Upload
doandien
View
213
Download
0
Embed Size (px)
Citation preview
Chapter III
MATERIALS AND METHODS
3.1. PLACES OF RESEARCH WORK
The isolation, characterization and identification of bacterial and fungal
pathogens were done in the Clinical Microbiology Research Laboratory of PRIST
University, East Campus, Vallam, Thanjavur district, Tamil Nadu. The isolation,
identification and characterization of mycobacteria were done at K.A.P.V.
Medical Hospital, Tiruchirapalli, Molecular methods such as PCR, AGE and
PAGE were done at Government Hospital for Chest Diseases (STDC),
Puducherry, RT-PCR was done at Center for Molecular Biology Methods,
Kovalam, Chennai, CD4 count was done at Medical College, Thanjavur, DNA
sequencing for Mycobacteria was done at Bioserve, Bangalore. The protein
sequencing was done at Molecular Medicine, New Delhi. Protein sequencing for
Candida was done at Xceleris Laboratory, Ahmadabad. Phytochemical studies
were done at Armats Biotek Private Ltd., Chennai. Antimycobacterial activity of
herbal extracts was done at Tuberculosis Research Centre, Chetpet, Chennai.
3.2. STUDY GROUP
250 biosamples were collected from 200 HIV positive patients who were
attending the HIV counselling and testing centers of both the two medical colleges
and hospitals of Tiruchirapalli and Thanjavur districts of Tamil Nadu, India
(Lat.10° N and Long.79° E). All the HIV positive patients in the age group
between 1 and 80 with symptoms state as inclusion criteria and also in the age
group of above 80 as exclusion criteria with or without symptoms.
3.2.1. Research design
The present investigation gives a view, which is ideally suited to study the
prevalence of opportunistic bacterial and fungal pathogens in the HIV+ patients.
A broad outline of the research design is presented (Fig.1).
Easy PDF Creator is professional software to create PDF. If you wish to remove this line, buy it now.
48
3.2.2. Instrumentation
In this work, isolation and identification of bacterial and fungal pathogens
were done in UV safety cabinet. PCR amplification of mycobacterial DNA was
done by using Automated Thermal Cycler. The stained gels of PCR amplified
DNA was done in UV Transilluminator and photographed using
Geldocumentation Unit. The amplified gene products were sequenced by DNA
sequencer. Real time PCR was done in RT-PCR System. The proteomics of
serum albumin protein was done by Nano LC-MS. The medicinal herbal plant
extracts were obtained by using Soxhelet Apparatus (Plate I).
3.2.3. Collection of biosamples
250 biosamples were collected from clinically symptomatic HIV+ patients
of various hospitals, medical colleges and social welfare organizations of
Thanjavur and Tiruchirapalli districts during the period from August 2008 to July
2009 (in a sterile container after aseptic precaution). All the patients were
thoroughly evaluated by detailed history, clinical examination and biochemical
parameters including CD4 count.
3.3. ASPECTS OF STUDY
To realize the objectives of the present study outlined in the introductory
chapter the following experiments or investigations were carried out.
? Isolation, identification and characterization of bacterial pathogens in HIV+
patients.
? Isolation and identification of fungal pathogens in HIV+ patients.
? Antibiotic sensitivity of bacterial pathogens.
? Proportional antibiotic sensitivity of mycobacteria.
? Antibiotic sensitivity of fungal pathogens.
Easy PDF Creator is professional software to create PDF. If you wish to remove this line, buy it now.
49
? Molecular characterization of isoniazid and pyrazinamide drug resistant
mycobacteria.
? Molecular characterization of Candida albicans.
? Phytochemical analyses of ten medicinal herbal plant extracts.
? Antibacterial activity of ten medicinal herbal plant extracts.
? Antifungal activity of five medicinal herbal plant extracts.
? Identification of effective compounds by thin layer chromatography and
bioautography.
? Characterization of purified compound by GC-MS and FT-IR analyses.
Standard procedures and protocols were followed in the microbial,
biochemical, phytochemical and molecular studies which are listed in Table 1.
TABLE 1 The list of standard procedures and protocols
S.No. Test Aspect Reference
I. CD4 Count To count the CD4 cells inblood, thereby detect theimmune status of the HIV+
patients
Kahan and Jani,2001
II. EXAMINATION OF BIOSAMPLES1. Microscopic examination
i. Staining techniquesa. Gram staining Identification of bacteria Aneja, 2003b. Acid fast staining
Ziehl Neelson staining Identification ofM. tuberculosis
Venkataraman andAlexander, 1987
Flourescent staining Identification ofM. tuberculosis
Blair et al., 1970
c. Lactophenol cotton bluestaining
Identification of fungi James Cappucinoand NatalieSherman, 2009
ii. Hanging drop method Motility of bacteria Aneja, 2003
Easy PDF Creator is professional software to create PDF. If you wish to remove this line, buy it now.
50
2. Macroscopic examinationi. Modified Petroff’s
methodCulture processing of sputum Allen and Baker,
1968ii. Culture techniques To isolate bacteria and fungi Aneja, 2003
III. Biochemical characterization1. Bacteria
1. Catalase test To detect the presence ofcatalase enzyme .
Kubica and Pool.,1960
2. Oxidase test To detect the presence ofoxidase enzyme
Cappucino andSherman, 2007
3. Indole test To detect the production ofindole.
Aneja, 2003
4. Methyl red test To detect the ability ofmicroorganisms to oxidizeglucose
Aneja, 2003
5. Voges proskauer To differentiate entericpathogens
6. Citrate utilization test To detect the utilization ofcitrate substrate.
Aneja, 2003
7. Urease test To detect the presence ofurease enzyme.
Aneja, 2003
8. Hydrogen sulphideproduction test
To detect fermentative natureof bacteria by H2S production
Cappucino andSherman, 2007
9. Sugar fermentation test Detect the fermentation ofsugars by pathogens.
Aneja, 2003
2. Mycobacteria1. Catalase test To detect the presence of
catalase enzymeKubica and Pool.,1960
2. Niacin test To detect the production ofniacin
Venkataraman andPrabakar, 1970
3. PNB test To detect the conversion of p-nitrobenzoic acid
Tsukamura andTsukamura, 1964
4. Nitrate reduction test To detect the utilization ofnitrate in the medium.
Angeby et al., 2002
IV. Antibiotic sensitivity testing of bacteria1. Disc diffusion method To detect the sensitivity of
isolated bacteriaBauer et al., 1996
Easy PDF Creator is professional software to create PDF. If you wish to remove this line, buy it now.
51
2. Proportional sensitivitymethod
To detecting the sensitivity ofisolated mycrobacteria.
Tripathy et al.,1970
V. Molecular Studies1. Isoniazid and Pylozinamide drug resistant Mycobacterium
tuberculosisa. Preliminary work Isolation of DNA Vijdea et al., 2008b. PCR Amplification of inhA, katG
and pncA genesCousins et al., 1992
c. Agarose gelelectrophoresis
To determination the size ofDNA of the clinical isolate
Helen et al., 2003
d. DNA sequencing To detection the nucleotidesequences of DNA of clinicalisolate
Altschul et al.,1997
e. RT-PCR To detect the molecular sizeof the DNA of the M.tuberculosis.
Heid et al., 1996
f. SDS-PAGE To determine the molecularsize of DNA of the resistantbacterial strain by band size
Zhang et al., 1992
2. Molecular characterization of Candida albicans1. Preliminary work Isolation of DNA Christine et al.,
19992. Agarose gel
electrophoresis [AGE]To purify of DNA Sambrook and
Russel, 20073. PCR To amplify DNA Christine et al.,
19994. DNA sequencing To determine the sequences
of DNA moleculeChristine et al.,1999
5. Phytogenetic analysis1. MEGA BLAST2. MEGA4
Analysis the similaritybetween the isolated strainand closely related strains
Christine et al.,1999
VI. Effect of medicinal herbal plants on the microbialpathogens1. Phytochemical analyses To detect the
phytocompounds ofmedicinal herbal plants
Trease and Evans,1996
2. Antimicrobial studiesa. Well diffusion for
bacteriaTo detect the antibacterialactivity of medicinal plants
Fazeli et al., 2007,
Easy PDF Creator is professional software to create PDF. If you wish to remove this line, buy it now.
52
b. Luciferase reporterphage assay forMycobacteria
To screen theantimycobacterial activity ofherbal extracts.
Sivakumar et al.,2007
c. Well diffusion assay forfungi
To detect the antifungalactivity of medicinal plantsagainst fungi
Zgoda and Porter,2001
d. TLC and bioautography To separate the antimicrobialcompounds
Wagner and Bladt,1996
3. Characterization analysesa. GC-MS Separation and
characterization of volatilecomponents in thephytochemical compounds.
Parasuraman et al.,2009
b. FT-IR Identification of functionalgroups of phytochemicalcompounds.
Sivaraju andKannan, 2010
3.4. CD4 COUNT PROCEDURE (Kahan and Jani, 2001)
20 µl of whole blood was added to a Partec test tube containing EDTA as
an anticoagulant. 20 µl of CD4 mAB PE was added and mixed gently and
incubated for 15 minutes at room temperature to protected from light, 800 µl of no
lyse buffer was then added and shaked or vortexed gently.
3.5. EXAMINATION OF BIOSAMPLES
3.5.1. Microscopic examination (Aneja, 2003)
Microbiological examination of pathogens was done using binocular
microscope (40X) following standard procedures and protocols (Table 1).
Staining techniques were performed to identify the bacterial, mycobacterial
and fungal pathogens namely gram’s staining, acid fast staining and lactophenol
cotton blue (LCB) staining respectively. Motility test was also examined for the
preliminary identification of the bacterial pathogens.
Easy PDF Creator is professional software to create PDF. If you wish to remove this line, buy it now.
53
3.5.2. Macroscopic examination on isolation of biosamples
3.5.2.1. Processing of samples (Allen and Baker, 1968)
Biosamples normally does not require processing for culturing bacteria and
fungi. Exceptionally they must be processed before handling them for HIV+
patients particularly sputum and urine was done by Modified Petroff’s Method.
3.5.2.2. Culture technique (Aneja, 2003)
The specific media used for the isolation of different bacteria were Nutrient
Agar. Lowenstein Jensen agar, Cetrimide agar, MacConkey agar, Mannitol salt
agar, blood agar, Chocolate agar, XLD agar and Eosine Methylene blue agar.
Specific media used for the isolation of different fungi were Rose Bengal agar,
Potato dextrose agar, Saborauds dextrose agar, Czapek dox agar, Chrom agar and
Brain heart infusion agar, which is prepared by the following standard procedures.
3.6. CHARACTERIZATION OF BACTERIA BY BIOCHEMICAL TESTS
(Aneja, 2003)
The following were biochemical tests performed for characterization of
identified bacterial pathogens namely Catalase, Oxidase, Indole, Methyl Red,
Voges Proskauer, Citrate Utilization, Urease, Hydrogen sulphide reduction and
Sugar fermentation tests (Aneja, 2003). The mycobacterial characterization
methods namely niacin test (Venkataraman and Prabakar, 1970) nitrate reduction
(Angeby et al., 2002) and PNB tests (Tsukamura and Tsukamura, 1964).
3.7. ANTIBIOTIC SENSITIVITY OF ISOLATED PATHOGENS
3.7.1. Antibiotic sensitivity test for bacterial pathogens (Bauer et al., 1966)
Krby-Bauer disc diffusion method is commonly employed for antibiotic
sensitivity tests for bacterial. The antibiotic discs were for used Amikacin,
Ampicillin, Carbennicillin, Cefixime, Ciprofloxacin, Norfloxacin, Rifampin,
Easy PDF Creator is professional software to create PDF. If you wish to remove this line, buy it now.
54
Streptomycin, Tetracycline and Vancomycin. The diameters of antibiotics
compared with standard charts.
3.7.1.1. Proportional sensitivity test method for mycobacteria (Tripathy et al.,
1970)
The antibiotic sensitivity of mycobacterial strains was done by proportional
sensitivity method. For the purpose of calculation, the number of colonies growing
on the drug-contianing medium was expressed as proportion of the estimated
number of colonies on the acidified drug-free medium.
Calculation
The CFU was calculated by using the following formula (Roberts et al.,
1991)
CFU =
No. of CFU (colony forming units) on drug slopes ×dilution factor
× 100No. of CFU (colony forming units) on drug free slopes ×dilution factor
1 (or) more than 1 per cent is resistant and less than 1 per cent is sensitive
(S) to concern drug (Always count the colonies from the higher dilution to lower
dilution (that is, S4, S3, S2, S1). If the colony count is more than 10 CFU only
consider for the calculation.
3.7.2. Antibiotic sensitivity tests for fungal pathogens (Bauer et al., 1966)
Antifungal activity test carried out by using the disc diffusion method. Ten
antibiotic discs namely amphotericin B, fluconazole, flucytosine, griseofulvin,
itraconazole, ketoconazole, miconazole, nystatin, trimethoprim and voriconazole
were placed upon the media isolated with inocula to detect the antibiotic
sensitivity of the isolated fungal organisms.
Easy PDF Creator is professional software to create PDF. If you wish to remove this line, buy it now.
55
3.8. MOLECULAR CHARACTERIZATION OF ISONIAZID RESISTANT
STRAIN OF M. tuberculosis
3.8.1. PCR amplification of isolated DNA (Cousins et al., 1992 and Helen
et al., 2003)
The isolated DNA was amplified using IS6110 primer in an automated
thermal cycler (Eppendorf). This confirms the template DNA of isolated DNA as
Mycobacterium tuberculosis.
3.8.1.1. Mycobacterial DNA extraction (Mani et al., 2003)
Mycobacterial DNA was extracted with the slight modifications of usual
DNA extraction method.
3.8.1.2. Isolation of inhA and katG gene from clinical isolate strain (Vijdea
et al., 2008)
The Mycobacterial DNA was extracted as stated above and the Taq
polymerase, dNTPs, MgCl2, Milli Q water, 10x Buffer, DMSO, Template forward
and reverse primer were used for amplification of each respective gene. The PCR
cycling parameters were 94°C for 5 minutes; followed by 40 cycles of 94°C for 1
minute, 57°C for 1 minute and 74°C for 1 minute and a final extension of 74°C for
5 minutes. The PCR was then kept at hold at 4°C for 15 minutes.
3.8.1.3 Agarose gel electrophoresis (Helen et al., 2003)
The amplified PCR product inhA and katG gene from clinical isolate strain
were run on 2 per cent agarose gel and purify the PCR product using PCR
purification kit (Invitrogen).
Easy PDF Creator is professional software to create PDF. If you wish to remove this line, buy it now.
56
3.8.1.4. PCR cleanup procedure
The amplified PCR products was purified using charges witch PCR clean
up kit (Invitrogen catalog No.CS12000) prior to given for DNA sequencing.
3.8.2. DNA sequencing analysis (isoniazid inhA and katG genes) (Williams
et al., 1995; Altschul et al., 1997)
The purified electrophoresed PCR product of inhA and katG genes were
and subjected to DNA sequencing analysis. The purified PCR product was directly
sequenced in an automated DNA Sequencer at Bioserve in Bangalore. The
nucleotide sequence obtained was analyzed using BLASTn Bioinformatics tool
available at National Center for Biotechnology Information to know the specificity
of PCR amplification and to identify the nucleotide variation. The sequence was
further subject for BLASTx to know the amino acid changes in comparison with
the wild type M. tuberculosis (H37Rv).
3.8.3. RT-PCR analysis (Marin et al., 2004)
It is necessary to add internal control (IC) in the reaction mix. IC allows the
user to determined and control the possibility of PCR inhibition. The internal
control (IC) (1 µl/reaction) was added and the result will be shown in the VIC/JOE
channel. The kit can be used for quantitative (or) qualitative Real-time PCR.
A positive control defined as 1 × 10-1 IU/ml. The positive control (1 × 10-1 IU/ml)
was taken as the starting high standard in the first tube. Respectively 36 µl of
molecular grade water was pipetted out into next three tubes. Three dilutions were
done. To generate a standard curve on the Real-time PCR system, all four dilution
standard should be used and defined as standard with specification of the
corresponding concentrations.
Easy PDF Creator is professional software to create PDF. If you wish to remove this line, buy it now.
57
RT-PCR protocol (Torres et al., 2003)
RT-PCR analysis was done by the method.3 µl Master Mix + 5 µl Sample / Positive control / Negative control
Reaction Plate / Tube
RT-PCR instrument
45°C for 10 minutes, 1 cycle
95°C for 15 minutes, 1 cycle
60°C for 60 seconds, 40 cycles
Fluorescence was measured at was measured at 60°C.
3.8.4. SDS-PAGE electrophoresis and nano LC/MS analysis
SDS-PAGE was used to separate the proteins from the clinically isolated
M. tuberculosis, the suspected protein spot of the gel was cut by a sterile surgical
blade and it was transferred to the 2 ml eppendorff tube containing 1.5 ml of milli
Q water. The protein sample was subjected to NANO LC/MS in the Center for
Molecular Medicine at New Delhi.
3.8.5. Phylogenetic analysis
Mycobacterial DNA was extracted from TB coinfected HIV patient. The
respective 16s RNA gene was isolated and amplified by using Taq polymerase,
dNTPs, MgCl2, Milli Q water, 10x Buffer, DMSO, template, forward and reverse
primer in the Thermal cycles. The PCR cycling parameters were 94°C for 5
minutes: followed by 40 cycles of 94°C, 57°C and 74°C for 1 minute and a final
extension of 74°C for 5 minutes. The PCR was then kept at hold at 4°C for 15
minutes. The amplified PCR product was withdrawn from thermal cycler and run
Easy PDF Creator is professional software to create PDF. If you wish to remove this line, buy it now.
58
on a 2 per agarose gel in TAE buffer. The ethidium bromide stained gels were
observed in a UV transilluminator and photographed using a Geldoc. The
amplified PCR product were purified using charge switch PCR cleanup kit
(Invitrogen catalog No.CSI2000). The purified PCR products was directly
sequenced in an automated DNA sequencer at Bioserve in Bangalore. The
nucleotide sequences were analyzed using CLUSTAL W algorithm.
3.9. MOLECULAR CHARACTERIZATION OF PYRAZINAMIDE
RESISTANCE STRAIN OF M. tuberculosis
PCR amplification of isolated DNA was done by using the method of
Cousins et al., 1992. Mycobacterial DNA extraction was done by using the
method of Mani et al., 2003. Isolation of pncA gene from TB confected HIV
patient was done by using the method of Vijdea et al., 2008. Agarose gel
electrophoresis was done by using the method of Helen et al., 2003. The amplified
PCR products were purified using charge switch PCR clean up kit (Invitrogen
catalog No.CS12000) prior to given for DNA sequencing. DNA sequencing
analysis (Pyrazinamide-pncA) was done by using the method of Williams et al.
(1995). RT-PCR analysis was done by using the method of Marin et al. (2004).
SDS-PAGE was done by using the method of Zhang et al. (1992).
Nano LC/MS process was done following the similar method for isoniazid
resistant strains described above. The protein sample was subjected to NANO
LC/MS in Center for Molecular Medicine at New Delhi. The amplified PCR
products were purified using charge switch PCR clean up kit (Invitrogen catalog
No.CS12000). The purified PCR product was directly sequenced in an automated
DNA Sequencer at Bioserve in Bangalore. The nucleotide sequence obtained was
analyzed using CLUSTAL W algorithm.
Easy PDF Creator is professional software to create PDF. If you wish to remove this line, buy it now.
59
3.10. MOLECULAR CHARACTERIZATION OF Candida albicans
3.10.1. PCR amplification of isolated fungi (Christine et al., 1999)
3.10.1.1. DNA isolation from cultured cells using master pureTM Yeast DNA
purification kit
The culture was harvested from 25 ml culture media after the 5 days of
incubation. The culture was rinsed with several volumes (original culture
volume)of 0.1 M MgCl2. The culture pellet was transferred to a chilled mortar and
grind the mycelium to a powder in the presence of liquid nitrogen. The powder
was transferred to microcentrifuge tube and continue with cell lysis and
precipitation of DNA.
3.10.1.2. Cell lysis and precipitation of DNA
The yeast cell lysis solution was thoroughly mixed to ensure uniform
composition before dispensing. 300 ml of Yeast Cell Lysis solution was added to a
microcentrifuge tube. The cells were suspended by either vortex mixing of
pipetting the cells repeatedly using a 1 ml capacity pipette tip. The suspended cells
were incubated at 65°C for 15 minutes. The samples were placed on ice for 5
minutes 150 ml of MPC protein precipitation reagent was added and vortex mixed
for 10 seconds. Pellet cellular debris was pelleted by centrifugation in a
microcentrifuge for 10 minutes at 10,000 rpm. The supernatant was transferred to
a clean microcentrifuge tube and 500 ml of isopropanol was added and mixed
thoroughly by inversion. The DNA was pelleted out by centrifugation in a
microcentrifuge for 10 minutes at 10,000 rpm. The supernatant was removed by
pipetting and discard. The pellet containing the DNA was washed with 0.5 ml of
70 per cent ethanol. The ethanol was carefully removed by pipetting and
discarded. The DNA pellet was briefly centrifuged and remaining ethanol was
removed. The DNA was suspended in 35 ml of TE Buffer. The DNA was stored at
4°C.
Easy PDF Creator is professional software to create PDF. If you wish to remove this line, buy it now.
60
3.10.1.3. RNase A treatment
The presence of RNase A may interfere with subsequent PCR amplification
of fungal genomic DNA. 1 ml of 5 mg/ml RNase A was added to the purified
DNA and it was incubated at 37°C for 30 minutes.
3.10.1.4. Quantitation and quality assessment of DNA
The DNA stock samples were quantified using Nanodrop
spectrophotometer at 260 and 280 nm using the convention that one absorbance
unit at 260 nm wavelength equals 50 µg DNA per ml. The UV absorbance was
checked at 260 and 280 nm for determination of DNA concentration and purity.
Purity of DNA was judged on the basis of optical density ratio at 260:280 nm. The
DNA ratio between 1.8 and 2.0 was considered to be of good purity.
Concentration of DNA was estimated using the formula.
Concentration of DNA (mg/ml) = OD 260 × 50 × dilution factor
3.10.1.5. Agarose gel electrophoresis (Sambrook and Russel, 2001)
The quality and purity of DNA were checked by agarose gel
electrophoresis. Agarose 0.8 per cent (w/v) in 0.5X TAE (pY 8.0) buffer was used
for submarine gel electrophoresis. Ethidium bromide (1%) was added @ 10 µl/100
ml. The wells were charged with 5 µl of DNA preparations mixed with 1 µl gel
loading dye. Electrophoresis was carried out at 80 V for 30 min at room
temperature. DNA was visualized under UV using UV transilluminator. The DNA
was used further for PCR.
3.10.1.6. Polymerase chain reaction (PCR)
Internal transcribed spacer (ITS) gene fragment was amplified by PCR
genomic DNA using ITS gene universal primers: IF and IR.
Easy PDF Creator is professional software to create PDF. If you wish to remove this line, buy it now.
61
Details of primers used for PCR
IF: 5′-TCC GTA GGT GAA CCT GCC G-3′
IR: 5′-TCC TCC GCT TAT TGA TAT GC-3′
PCR was carried out in a final reaction volume of 25 µl in 200 µl capacity thin
wall PCR tube. PCR tubes containing the mixture were tapped gently and spin
briefly at 10,000 rpm. The PCR tubes with all the components were transferred to
thermal cycler. The PCR protocol designed for 30 cycles for the primers.
3.10.1.7. Visualization of PCR product
To confirm the targeted PCR amplification, 5 µl of PCR product from each
tube was mixed with 1 µl of 6X gel loading dye and electrophoresed on 1.2 per
cent agarose gel containing ethidium bromide (1 per cent solution @ 10 µl/100
ml) at constant 5V/cm for 30 min in 0.5 X TAE buffer. The amplified product was
visualized as a single compact band of expected size under UV light and
documented by gel documentation system (Gene Genius, SynGene Bio Imaging
System, UK).
3.10.1.8. Purification of PCR product
Amplified PCR product was purified using Exosap enzyme as per the
protocol given in table 3 and further used for sequencing reaction.
3.10.2. Sequencing of purified DNA
Sequencing of Purified Internal Transcribed Spacer (ITS) Gene Segment
The concentration of the purified DNA was determined and was subjected
to automated DNA sequencing on ABI 3730×1 genetic analyzer (Applied
Biosystems, USA). Sequencing was carried out using BigDye® Terminator v3.1
Cycle sequencing kit following manufacturers instructions.
Easy PDF Creator is professional software to create PDF. If you wish to remove this line, buy it now.
62
Cycle sequencing
Cycle sequencing was performed following the instructions supplied along
with BigDye® Terminator v3.1 Cycle Sequencing Kit. The reaction was carried
out in a final reaction volume of 20 µl using 200 µl capacity thin wall PCR tube.
The cycling protocol was designed for 25 cycles with the thermal ramp rate of 1°C
per second. After cycling, the extension products were purified and mixed well in
10 µl of Hi-Di formamide. The contents were mixed on shaker for 30 minutes at
300 xg. Eluted PCR products were placed in a sample plate and covered with the
septa. Sample plate was heated at 95°C for 5 min, snap chilled and loaded into
autosampler of the instrument.
Electrophoresis and data analysis was carried out on the ABI 3730xl
Genetic analyzer using appropriate Module, Basecaller, Dyeset/Primer and Matrix
filers.
3.10.3. Sequence analysis by bioinformatics tools
The obtained sequenced data of forward and reverse reactions were used to
great a consensus sequence of large subunit DNA (LSU DNA). Both end of the
sequence was verified with the chromatogram file and edited if required. The
sequence was converted into FASTA format and saved in notepad. Internal
transcribed spacer (ITS) gene sequence were used to carry out BLAST (Basic
local alignment search tool) with nr database of NCBI Genbank using
MEGABLAST algorithm. The BLAST data was arranged in maximum percentage
identify and first ten sequence was selected and exported in FASTA format. Based
on maximum identity score and query coverage the best highly identical 10
sequences were selected and aligned using multiple alignment software program
ClustaW (MEGA4 tool). The evolutionary history was inferred using the
Neighbour-joining method. The bootstrap consensus tree inferred from 500
replicates is taken to represent evolutionary history of the taxa analyzed. The
Easy PDF Creator is professional software to create PDF. If you wish to remove this line, buy it now.
63
evolutionary distances were computed using the Kimura 2-parameters method.
Phylogenetic analysis was conducted in MEGA4.
Software tools
3.10.3.1. BLAST
BLAST® (Basic Local Alignment Search Tool) is a set of similarity search
programs designed to explore all of the available sequence databases regardless of
whether the query is protein or DNA. The BLAST programs have been designed
for speed, with a minimal sacrifice of sensitivity to distant sequence relationships.
The scores assigned in a BLAST search have well-defined statistical
interpretation, making real matches easier to distinguish from random background
hits. BLAST uses a heuristic algorithm that seeks local as opposed to global
alignments and is therefore able to detect relationships among sequences that share
only isolated regions of similarity (Altschul et al., 1990).
Algorithm: MEGABLAST
The best way to identify an unknown sequence is to see if that sequence
already exists in a public database. If the database sequence is a well-characterized
sequence, then one will have access to a wealth of biological information
MEGABLAST, discontiguous-megablast and blast all an beused to accomplish
this goal. However, MEGABLAST is specifically designed to efficiently find log
alignment between very similar sequences and thus is the best tool to use to find
the identical match to your query sequence. In addition to the expect value
significance cut-off, MEGABLAST also provides an adjustable percent identity
cut-off for the alignment, which provides cut-off in addition to the significance
cut-off threshold set by expect value.
Database “nr”
Easy PDF Creator is professional software to create PDF. If you wish to remove this line, buy it now.
64
All GenBank + EMBL + DDBJ + PDB sequences (but no EST, STS, GSS,
or phase 0, 1 or 2 HTGS sequences). No longer “non-redundant” due to
computational cost.
3.10.3.2. MEGA4
MEGA is an integrated tool for conducting automatic and manual sequence
alignment, inferring phylogenetic trees, mining web-based databases, estimating
rates of molecular evolution and testing evolutionary hypotheses.
3.11. EFFECT OF MEDICINAL HERBAL PLANTS ON THE MICROBIAL
PATHOGENS
3.11.1. Collection of samples
Fresh herbal leaves of Acalypha indica L., Adhatoda vasica L., Calotropis
procera L., Datura metel L., Ocimum basilicum L., Solanum trilobatum L.,
Tylophora indica M., Vitex negundo L. and Withania somnifera Dunal and root
parts of Acorus calamus L. were collected from the herbal garden of PRIST
University, Thanjavur and were identified with the help of a botanist (Plaet II(A)).
The leaves and root parts of respective medicinal plants were washed with distilled
water separately and used for this study.
3.11.1.1. Medicinal herbal plants
Acalypha indica L.
Kingdom : Plantae
Order : Malpighiales
Family : Euphorbiaceae
Genus : Acalypha
Species : indica
Common Name : Kuppai meni.
Easy PDF Creator is professional software to create PDF. If you wish to remove this line, buy it now.
65
Acorus calamus L.
Kingdom : Plantae
Order : Acorales
Family : Acoraceae
Genus : Acorus
Species : calamus
Common Name : Sweet flag, sweet sledge.
Adhatoda vasica L.
Kingdom : Plantae
Order : Lamiales
Family : Acanthaceae
Genus : Adhathoda
Species : vasica
Common Name : Adulsa(vasaka)
Calotropis procera L.
Kingdom : Plantae
Order : Gentianales
Family : Apocynaceae
Genus : Calotropis
Species : procera
Common Name : Sodom apple
Datura metel L.
Kingdom : Plantae
Phylum : Tracheophyta
Class : Magnoliopsida
Easy PDF Creator is professional software to create PDF. If you wish to remove this line, buy it now.
66
Order : Solanales
Family : Solanaceae
Genus : Datura
Species : metel
Ocimum basilicum L.
Kingdom : Plantae
Order : Lamiales
Family : Lamiaceae
Genus : Ocimum
Species : basilicum
Common Name : Sweet basil
Solanum trilobatum L.
Taxonomic hierarchy of Solanum trilobatum L.
Kingdom : Plantae
Phylum : Tracheophyta
Subphylum : Euphyllophytina
Class : Magnoliopsida
Order : Solanales
Family : Solanaceae
Genus : Solanum
Species : trilobatum
Tylophora indica M.
Taxonomic hierarchy of Tylophora indica L.
Kingdom : Plantae
Order : Gentianales
Easy PDF Creator is professional software to create PDF. If you wish to remove this line, buy it now.
67
Family : Apocyanaceae
Genus : Tylophora
Species : indica
Common name : Indian ipecac
Vitex negundo L.
Kingdom : Plantae
Order : Lamiales
Family : Verbenaceae
Genus : Vitex
Species : negundo
Common Name : Nirgund
Withania somnifera D.
Taxonomic hierarchy of Withania somnifera L.
Kingdom : Plantae
Division : Angiosperma
Class : Dicotyledoneae
Order : Tubiflorae
Family : Solanaceae
Genus : Withania
Species : somnifera Dunal
3.11.2. Preparation of medicinal herbal plant extracts
The leaves of nine medicinal plants and root materials of one plant were
carefully washed with tap water, rinsed with distilled water and air dried for one
hour. After washing the roots were cut into small pieces. Then the leaves and
small root segments were dried at room temperature. After drying process, they
were ground into powder and stored at room temperature in sterile pockets.
Easy PDF Creator is professional software to create PDF. If you wish to remove this line, buy it now.
68
3.11.3. Extraction method (Plate IIB-D)
The medicinal herbal plant leaves and root extracts were obtained by
soxhlet apparatus. For this extraction, four different solvents such as hexane,
ethylacetate, diethyl ether and methanol were used. After extraction, the solvent
was removed by condensation of the extracts.
3.11.4. Phytochemical analysis (Trease and Evans, 1996)
The herbal extracts were subjected to various phytochemical tests
separately. Different qualitative chemical tests were performed for determining the
phytoconstituents present in the plant extracts. Phytochemical analysis were done
according to the procedure of phytochemical methods. Methanolic extract of
plants were used for qualitative phytochemical analysis. The results of the
analyses of ten herbal extracts were tabulated.
Detection of tannins: 0.5 g of the dried powdered sample of each plant was
separately added with 20 ml of water and boiled in a test tube and filtered. A few
drops of 0.1 per cent of ferric chloride solution was added and observed for
brownish green or blue black colouration which indicated the presence presence of
tannin.
Detection of saponins
The 50 mg of each plant extract was diluted with distilled water and made
upto 20 ml. The suspension was shaken in a graduated cylinder for 15 mins. A 2
cm layer of foam indicated the presence of saponins.
Detection of flavonoids
Shinoda’s test: In a test tube containing 0.5 ml of extract 5-10 drops of diluted
HCl and small piece of ZnCl or magnesium were added and the solution was
boiled for few minutes. Appearance of reddish brown colour indicated the
presence of flavonoids.
Easy PDF Creator is professional software to create PDF. If you wish to remove this line, buy it now.
69
Alkaline reagent test: A few drops of diluted NaOH was added to the extract. An
intense yellow colour was produced and become colourless after of few drops of
diluted acid indicated the presence of flavonoids.
Detection of Terpenoids: 0.5 g of crude powder was dissolved in 5 ml of
methanol. 2 ml of the extract was treated with 1 ml of 2, 4-dinitrophenyl hydrazine
dissolved in 100 ml of 2 M HCl. A yellow-orange colouration was observed as an
indication of terpenoids.
Detection of Glycosides: Each plant extract of about 50 mg was hydrolysed with
concentrated HCl for 2 hours on a water bath, filtred and the hydrolysate was
subject to the test.
Borntrager’s test: To 2 ml of filtrate hydrolysate, 3 ml of chloroform was added
and shaken. Chloroform layer was separated and 10 per cent ammonia solution
was added to it. Appearance of pink colouration suggested the positive response
for anthraquinones indicated the presence of phenolic compounds.
Detection of Phenolic compounds
Ferric chloride test: 50 mg of each plant extract dissolved in 5 ml of distilled
water. To this, few drops of neutral 5 per cent ferric chloride solution was added.
A dark green colour indicated the presence of phenolic compounds.
Gelatin test: 50 mg of the extract dissolved in 5 ml of distilled water, 2 ml of 1
per cent solution of gelatin containing 10 per cent sodium chloride was added to it.
White precipitate indicated the presence of phenolic compounds.
Lead acetate test: 50 mg of the extract was dissolved in 5 ml of distilled water.
To this 3 ml of 10 per cent lead acetate solution was added. A bulky white
precipitate formed as an end point.
3.11.5. Detection of fixed oils and fats
Saponification: A few drops of 0.5 N alcoholic potassium hydroxide solution was
added to a small quantity of extract along with a drop of phenolphthalein. The
Easy PDF Creator is professional software to create PDF. If you wish to remove this line, buy it now.
70
mixture was heated on a water bath for 2 hours. The formation of foam as a partial
neutralization of alkali indicated the presence of fixed oils and fats.
3.11.6. Detection of Alkaloids
Mayers Test: 50 mg of solvent free extract was stirred with few ml of diluted HCl
and filtered. To 1.2 ml of filtrate, 0.1 ml of Mayer’s reagent was added. A white
creamy precipitate indicated the presence of alkaloids.
Wagners Test: 5 g of the powdered sample was extracted by boiling in 50 ml of
distilled water in a water bath for 30 min. It was then filtered into a test tube and
the filtrate collected. The filtrate was tested with Wagner’s reagent and results
compared to blanks.
Hagers Test: 5 g of the powdered sample was extracted by boiling in 50 ml of
distilled water in a water bath for 30 min. It was then filtered into a test tube and
the filtrate collected. The filtrate was tested with Hager’s reagents and results
compared to blanks.
3.11.7. Detection of Carbohydrates
The extract (100 mg) was dissolved in 5 ml of water and filtered. The
filtrate was subjected to the following tests.
a) Molish’s test: To 2 ml of filtrate of each plant extract, 2 drops of alcoholic
solution of α-napthol was added. The mixture was shaken well and 1 ml of
concentrated sulphuric acid was added slowly along the sides of the test tube
and allowed to stand. The formation of violet ring indicated the presence of
carbohydrates.
b) Fehling’s test: One ml of each Fehling’s solution A and B was mixed with the
2 ml of plant extract and boiled on water bath. An yellow red precipitate was
formed indicated the positive reaction of Fehlings test.
Easy PDF Creator is professional software to create PDF. If you wish to remove this line, buy it now.
71
c) Benedict’s test: One ml of benedicts reagents was added with 2 ml of plant
extract and boiled in a water bath for 2 minutes. The formation of brick red
precipitate indicated the positive reaction of benedicts test.
3.11.8. Detection of proteins and aminoacids
The 100 mg each plant extract was dissolved in 10 ml of distilled water and
filtered through Whatmann no:1 filter paper. The filtrate was subjected to proteins
and amino acids determination.
Millon’s test: To 2 ml of filtrate, few drops of Millon’s reagent was added. A
white precipitate indicated the presence of proteins.
Biuret test: An aliquot of 2 ml of filtrate was mixed with one drop of 2 per cent
copper sulfate. To this 1 ml of ethanol (95%) was added followed by excess of
potassium hydroxide pellets. The pink colour in the ethanolic layer indicated the
presence of proteins.
3.11.9. Detection of Phytosterols
Libermann-Burchard test: 50 mg of plant extract was mixed with drops of acetic
anhydride and sulfuric acid. The appearance of a blue-green colour is a positive
test for cholesterol.
3.11.10. Antimicrobial activity of herbal extracts
3.11.10.1. Well diffusion assay for antibacterial activity (Fazeli et al., 2007)
Agar well diffusion assay is used widely to determine the antibacterial
activity of crude extract containing unknown components (Perez et al., 1990). 24
hours growing culture of bacterial pathogens namely E. coli, S. typhi,
P. aeruginosa, E. aerogenes, S. aureus, K. pneumoniae, P. vulgaris, C. botulinum,
S. pneumoniae and S. dysentriae were swabbed separately on the nutrient agar
media containing plates, different extracts namely hexane, ethyl acetate and
Easy PDF Creator is professional software to create PDF. If you wish to remove this line, buy it now.
72
methanol with three types of concentrations used viz., 100 µg (10 µl), 250 µg (25
µl) and 300 µg (30 µl).
3.11.10.2. Well diffusion assay for antifungal activity (Zgoda and Porter,
2001)
Agar well diffusion assay is also used to determine the antifungal activity
of medicinal herbal plants. Three types of solvents have been used namely
ethylacetate, diethyl ether and methanol. Various fungal pathogens namely
C. albicans, A. niger, A. flavus, A. terreus, H. capsulatum, P. marneffei,
B. dermatidis, C. neoformans, F. moniliforme and F. solani were swabbed
separately on the SDA media containing plates. The wells about 10 mm in
diameter were made by using cork borer. Different concentrations of plant extracts
namely 100 µg (10 µl), 500 µg (50 µl) and 1000 µg (100 µl).
3.1.10.3. Screening of antimycobacterial activity of Withania somnifera
extracts (Sivakumar et al., 2007)
Antimycobacterial activity of W. somnifera was done by Luciferase
reporter phage assay. The procedure (Riska et al., 1999) was optimized and
modified by Dr. Vanaja Kumar Tuberculosis Research Centre. Chetpet. Chennai,
India. Fifty-microliter bacterial suspension of M. tuberculosis equivalent to
MacFarlands No.2 standard was added to 400 µl of G7H9 with and without the
test compound. Hexane and methanolic extracts of W. somnifera separately added
with 1 per cent DMSO (Vehicle solvent). Two different concentrations of hexane
and methanol extracts were used viz., 100 and 500 µg. For each sample, two drug-
free controls and two drug concentrations were prepared and this set up was
incubated for 72 h at 37°C. After incubation 50 µl of the high titer Luciferase
reporter phage (phAE 129) and 40 µl of 0.1 M CaCl2 were added to all the vials
and this setup was incubated at 37°C for 4 h. After incubation 100 µl of the
Easy PDF Creator is professional software to create PDF. If you wish to remove this line, buy it now.
73
mixture was taken from each tube into a luminometer cuvette and equal amount of
working D-luciferin (0.3 mM in 0.05 M sodium citrate buffer. pH 4.5) solution
was added. The RLU was measured after 10 s of integration in the Luminometer
(Monolight 2010). Duplicate readings were recorded for each sample and the
mean and standard deviation values were calculated. The percentage reduction in
the RLU was calculated for each test sample and compared with control.
3.11.10.4 Thin layer chromatography (TLC) (Wagner and Bladt, 1996)
In order to separate antimicrobial principles, the crude leaf extracts showed
high antibacterial and antifungal activity was subjected to thin layer
chromatography. The separation of the compound depends on the usage of the
solvent. Here the solvent used were 5 and 10 per cent methanol in chloroform.
1 mg/ml concentration of the effective plant extract was spotted on the TLC plates
and dried. It was then run with both ultraviolet and iodine chamber. Thin layer
chromatography was performed on Merck TLC F254 plates, with chloroform:
Methanol (95:5) as mobile phase. The separated components were visualized
under visible and ultraviolet light (254 and 360 nm). The Retention Factor (RF)
value was calculated by using the following formula.
Distance traveled by soluteRF = _________________________
Distance traveled by solvent.
The compounds from the spots were scrabbed and used for further screening.
3.11.10.5. Bioautography (Beague and Kline, 1972)
Bioautography is a rapid aid in the bioassay guided isolation and
fractionation of antibacterial compounds and its fractions. In this approach, the
activity of plant extracts against the pathogens are determined on chromatograms
in accordance with the bioautography procedure. Developed chromatography
plates of crude extract was dried overnight sprayed with a suspension of actively
Easy PDF Creator is professional software to create PDF. If you wish to remove this line, buy it now.
74
growing cells of bacteria and fungi and incubated at 37 and 24°C respectively in a
chamber at 100°C relative humidity for 18 hrs. Plants were sprayed with MTT-3
(4,5-Dimethyl thiazol-2.5- Diphenyl tetrazolium Bromide) (5 mg/ml). Clear zones
on the chromatogram indicate inhibition of growth after incubating for hours at
37°C. The method was chosen for its simplicity, low cost, accuracy and rapid
result that make it ideal for bioassay guided isolation (Eloff, 1998).
3.11.11. Characterization studied on herbal plants
3.11.11.1. Gas chromatography and mass spectrometry of purified TLC
compound of effective antibacterial and antifungal herbal plants
(Parasuraman et al., 2009)
GC-MS techniques involves the separation and characterization of volatile
components in a test sample of phytochemical compound. Instrument control
parameter were shown in Fig. Here the compounds from both the herbal extracts
of W. somnifera and A. India L. acquired through TLC were tested in this GC-MS
analysis. In this analysis, a capillary column was used. An inert gas helium was
used as a carrier gas. The components of a compound was evaporated in the
injector of GC equipment and segregated in the column by adsorption and
absorption technique with suitable temperature of oven about 325°C controlled by
software. The GC column was heated in the oven, each and every component of
the test compound was eluted from the column, the time termed as Retention Time
(RT) was noted. The eluted components were tested in the mass detector. The
spectrum of the unknown components were compared with the spectrum of known
components stored in the NIST library, thus the name, molecular weight and
structure of the components of the test compound were ascertained. It is not
possible to make an accurate identification of a molecules by gas chromatography
or mass spectrometry alone. The mass spectrometry normally requires a very pure
sample while gas chromatography using a traditional detector detects multiple
Easy PDF Creator is professional software to create PDF. If you wish to remove this line, buy it now.
75
molecules that happen to take the same amount of tie to travel through the column
have the same retention time which results in two or more molecules to co elute.
Therefore when an identifying mass spectrum appears at a characteristic retention
time in a GC-MS analysis, it typically tends to increased certainty that analyte of
interest was in the sample.
3.11.10.2. FT-IR analysis of effective compound (Sivaraju and Kanna, 2010)
FT-IR analyses were done by the procedure. ATR model FT-IR
Spectrophotometer (Bruker Co., Germany) was used for the analysis of the
methanolic crude extracts of A. indica and W. somnifera. The compound acquired
through TLC was subjected to FT-IR. The samples was individually milled with
potassium bromide (KBr) to form a very fine powder. The powder was then
compressed into a thin pellet which can be analyzed. The spectrum was recorded
using Attenuated Total Reflectance (ATR) technique beach measurement.
Easy PDF Creator is professional software to create PDF. If you wish to remove this line, buy it now.