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Global Journal of Patholog y and Microbiology, 2013, 1, 17-23 1

E-ISSN: 2310-8703/13 © 2013 Pharma Profess ional Services

The Practice of Basic Principles in Routine DiagnosticBacteriology for Better Diagnostic Value: A Review

J.O. Isibor *

Department of Microbiology, Faculty of Natural Sciences, Ambrose Alli University, Ekpoma, Edo State,Nigeria

Abstract: The routine medical bacteriology laboratory is saddled with the responsibility of diagnosing the etiologicalagents of diseases that afflict humans. The ability to control such bacterial infections is largely dependent on the ability todetect the etiological agents and to reliably identify organisms of clinical significance in a cost effective and timelymanner. Such services to the hospital patients are usually urgently required by the clinician for the benefit of the endreceiver; the patient. Quick as the results may be so desired, basic principles and standard operational procedures stillneed to be followed without sacrificing efficiency and correct diagnosis at the altar of speed. The bacteriologist needs toadhere strictly to aseptic techniques while carrying out his diagnostic procedures. The benefits of newer diagnostictechniques such as sequence analysis of the 16S rRNA gene, notwithstanding, the bacteriologist still need to view aGram-stained smear of the specimen microscopically, culture the clinical specimens for purposes of isolating thecausative agent of infection, as well as determine the antibacterial susceptibility pattern, without which antibiotic therapycannot be effectively instituted. These procedures, carefully controlled, have their indispensible value in routinediagnostic bacteriology. Although the newer technologies are reported to be reliable and rapid, users must be mindful ofthe common cliché, “garbage - in - garbage - out”! In addition, newer technologies are however not readily within theeconomic reach of most health facilities the world over. This review highlights some important and indispensible

principles required by the practicing bacteriologist for effective identification and diagnosis of bacterial infections.

Keywords: Laboratory diagnosis, bacteriology, clinical specimens, valuable techniques.

1. INTRODUCTION

A major role played by health institutions all over the

world is that of rendering effective and efficient health

care service to those requiring such services. Through

a well coordinated team work, health professionals

from various disciplines contribute their expertise to

effect a holistic treatment for those in need. The health

status of any individual is usually determined by three

main factors, viz, the individual (host), the diseaseetiology (pathogen) and the environmental factors.

Whenever environmental conditions are so favourable

for the pathogen to thrive in, and the host becomes

vulnerable, disease or infection sets in. The presence

of an infection in a host oftentimes heralds the

production of symptoms, although there may be an

asymptomatic infection. In addition to the doctor’s

clinical assessment of the patient, laboratory

investigations are meant to complement, and in most

cases, direct the course of effective treatment for the

patient.

The purpose of this review therefore, is to highlight

those basic diagnostic techniques often overlooked by

bacteriologists in their routine bench work.

Understanding these principles could go a long way in

*Address correspondence to this author at the Department of Microbiology,Faculty of Natural Sciences, Ambrose Alli University, Ekpoma, Edo State,Nigeria; Tel: + (234)803 5515110; E-mail: [email protected]

assisting the clinicians, the bacteriologist and the

patient who is at the receiving end.

2. THE CLINICAL SPECIMEN

According to Caroll [1], laboratory procedures used

for diagnosing disease conditions cut across

microscopic examination of patients’ specimens, the

detection of antigen from the infecting agent by

immunologic assay, nucleic acid hybridization studiesto detect pathogen-specific genes in patients

specimens, analysis of patients’ specimens and using

PCR techniques and serological detection o

antibodies. Serology, however, has its pitfalls [2]. In a

these, the bacteriologist must realize that irrespective

of the technique used, the result of diagnosis can be

only as reliable and correct as the original specimen

submitted for investigation will allow.

Whenever possible and before antibiotic therapy is

instituted, it is most desirable to aseptically collec

enough representative specimens for prompexamination. Here, emphasis must be laid on the

requirement that in handling any clinical specimen, the

bacteriologist must engage his laboratory work unde

aseptic conditions. Aseptic techniques involve

developing both manual dexterity in safely handling

microorganisms and mental dexterity in thinking ahead

about what one is doing with the microorganisms. The

concept of aseptic technique began with the pioneering

work of Dr. Ignaz Phillip Semmelweis, a Hungarian



18 Global Journal of Pathology and Microbiology, 2013, Vol. 1, No. 1 J.O. Isibo

physician of German extraction. Fondly referred to as

the “Saviour of Mothers”, he discovered that the

incidence of puerperal fever could be drastically

reduced by the use of hand disinfection in obstetrical

clinics [3]. He proved the capability of reducing

mortality from about 10% - 35% to below 1% through

hand washing with chlorinated lime solution. Other

basic safety procedures, often disregarded by routinebench workers include, (a) the need to wear laboratory

coats and gloves, (b) the provision of discard jars

containing appropriate concentration of disinfectants for

collecting used slides, contaminated pipettes etc, (c)

the need for proper decontamination of the laboratory

bench with suitable disinfectant before and after

laboratory work. In carrying out laboratory procedures

such as biochemical testing of isolates (e.g. testing

Staphylococcus aureus for presence of catalase

enzyme), special precautions must be taken to prevent

the contamination of the skin or workbench by aerosols

generated by the reaction. For example, at the point of

transferring a test colony of S. aureus onto a few drops

of hydrogen peroxide placed on a glass slide, rather

than leave the entire preparation exposed, the slide

could be placed in a Petri dish, while the cover is used

to shield the vulnerable parts of the body from the

aerosol generated in a positive catalase reaction.

3. COOPERATION BETWEEN THE DOCTOR’S

CLINIC AND THE BACTERIOLOGIST’S LABORA-

TORY

This important necessity is sometimes taken forgranted by practitioners, resulting to these personnel

working at cross-purposes to the detriment of the

patient’s health. The channel of communication is often

strengthened when collective attention is focused on

the patient. The prompt collection of sufficient and

representative amount of the patients’ clinical

specimen(s) is a sine qua non to a successful

laboratory diagnosis. It must also be stressed that it is

essential to view any specimen microbiologically. In

order words, the possibility of encountering any type of

microbe from the patient’s specimen may not be ruled

out. Hence, the provision of enough clinical historyalongside the laboratory request-form could help

prevent what would otherwise result in a futile exercise;

indeed, a wild-goose chase!

4. WHEN DO WE CONSIDER AN INFECTION

SIGNIFICANT?

The infectious agents encountered in the practice of

medical microbiology are legion and no single test will

permit isolation or characterization of all the potential

pathogens. It is therefore imperative for one to be

conversant with the human microbiota in order to be

able to adjudge which is a likely pathogen, or a

member of the resident flora. There is need for caution

here! An infection is said to occur whenever a microbe

successfully invades the primary defense armour of the

human body, attaches to and multiply within sus

ceptible cells, sometimes spreading to other organsand tissues of the host’s body. Many infections are

caused by organisms that are permanent or transien

members of the normal flora. For example, coagulase

negative S. aureus, hitherto regarded as a commensa

when living on the human skin, has been isolated from

patients suffering from bacterial meningitis [4]. The

incidence of significant bacteriuria involving coagulase

negative S. aureus has also been reported by Souveni

et al. [5]. Escherichia coli, a normal resident of the

gastrointestinal tract of healthy individuals, are the

most frequently documented uropathogens [6], just as

it usually can be isolated from infected wounds [7, 8]The relative numbers of specific organisms found in a

culture are important when members of the norma

flora are the cause of infection. The lesson here is tha

the presence of an organism in an anatomical site

other than its “normal” residence must be furthe

investigated by the bacteriologist. “In the early forties, i

was important to know the source of a culture, for its

identification might depend on whether it was isolated

from above (Klebsiella) or below (Escherichia) the

umbilicus. Today we are not surprised to isolate an

infectious organism (bacteria) from a respiratory site o

vice versa”. In urine bacteriology, the number oleucocytes, erythrocytes, casts, and bacteria/ml are

counted and the bacteria identified. Normally, there wil

be none or less than 20/mm3 of leucocytes or othe

cells and a colony count of less than 104 organisms pe

milliliter. The presence of 104/ml of a single type o

enteric Gram-negative rod is strongly suggestive o

urinary tract infection, especially in men, while young

women with acute dysuria and urinary tract infection

will have 102 – 10

3/ml. Sometimes urine culture may be

negative in the presence of clinical signs of urinary trac

infection. In this case the possibility of an undisclosed

disease condition, an obstruction in the urinary tract otuberculosis of the bladder should be further investi

gated. On the other hand, a bacterial count of the same

type of organism from two consecutive specimens

exceeding 105/ml indicates an ongoing urinary trac

infection and a significant bacteriuria [1, 9, 10].

5. THE MEDIA ROOM AND CULTURE MEDIA

This aspect of microbiological work, by my

estimation, is the hub around which the wheel o



The Practice of Basic Principles in Routine Diagnostic Bacteriology Global Journal of Pathology and Microbiology, 2013, Vol. 1, No. 1 1

diagnostic bacteriology revolves. Regrettably, the

“kitchen”, as the media room is sometimes colloquially

referred to, is oftentimes left in the hands of amateur

“cooks”, with resultant poor outcomes. Just as the

media room and facilities therein are expected to be in

top sanitary condition, so also are the processes

involved in media preparation. Problems may arise

when control strains do not give expected results oncertain prepared media, and if such media are

inoculated with specimens that are not easily

obtainable e.g. cerebrospinal fluid from a neonate.

Since all culture media mimic the natural and essential

nutrients which are present in the living tissue and

which we try to provide for within a Petri dish, adequate

and correct measurements of the dehydrated powder,

distilled water, and additives and other supplements

must be strictly carried out according to the

manufacturers’ recommendations. The need for

specimen preservation must not be overlooked. For

instance, if urine specimen cannot be examined

immediately after collection, it can either be refrigerated

at 4oC or preserved in 1.8% boric acid. The acid is

dissolved in the specimen by shaking. Numerous

techniques have been developed for the examination of

urine samples [11-13]. Their usefulness however,

varies according to the facilities available to the

bacteriologist. For more precise interpretation,

however, a formal plating technique (using a standard

inoculating loop) must be used, as this gives adequate

qualitative and quantitative information [14-16].

Cysteine Lactose Electrolyte Deficient (CLED) agar,blood and MacConkey’s agars have been used

successfully by various laboratories as culture media

for urine specimens. The precise composition of a

satisfactory medium depends on the species being

cultivated due to the great variety of nutritional

requirement. However, depending on the nature of

experiment some desired adjustments in the

composition of culture media ingredients could be

made [17].

6. SELECTIVE USE OF CULTURE MEDIA

The bacteriologist appears to me like a detective

who sets out to disclose the identity of microorganisms

hidden within a clinical specimen. Without the luxury of

the more complex molecular and immunological

techniques, he could provide a presumptive

identification of the causative agent of an infection

merely on the basis of microscopic examination of

specimens and studying the growth and biochemical

characteristics of isolated microorganisms. Thus,

knowledge of diverse numbers of bacteria makes fo

easy identification of isolates from clinical specimens

Frequently a medium is used to select and grow

specific microorganisms or to help identify particula

specie present in a specimen from nature. Fo

example, the use of blood agar, eosin methylene blue

agar, MacConkey agar and mannitol salt agar as

selective and differential media for isolating Strepto

coccus pyogenes (a – haemolytic streptococcus)

faecal E. coli which produce a metallic sheen, lactose

fermenting E. coli which appear as red colonies, and S

aureus respectively, has practical utility when

investigating clinical specimens in diagnostic

bacteriology [16].

7. THE PATHOGEN E. COLI O157:H7

E. coli O157:H7 is an emerging pathogen of much

public health concern and therefore regular screening

for the presence of this pathogen in man and his

immediate environment is an important proactive

measure for early detection of infection and the

establishment of necessary control measures. Mostly

due to the need to cut cost in the management o

laboratory services, most bacteriologists do no

routinely search for E. coli O157:H7 together with othe

enteric pathogens in their isolation procedures [18]. Fo

instance, for the differential isolation of this organism

from suspected specimens, the less costly MacConkey

agar is used. According to Raji et al. [19], the majo

problem with E. coli O157:H7 is that it is not detectedby the usual methods used to isolate and identify

“traditional” enteric bacterial pathogens. MacConkey

agar, which contains lactose as fermentable sugar is a

suitable indicator medium for isolating almost all strains

of E. coli which can ferment lactose.

The successful differential isolation of E. col

O157:H7, however, requires the incorporation o

sorbitol (rather than lactose) as fermentable sugar into

the MacConkey agar medium. The diagnostic value o

this is that while sorbitol is fermented by all other E. col

strains after 24hr incubation, E. coli O157:H7 serotype

doesn’t. In view of the health conditions associated with

infections with this pathogen it might be worthwhile i

physicians in developing countries, query E. co

O157:H7 infection whenever the patient experiences

bloody diarrhea, and microbiology laboratories should

consider routinely searching for E. coli O157:H7 using

sorbitol MacConkey agar supplemented with cefuxime

tellurite.




8. THE PURE CULTURE CONCEPT

The early pioneers of bacteriology realized too well

the importance of working with a pure culture. This

aspect of work requires much ingenuity and patience

but its practicability is most times compromised in most

busy hospital laboratories. In their natural microbiota,

bacterial organisms live as mixed populations ofdifferent species. Many difficulties in identification arise

as a result of the use of an impure culture as starting

material. As a “detective”, the onus rests upon the

bacteriologist to skillfully identify the specie(s) of

interest. Therefore, before an organism can be

identified, it must be obtained as a ‘pure culture’ such

that when replated, the descendants of a single colony

will be like the parent. It is a futile exercise to attempt to

carry out any characterization tests on an impure

culture. Oftentimes, many isolations are made on

selective or differential media which themselves

contain substances that suppress or inhibit the growthof some organisms. It is good practice, therefore, to

replate the colonies picked from a selective medium

onto a non-inhibitory and preferably an indicating

medium, before a culture can be assured to be pure.

9. PERFORMANCE AND CONTROL OF ANTIBIOTICSUSCEPTIBILITY TESTS

Antibiotic susceptibility testing, like antibiotic usage,

has fallen into misapplication by many laboratory

scientists. It is doubtful if tests on the same organism,

done in different laboratories, would result in consistentrecommendations for treatment. Antibiotic susceptibility

testing is supposed to act as a guide to antibiotic

therapy, leading to continuance or alteration of therapy.

It is usually designed to determine which antibiotics are

likely to be effective in curing the infections in question.

The classical disk-diffusion technique is a convenient

and most commonly used method which requires that a

source of antimicrobial agent is applied to the surface

of a well-dried agar medium (usually dried at 37oC for

20 to 30 minutes in an incubator.) During incubation,

antibiotic diffuses radially from the disk into the

medium, inhibiting the growth of a sensitive organismto a degree partially dictated by the susceptibility of the

organism. Other factors that could affect the zone of

inhibition around the antibiotic disk have been well

documented by Brown and Blowers [20]. It is important

that the bacteriologist clearly defines and apply the

criteria for reading and interpreting results of the

antibiotic susceptibility tests. It is a far cry from the

normal practice if a ‘that – zone – looks – about – the

right – size’ attitude is adopted by the bacteriologist. It

is a basic scientific principle that tests should be

controlled, and no matter how urgent test results are

required for use by the clinician, efficiency and

accuracy in the test procedures must not be

compromised. For example, in interpreting the correc

zone size of a disk-diffusion test, Cremer [21] regards

an organism as sensitive (implying that the infection

should respond to treatment with normal doses oantibiotic under normal circumstances) when a zone

radius is the same size as or is larger than the contro

or is not smaller by more than 3mm. A zone that is

more than 3mm in radius but is smaller than the contro

by more than 3mm in radius is reported as moderately

sensitive, while a zone of radius of 2mm or less is

reported as resistant . The approved performance

standard for antimicrobial susceptibility testing has

been documented [22] and remains a useful manual fo

the practising bacteriologist.

Despite the usefulness of antimicrobial susceptibilitytesting, antibiotic resistance has over the years

remained an intractable and worrisome problem fo

medical personnel and patients. Many reasons have

been attributed to this phenomenon [23-26]. Correc

identification of the causative organism of bacteria

infection as well as adopting proper procedures fo

carrying out susceptibility testing will no doubt assist in

curbing antibiotic resistance. Although some physicians

may simply be interested in the Gram result and

corresponding antibiogram in order to initiate antibiotic

management of an infected patient, it is important tha

organisms are correctly identified so as to have correc

epidemiological data of causal agents in a given

disease state, as well as for infection control purposes.

10. NEWER TECHNOLOGIES VERSUS TRADITIONAL METHODS OF BACTERIAL IDENTIFICATION

Many scientists would no doubt sometimes pause

for a while to contemplate the depth of inspiration, the

uncommon wisdom and scientific knowledge displayed

by pioneers such as Louis Pasteur, Robert Koch

Antony Van Leeuwenhoek, Richard Petri, Fanny andWalter Hesse and a host of others who laid the

foundation for the discipline of microbiology. Before the

advent of advanced automated technologies fo

bacterial identification, bacteriological diagnosis has

been traditionally based on bacterial culture, the

observation and analysis of phenotypic expression

such as shape and colour, and biochemical testing o

metabolic end products characteristic of the studied

microbes. For example, specific media were devised




for recognizing production of acid and/or gas under

appropriate and selective conditions. However, these

phenotypic methods may not be so reliable since they

depend on the expression of the chosen characters

which may vary as a result of environmental factors or

strain variations. The Gram staining technique had

been in use and still remains a useful practical tool for

bacterial identification and classification and can offervaluable information for clinical diagnostics including

aiding in deciding what antibiotic disk to apply for

testing bacterial susceptibility to antibiotics. There is

need to understand the principle of the Gram staining

reaction in order to control it and achieve better results.

In the staining procedure, a crystal-violet iodine

complex is formed within the protoplast of the stained

cells. Gram-positive bacteria resist decolourization and

retain the dye complex, while Gram-negative cells take

up the counterstain after decolourization.

Factors that influence the staining process include,1) differences in cell composition, 2) pH of the medium,

and 3) permeability of the bacterial cell wall. The cell

wall of Gram-positive has a higher peptidoglycan and

lower lipid content than Gram-negative bacteria. Gram-

positive bacteria are able to retain basic dyes (such as

crystal violet and methyl violet) at a higher pH than the

Gram-negative species, showing an isoelectric point of

pH 2-3 as compared with pH 4-5. The more acidic

character of their protoplasm, which is enhanced by

treatment with iodine, may partly explain the stronger

retention of basic dyes by Gram-positive cells. It is also

known that decolourization with either acetone or

ethanol dissociates the lipid layer from the Gram-

negative cells, leading to the primary stain diffusing

freely out of the Gram-negative cells, but not from the

Gram-positive cells, whose cell walls are less

permeable. Also, the decolourizer causes dehydration

in the thicker Gram-positive cell walls, closing the pores

as the cell wall shrinks during dehydration. This

effectively blocks the diffusion of the crystal violet-

iodine complex, and the bacterial cells remain stained.

Gram positivity is a feature of relatively young

bacterial cells; as they age, the cells lose this

characteristic and apparently become Gram-negative.

Consequently, younger cells about 24 hours old are

preferably stained. Genuinely Gram-negative bacteria

do not retain the primary stain which is easily removed

by the decolourizer. Also, the length of decolourization

is critical in differentiating Gram-positive bacteria from

Gram-negative ones, as excessive decolourization will

remove all the stain from Gram-positive cells rendering

them false Gram-negative. Preston and Morrell’s

modification of Gram’s Method is reported as giving

reliable results without the need for taking great care in

adjusting the duration of decolourization [27]. It would

be advisable as a matter of policy for workers in each

laboratory to adopt a standard Gram staining

procedure, but not without controlling the staining

technique. Recommended quality control cultures fo

assessing the correctness of the Gram stainingtechnique include Escherichia coli NCTC 10418 (pink

to red Gram-negative bacilli), Oxford Staphylococcus

aureus NCTC 6571 (blue to purple Gram-positive

cocci), and haemolytic Streptococcus group A NCTC

8198 (blue to purple Gram-positive cocci).

In practice traditional microbiological techniques can

take days to provide results and there are limitations in

identifying certain species of microorganisms. The

nucleic acid amplification technology has howeve

opened a new horizon for microbial detection and

identification. Unlike the phenotypic methods earliementioned, the nucleic acids involved in the expression

of a character will remain present in the organism and

even though environmental factors have suppressed

expression they remain a more reliable material fo

microbiological analysis. In addition, molecula

methods become very relevant where culture

procedures fail to identify the causal organism due to

one or more of the following reasons; (i) prior antibiotic

therapy, e.g. treatment of acute meningitis with

intravenous benzyl penicillin, (ii) where specialized cel

culture techniques are required, e.g. Chlamydia spp

and Coxiella burnetti, (iii) where the organism is slowgrowing, e.g. Mycobacterium spp, (iv) where the

organism is fastidious in nature, such as the HACEK

group of organisms in the case of endocarditis [28]

The HACEK group of bacteria includes Haemophilus

parainfluenzae, Aggregatibacter species

( Aggregatibacter aphrophilus, Aggregatibacter actino

mycetemcomitans, and Aggregatibacter segnis)

Cardiobacterium species (Cardio-bacterium hominis

and Cardiobacterium valvarum), Eikenella corrodens

and Kingella species.

Other sophisticated techniques including scanning

electron microscopy [29], fatty acid analysis [30]

sequence analysis `of the bacterial 16S rRNA gene

[31, 32] have been used successfully for bacteria

identification. Whereas a pure culture is required fo

proper identification and characterization using the

traditional cultural methods, these newer technologies

are reported to be rapid, reliable and may not require a

pure culture to be able to identify bacterial agents [33]

Scanning election microscopy aids in detecting and

identifying morphological features of bacterial cells [34]




Fatty acids abound in bacterial cell membranes and

their profiles which can be determined by gas

chromatography, distinguish the various bacteria on the

basis of their physical properties [35]. According to

Eren et al. [31], the regularity and constancy of the 16S

rRNA gene in bacterial cells make it useful for detecting

bacterial cells in natural specimens and confirming

phylogenetic relationship between species.

Strategies based on polymerase chain reaction

(PCR) have been reported to be faster and highly

sensitive for microbiological identification [36], but they

are not optimized for resource-limited environments.

Pure PCR products of the gene can be sequenced and

aligned against bacterial DNA database for purposes of

identification [32]. Although the PCR operates on the

basis of three simple reactions i.e., denaturation of the

DNA double strands, annealing of the oligonucleotide

primers, and extension of the primers, different

modifications or protocols have been developed tomeet specific purposes. Different criteria dictate the

appropriate protocol to employ for bacterial diagnosis.

If speed is an important factor of the assay, e.g. the

detection of meningococcal DNA in cerebrospinal fluid

from children with suspected meningitis, employment of

the real-time assays should be adopted [37]. Where

multiple targets are involved as in the determination of

multiple respiratory pathogens from sputum, the

multiplex PCR format should be employed [28]. It must

be noted that PCR protocols may be affected by

contamination from various sources, leading to false

results; hence there is utmost need to control thereactions.

Ralph Weissleder, and colleagues at

Massachusetts General Hospital in Boston, USA, has

pioneered the magneto-DNA nanoparticle system for

rapidly identifying bacteria. This hybridization assay

involving probes which target bacterial 16S rRNAs was

designed to detect amplified target DNAs using a

miniaturized nuclear magnetic resonance (NMR)

device the size of a microscope slide. The system is

reported to be robust, rapid and is able to

simultaneously diagnose a panel of 13 bacterial

species in clinical specimens within 2 hrs compared

with conventional culture (procedural time, 3-5 days). In

view of the high sensitivity exhibited by the system

(down to single bacteria), it could potentially be used

for the early diagnosis or detection of rare pathogens in

dilute samples [38].

CONCLUSION

These advanced techniques though attractive are,

unfortunately, not within the common reach of many

laboratories, especially those in the developing

countries of the world. The Gram staining techniques

microscopy and the culture of clinical specimens fo

purposes of isolating the causative agent of infection

still have their indispensible value in routine diagnostic

bacteriology. Robert Koch (1843 – 1910) did not have

to wait for Dr. Kary Banks Mullis to invent the PCR

technology before elucidating his postulates for provingthe etiology of microbial infections. What is required

therefore, is for the bacteriologist to exercise much

caution and wisdom as an “investigating detective”, and

work on his or her specimens painstakingly, adhering

to all safety precautions while applying aseptic

techniques to arrive at useful and reproducible results

It is a very wise decision, for instance, never to hastily

discard clinical specimens or inoculated media until a

tests have been satisfactorily completed and results

issued out to the clinician. Had Alexander Fleming

hurriedly disposed his ‘contaminated’ agar plate

without giving it a little further thought, humanity would

probably have had to wait for a longer period of time

before reaping the benefits of Penicillin!.

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http://dx.doi.org/10.1038/nnano.2013.70

Received on 22-10-2013 Accepted on 01-11-2013 Published on 19-11-2013

DOI: http://dx.doi.org/10.14205/2310-8703.2013.01.01.4

© 2013 J.O. Isibor; Licensee Pharma Professional Services.This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License(http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted, non-commercial use, distribution and reproduction inany medium, provided the work is properly cited.

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