them. Plates were incubated at 37°C for 24 hours and the inhibition zones were

observed for metronidazole, metronidazole H, tinidazole, amoxicillin +clavulanic

acid, cefaxime, amoxicillin, ciprofloxacin, erythromycin, co-trimoxazole, tetracycline,

azithromycin, miconazole, rifampicin (Jorgensen et al., 2007).