Automation and Basic Techniques in Medical Microbiology

213 Pages·2022·4.877 MB·English

by  Santi M. Mandal, Debarati Paul

Santi M. Mandal · Debarati Paul
Automation and
Basic Techniques
in Medical
Microbiology
Automation and Basic Techniques in Medical
Microbiology
(cid:129)
Santi M. Mandal Debarati Paul
Automation and Basic
Techniques in Medical
Microbiology
SantiM.Mandal DebaratiPaul
CentralResearchFacility AmityInstituteofBiotechnology
IndianInstituteofTechnologyKharagpur AmityUniversityUttarPradesh(AUUP)
Kharagpur,WestBengal,India Noida,UttarPradesh,India
Bothauthorscontributedequally
ISBN978-1-0716-2371-8 ISBN978-1-0716-2372-5 (eBook)
https://doi.org/10.1007/978-1-0716-2372-5
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The book is dedicated to
God
for His provision during the toughest Covid
times
and
our beloved parents and family
who have been constant sources of
motivation.
Preface
In this handbook every endeavour has been made to make a comprehensive
one-volume reference text and practical book which is of value to practising
microbiologists throughout their undergraduate, postgraduate, and subsequent
careers.
During the preparation of the text the interest of students has been taken into
consideration suchthatthey getanoverview ofsafetyguidelineswhileworkingin
the laboratory before embarking on actual experiments and handling various
instruments. Students have been instructed about how they should follow the
protocols and write lab manuals with precise documentation.Codeof conduct ina
microbiologylaboratorysetuphasbeenstatedtomakethemawareoftheirrolesand
responsibilities while performing various experiments. There is a summary of
hazards that may be encountered in the microbiology laboratory, intending at
acquainting the students with some essential elements needed to maintain a safe
workingpractice,andtoadvisestronglyontheimportanceofconsultingwithsenior
membersofthelaboratoryortheappointedsafetyofficer,andofconsultingcompre-
hensive specialist texts which should be readily accessible to laboratory users. In
particulartheadviceofsafetyofficersshouldbesought,sincebyvirtueoftheiroffice
theyhaveavailablethelatestinformation.
Hazardsassociatedwithparticularchemicalsandproceduresarenotedinthetext
asfarasinformationisavailable.
Classicalknowledgehasbeenpresentedalongwiththeupdatesinthefieldwith
applicationofmicrobiologyasthefocalpoint.Samplepreparationandinterpretation
of data thus obtained have been introduced making this handbook exceptional
amongstexistingbooksofmicrobiology.
The book provides a bridge between the theory books provided by current
standard undergraduate textbooks and the wider specialist fields of research litera-
ture.Furthermore,itisourintentiontoreflectonresearchdevelopmentsinthearea
of medical microbiology, since we consider that the teaching of concepts must
closely integrate mechanistic theory with the strategy and methodology of experi-
mentationanddatathereof.
Theuseofroboticsandautomationinmedicalbiologyhasbeendescribedinthe
first chapter, stating its history of origin and advancements and implications in
modern microbiology. The chapter describes the history and types of different
vii
viii Preface
auto analysers used in medical microbiology labs, along with their uses and
limitations. The differences between semi-automated and fully automated systems
havebeenhighlightedalongwiththeirnumerousapplications.
Manualandautomatedcharacterizationofmulti-antibiotic-resistant(MAR)bac-
teria have been described in the next section. The types of resistance shown by
microbeshavebeendescribedalongwiththenecessityandimplicationsofantibiotic
susceptibility testing. Qualitative tests suchas thedisc diffusion method by Kirby-
Bauer; quantitative tests including minimum inhibitory concentration (MIC); and
automatedtestsforresistance,e.g.turbidimetricdetectionandmoleculartechniques
namely multiplex PCR and RAPD, have been detailed. The use of other processes
for the detection of MARs has been described (Padlock PCR and microarray
analysis, quantitative real-time PCR), and these may be applicable to all microbial
strainsirrespectiveofpathogenesis.
Currentlywholegenomesequencingfordetectionofpathogensandforstudyof
microbiota of healthy and unhealthy people has led to several important diagnoses
about disease-causing agents and has immensely assisted in patient care. As com-
paredtoothertechniquesofclinicalmicrobiologythatgivepartialinformation,such
asPCR-basedtechniquesdiscussedpreviously,theWGS-basedtechniquesarefaster
and have the potential to generate wholesome datasets. Few WGS methods have
beendiscussedandtheirapplications,andbottleneckshavebeendiscussed.
The next section includes the modern advanced techniques used for studying
microbial and other cells. The principles of spectrometry, spectroscopic methods,
andtheinterpretationofspectraarenowtreatedtogetherinChaps.5and6.Themost
significant addition to this chapter is a more detailed coverage, with examples of
spectra from different types of spectroscopy which is considered alongside with
usage of laboratory equipment. The assistance of the company representatives of
very many manufacturers of chemicals and of laboratory, spectroscopic, and chro-
matographicequipmenthasbeeninvaluable.
TheuseofMALDI-TOFasanaidforbacterialidentificationandidentificationof
toxins produced by microbes that can in turn be used to characterize pathogenic
bacteriahasbeendiscussedindetailinthesixthchapter.Descriptionofapplications
andlimitationsofthistechniquehavebeenincludedtoapprisereadersofthelatest
techniquesinthisupcomingfield.
Thefollowingsectiondescribesaverypopularandimportanttechniqueusedfor
immune assays for the detection of various microbial strains based on antigen and
antibodyinteractions.ThevarioustypesofELISA(enzyme-linkedimmunosorbent
assay)techniqueshavebeenvividlydescribed,andaprotocolhasbeensuggestedfor
students to perform this assay. The advanced forms of this assay have also been
described, e.g. radioimmunoassay (RIA) and automated ELISA. The significance,
advantages,andlimitationshavealsobeendetailedforgettinganoverallperspective
ofthetopic.
Isolation of normal microbiota from human body and microbial identification
usingvariousbasicandautomatedtechniqueshavebeendescribed with methodol-
ogy and applications. The progress made in this area in this current era of modern
technologieshasbeenfocussedon.
Preface ix
Immunotechniques apart from ELISA have been described that will help the
reader to understand the very basics and importance of such techniques in clinical
microbiology.
The advances in microscopy chapter would then encompass advances in this
techniquefromtheoldesttothelatesttechnologyandfromthesimplestdesignsto
the intricate modern engineering and progress in electronics and other fields. The
chapter also encompasses sample preparation for electron microscopy which will
helpthereadertocarryoutexperiments,aftergettingawholesomeknowledgeofthe
instrumentationandtheirapplicability.
Weareindebtedtotherefereeswhomadevaluablecommentsonthemanuscript
whichundoubtedlyhelpedtoimprovethefinaltext.Theauthorsaregratefultotheir
colleagues in academia, industry, and government who have made useful
suggestionsforimprovingthishandbook,bothasabookandasareferencesource.
Theauthorsareespeciallythankfultoallthecontributors,whosecombinedexpertise
hasmadethisuniquetextbookpossible.
Kharagpur,WestBengal,India SantiM.Mandal
Noida,UttarPradesh,India DebaratiPaul
Acknowledgements
For help in preparing this book Automation and Basic Techniques in Medical
Microbiology, it is a pleasure to thank our Founder President Prof (Dr) Ashok K
ChauhanandourChancellorandViceChancellor,AmityUniversity,UttarPradesh,
Noida.Withouttheirprovisionandzeal,itwouldnothavebeenpossible.
WealsosincerelythanktheHeadoftheDeptofBiotechnology,IITKharagpur,
forsupportandencouragement.
TheauthorsarealsogratefultoDrNeelamSingh,bothforinitialconceptualiza-
tionandmotivation.
Heartfelt thanks are extended to our colleagues, students, handling/review
editors, and reviewers for their input for improving the content and designing of
thisbook.
xi
Contents
1 GoodLaboratoryPractices. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.2 BasicRecordandLabNoteBook. . . . . . . . . . . . . . . . . . . . . . 3
1.3 LaboratorySafetyEquipment. .. . . . . .. . . . . .. . . . .. . . . . .. 4
1.4 BiosafetyLevelsandPractices. . . . . . . . . . . . . . . . . . . . . . . . . 4
2 AutomationinMedicalMicrobiology. . . . . . . . . . . . . . . . . . . . . . . 7
2.1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
2.2 ApplicationsofAutomation. . . . . . . . . . . . . . . . . . . . . . . . . . . 7
2.3 AdvantagesandDisadvantages. . . . . . . . . . . . . . . . . . . . . . . . 9
2.3.1 AdvantagesofUsingAuto-analysers. . . . . . . . . . . . . 9
2.3.2 DisadvantagesofAutomation. . . . . . . . . . . . . . . . . . 9
2.4 TypesofAuto-analysers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
2.5 HistoryofAuto-analysers. . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
2.6 LaboratoryAutomationandTotalLaboratoryAutomation. . . . . 14
2.7 TypesandApplicationsofAuto-analysersinMicrobiology. . . . 14
2.7.1 MicrobiologicalSpecimenProcessor. . . . . . . . . . . . . 14
2.7.2 RoutineBiochemistryAnalysers. . . . . . . . . . . . . . . . 15
2.7.3 Immunology-BasedAnalysers. . . . . . . . . . . . . . . . . . 15
2.7.4 HaematologyAnalysers. . . . . . . . . . . . . . . . . . . . . . 16
2.7.5 CellCounter. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
2.7.6 Coagulometer(s). . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
2.7.7 AdditionalInstrumentforHaematology-Based
Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
2.7.8 OtherMiscellaneousAnalysers. . . . . . . . . . . . . . . . . 17
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
3 ManualandAutomatedCharacterizationofMulti-antibiotic-
Resistant(MAR)Bacteria. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
3.1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
3.2 TypesofAntibioticSensitivityTests. . . . . . . . . . . . . . . . . . . . 20
3.2.1 Kirby-BauerDiscDiffusionMethod. . . . . . . . . . . . . 20
3.2.2 TheMinimumInhibitoryConcentration(MIC)
Method. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
xxiiiiii