The methods used for detection and measurement of biofilms produced on medical devices.
\r\n\tThis book aims to cover following topics: i) the effects of long-term environmental change past events on turtle diversification and their evolutionary responses to climate change, ii) responses (developmental, physiological and behavioural) of extant species to environmental stressors, iii) impacts of changing environmental conditions on life history traits (growth patterns, sexual maturation, reproduction, longevity), iv) thermal environment change, biogeographic distribution and ecological niche modeling, v) environmental variation, population and community dynamics, and population level-response modeling, and vi) impacts of future global environmental change (climate change, alongside habitat destruction and fragmentation and overexploitation,) on population future trend and viability and their implications for informing adaptive conservation management strategies.
",isbn:null,printIsbn:"979-953-307-X-X",pdfIsbn:null,doi:null,price:0,priceEur:0,priceUsd:0,slug:null,numberOfPages:0,isOpenForSubmission:!1,hash:"ec7c5f39f89066d7d788873d669bf740",bookSignature:"Prof. Mohammed Znari and Dr. Mohamed Naimi",publishedDate:null,coverURL:"https://cdn.intechopen.com/books/images_new/8155.jpg",keywords:"Climate-mediated species diversification, turtle fossil record, evolutionary adaptation, Environmental stressors, growth, sexual maturation and reproduction, fluctuating asymmetry, homeostatic adaptations, ecological niche modelling , population and community dynamic, conservation strategies",numberOfDownloads:null,numberOfWosCitations:0,numberOfCrossrefCitations:0,numberOfDimensionsCitations:null,numberOfTotalCitations:null,isAvailableForWebshopOrdering:!0,dateEndFirstStepPublish:"August 29th 2019",dateEndSecondStepPublish:"September 19th 2019",dateEndThirdStepPublish:"November 18th 2019",dateEndFourthStepPublish:"February 6th 2020",dateEndFifthStepPublish:"April 6th 2020",remainingDaysToSecondStep:"2 years",secondStepPassed:!0,currentStepOfPublishingProcess:5,editedByType:null,kuFlag:!1,biosketch:null,coeditorOneBiosketch:null,coeditorTwoBiosketch:null,coeditorThreeBiosketch:null,coeditorFourBiosketch:null,coeditorFiveBiosketch:null,editors:[{id:"223073",title:"Prof.",name:"Mohammed",middleName:null,surname:"Znari",slug:"mohammed-znari",fullName:"Mohammed Znari",profilePictureURL:"https://mts.intechopen.com/storage/users/223073/images/system/223073.jpg",biography:"Mohammed Znari, has PhDs in Ecology (from Pierre & Marie Curie University, Paris, France, 1988 and Cadi Ayyad University, Marrakech, Morocco, 1999). He is a full Professor of ecology and conservation biology at the Faculty of Science – Semlalia, Marrakech. His research interests are morphometrics, systematics and molecular phylogeography, physiological ecology, ecology and life history along with conservation ecology in various taxa including turtles, steppe-land birds and mammals. He has been involved in several international projects and obtained several international fellowships and research-conservation grants. He is also the curator of vertebrate zoology at the Natural History Museum of Marrakech. He was a member of the International Committee of the World Congress of Herpetology and he is currently a vice-President of the Moroccan Herpetological Society. He has produced dozens of publications in peer-reviewed journals and contributions in international scientific meetings.",institutionString:"Cadi Ayyad University",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"1",totalChapterViews:"0",totalEditedBooks:"0",institution:null}],coeditorOne:{id:"292272",title:"Dr.",name:"Mohamed",middleName:null,surname:"Naimi",slug:"mohamed-naimi",fullName:"Mohamed Naimi",profilePictureURL:"https://mts.intechopen.com/storage/users/292272/images/system/292272.jpg",biography:"Mohamed NAIMI has a PhD in Animal Biology, Ecology, Conservation Biology and Environment (Cadi Ayyad University –UCA- Marrakech, Morocco). He has been an Assistant professor at Sultan Moulay Slimane University - SMSU - Beni-Mellal, Morocco since 2016. He is a member of the Polyvalent Laboratory for Research and Development (LPVRD) at SMSU and associate member of the Biodiversity and Ecosystems Dynamics Laboratory and the Natural History Museum of Marrakech –UCA-. His research interests are trophic ecology, developmental and reproductive biology, morphometry and physiological ecology (on various taxa, including aquatic animals, herpetofauna and terrestrial mammals). He has participated in several international projects and received different scholarships. He has produced several papers in peer-reviewed journals and communications in international scientific meetings. Before joining the SMSU, he held several positions in the external services of the Marine Fisheries Department in Morocco.",institutionString:"University of Sultan Moulay",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"0",totalChapterViews:"0",totalEditedBooks:"0",institution:null},coeditorTwo:null,coeditorThree:null,coeditorFour:null,coeditorFive:null,topics:[{id:"5",title:"Agricultural and Biological Sciences",slug:"agricultural-and-biological-sciences"}],chapters:null,productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"},personalPublishingAssistant:{id:"250240",firstName:"Nino",lastName:"Popovic",middleName:null,title:"Mr.",imageUrl:"https://mts.intechopen.com/storage/users/250240/images/7088_n.png",email:"nino@intechopen.com",biography:"As an Author Service Manager my responsibilities include monitoring and facilitating all publishing activities for authors and editors. From chapter submission and review, to approval and revision, copyediting and design, until final publication, I work closely with authors and editors to ensure a simple and easy publishing process. I maintain constant and effective communication with authors, editors and reviewers, which allows for a level of personal support that enables contributors to fully commit and concentrate on the chapters they are writing, editing, or reviewing. I assist authors in the preparation of their full chapter submissions and track important deadlines and ensure they are met. I help to coordinate internal processes such as linguistic review, and monitor the technical aspects of the process. As an ASM I am also involved in the acquisition of editors. 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Surface proteins and polysaccharide intercellular adhesions (PIA) play a role in the biofilm production and development. It is hard to treat biofilm-embedded bacteria than planktonic forms. Biofilm producer microorganism causes biofilm-related infections such as indwelling and medical device-related infections such as endocarditis, urinary tract infections, septic arthritis, chronic rhinosinusitis, ocular infections, wound infections, etc. The results of biofilm produced on indwelling medical devices are recurrent, untreatable infections and failure of medical device. To overcome chronic and recurrent infections, it is important to detect biofilms of microorganisms, maturation and dispersion, and determine antibiofilm and antibacterial activity of agents against biofilm and bacteria within biofilm, respectively [1]. Identification of genes involved in biofilm formation and measurement of gene expression as a result of antibiofilm and antibacterial activity of agents can be advantageous with carrying out high-throughput screens using microtiter plate assay system.
The standard antimicrobial susceptibility tests such as broth macrodilution and microdilution methods that are routinely used in laboratories and published by Clinical Laboratory Standards Institute (CLSI), National Committee for Clinical Laboratory Standards (NCCLS), and European Committee on Antimicrobial Susceptibility Testing (EUCAST) could never yield accurate results in biofilm producer microorganisms, due to being appropriate for the detection of antimicrobial activity of agents against planktonic microorganism [2].
There are several methods which have been used by clinical microbiologist for detection and measurement of microbial biofilms in response to agents (Tables 1
Method | Action of application | Aim |
---|---|---|
Roll plate | Extraluminal biofilm detection | Growth of biofilm-embedded bacteria |
Sonication, vortex, and plate counting | Intraluminal and extraluminal biofilm detection | Growth of biofilm-embedded bacteria |
Acridine orange staining | Extraluminal biofilm detection | Direct investigation of biofilm produced on catheter by microscopy |
Streak plating of alginate swab | Investigation of biofilm produced on indwelling catheter | Growth of biofilm-embedded bacteria |
The methods used for detection and measurement of biofilms produced on medical devices.
Method | Aim |
---|---|
Tube method (TM) | Qualitative detection by observing biofilm lined on bottom and walls of tube |
Congo red agar (CRA) | Qualitative detection by observing colony color change |
Microtiter plate (MtP) | Quantitative detection of biofilm by microplate reader (microELISA) |
Real-time PCR | Detection of biofilm genes |
Conventional PCR | |
Multiplex PCR |
The methods used for detection of biofilm.
Method | Application | Target |
---|---|---|
Microtiter plate (MtP) | Measurement of biofilm produced on walls of wells in response to agent | Measures the effect of agents against biofilm production |
Microtiter plate (MtP) (MBEC) | Measurement of biofilm remained on walls of wells in response to agent and detecting MBEC of agents | Measures the effect of agents against mature biofilm formed on walls of wells |
Vortex and plate counting | Plate counting of biofilm-embedded bacteria and detecting bMBC of agents | Screens antimicrobial activity of agents against biofilm-embedded bacteria |
Checkerboard assay | Plate counting of biofilm-embedded bacteria and FIC indexes are calculated | Screens antimicrobial activity of combination of agents |
Sonication, vortex, and plate counting | Plate counting of biofilm-embedded bacteria and detecting bMBC of agents | Screens antimicrobial activity of agents against biofilm-embedded bacteria |
Quantitative PCR | Measurement of specific biofilm gene expression | Monitors expression of biofilm genes in response to agents |
Mass spectrometry (MS) | Measurement of exoenzymes located in biofilm matrix | Monitors expression of bacterial proteins in response to agents |
The screening methods for antibiofilm and antimicrobial activity of agents against biofilm producer bacteria.
Instruments | Culture dynamics | Substratum | Method |
---|---|---|---|
Modified Robbins device | Batch culture | Silastic disks | Viable counting |
Calgary biofilm device | Batch culture | Plastic polycarbonate pegs | Viable counting, after pegs are sonicated |
Disk reactor | Batch culture | Teflon coupons | Direct or viable counting, after coupons are sonicated, vortexed, and homogenized |
CDC biofilm reactor | Continuous culture | Plastic connectors | |
Perfused biofilm fermenter | Continuous culture | Cellulose-acetate filters | Viable counting, after filters are shaken in sterile distilled water |
Model bladder | Continuous culture | Urinary catheters | Examining directly by SEM or TEM or analyzing chemically |
Flow cell | Continuous culture | Chambers with transparent surfaces | Examining by confocal laser scanning microscopy |
Instruments used to produce biofilm and examine biofilm process.
CDC biofilm reactor, Centers for Disease Control biofilm reactor; SEM, scanning electron microscopy; TEM, transmission electron microscopy.
The aim of this chapter is to overview certain methods used by biofilm detection and antibacterial and antibiofilm researches such as tube method (TM), Congo red agar (CRA) method, microtiter plate (MtP) assay, plate counting of biofilm-embedded bacteria (sessile bacteria), PCR, mass spectrometry (MS), confocal laser scanning microscopy (CLSM), etc.
Complexity and dynamics of biofilms can be observed by biofilm imagining optical technology including light microscopy, SEM, TEM, and CLSM. These techniques are used to visualize 3D structure and check the existence of biofilm [4].
Light microscopy is the easiest, cheapest, most simple, convenient and fastest method to quantitatively observe the morphology of microorganisms adhered to surfaces and to semiquantitatively estimate the amount of microorganism attached on surface (exist, absent, abundant, rare, etc.). Microorganisms including
Images of cells and cell structures such as protein and nucleic acid are obtained by electrons at high magnification and resolution. Monitoring of components of cell can be done directly in TEM by negative staining. Due to photons and electrons penetrating cells poorly, thin section of cell cut is stabilized and stained by certain chemicals with the treatment of osmic acid, permanganate, uranium, lanthanum, or lead salts. These stains contain high atomic weight. Due to stains having high atomic weight, contrast is accelerated by electron dispersion from sample. If observation of outer structure of cells will be done, it is not important whether the section of cell is thin or thick.
Due to inadequate stabilization of polysaccharides is done by the conventional fixatives such as aldehydes, glutaraldehyde, paraformaldehyde, and osmium tetroxide, water content of biofilm is eliminated by graded dehydration with alcohol after this postfixation step. After sample is infiltrated with resin, sample is embedded in gelatin capsule and headed for polymerization. Then, thin section taken is poststained with uranyl acetate and lead citrate.
Exopolysaccharide constituents are not observed with its own electron dense and staining poststains such as uranyl acetate and lead citrate with TEM, due to not only having high electron translucent, but also contrast is not developed by conventional poststains. According to the studies, glycocalyx of
The SEM image of
To visualize 3D images of cell sample is coated with heavy metals such as gold. Electrons released from metal coating of sample are caught by SEM for image production. The procedure of SEM is similar to TEM except for some additional chemicals (gold), lacking infiltration, embedment in resin, polymerization, and thin section staining with lead citrate and uranyl acetate [5]. As in the steps of TEM method, postfixation and dehydration steps of SEM are similar to TEM. The step is applied after dehydration step is drying and coating sample with gold in the processing for SEM, rather than infiltration with resin, embedment in gelatin capsule, and staining with lead citrate and uranyl acetate in the processing for TEM. After dehydration process with graded alcohol, sample is dried and coated with gold palladium [5]. After all these steps are done, sample is observed in SEM (Figure 2).
The TEM image of
Biofilms formed on flow cells of which surface are transparent can be observed by confocal laser screening microscopy (CLSM). Three-dimensional (3D) morphology and physiology of biofilms can be screened by CLSM [2]. Thick samples such as biofilms and microorganisms localized in the depth such as biofilm-embedded microorganisms need to be observed by CLSM (Figure 3).
Bacterial community embedded in a biofilm matrix visualized by CLSM. Each bacterium observed with a distinct color located at different depths of biofilm [
For observation of biofilm with confocal microscopy and related methods, biofilm must be fluorescent as a result of fluorescent molecules such as green fluorescent protein (GFP) that is fluorescent protein expressed by biofilm producer microorganism within biofilm (gene of cell interested is tagged by gene cassette encoding GFP) or staining components of heterogeneous mass of biofilm with fluorescence or fluorescence-labeled dyes [2]. Stains such as lectins target extracellular matrix, whereas certain fluorophores target extracellular DNA (eDNA) to visualize eDNA content of biofilm matrix [2, 7].
By scanning laser light across the sample, deep penetration of excitated energy is provided. As a result of fluorescence of biomolecules such as GFP or chlorophyll that are intrinsic fluorophores or molecules signed by exogenous probes such as fluorescent-labeled antibodies detected by photomultiplier, 3D digital image is formed. Observation of biofilms that are multilayered and have complex 3D structures requires additional resolution [2]. Images of each layers of biofilm obtained are combined by computer for construction of digital 3D images of whole biofilm.
Biofilm producer microorganisms can be manipulated genetically by tagging of microbial gene of interest by gene cassette encoding GFP as a reporter gene (
Idea about gene activity in biofilms is given by confocal microscopy applied to 3D localization of nonenzyme reporter systems such as GFP. Growth phase and activity of bacteria embedded in biofilms can be defined by promoter-reporter systems that is designed and fluoresced just in living dividing cells. In situ cellular growth activity of bacteria embedded in biofilms is determined by measuring ribosome-hybridization-signal intensity, due to synthesis rate and content of ribosome correlated with the growth rate (especially in exponential phase). Expression cassette that is active only in growing cells, labeled by GFP and controlled by rRNA promoter, can be constructed to monitor growth phase and activity of bacteria within biofilm [2, 10].
Specific microorganisms present in a heterogeneous biofilm community can be identified by the probes of fluorescent in situ hybridization (FISH) method. GFP that is translated enables procedure not to require fixation or staining. Fluorescent-labeled microorganism within biofilm can also be examined by FISH. DNA probes designed to hybridize 16S rRNA of microorganism integrated to either fluorescent dye such as FITC or Rhodamine or enzyme such as horseradish peroxidase. The advantage of probes conjugated with horseradish peroxidase is not to destroy microorganism within biofilm. The growth rate of microorganism within biofilm can be determined by FISH method, due to the amounts of ribosomes existing in a microorganism that is directly proportional to growth activity of microorganism. Probe must be designed to label conserved region of only a single species (Figure 4) [2, 12].
Fluorescence image of 28 distinct
Roll plate method is applied for the detection of possible microbial colonization having a potential to develop indwelling device-associated infection on the outer surface of cylindrical materials such as catheters and vascular grafts. Microorganism colonize on external surface of catheter is detected by roll plate method, instead of microorganism colonize on intraluminal site of catheter. Material is touched and rolled on the surface of medium [3].
Congo red agar (CRA) method that is a qualitative assay for detection of biofilm producer microorganism, as a result of color change of colonies inoculated on CRA medium, is described by Freeman et al. The CRA medium is constructed by mixing 0.8 g of Congo red and 36 g of sucrose to 37 g/L of Brain heart infusion (BHI) agar. After incubation period that was 24 h at 37°C, morphology of colonies that undergone to different colors is differentiated as biofilm producers or not. Black colonies with a dry crystalline consistency indicate biofilm producers, whereas colonies retained pink are non-biofilm producers (Figure 5) [13].
CRA method applied on CRA medium. Black crystalline colonies of biofilm producer cell and pinkish-red colonies of biofilm nonproducer cell.
Tube method (TM) that is a qualitative assay for detection of biofilm producer microorganism, as a result of the occurrence of visible film, is described by Christensen et al. [14]. Isolates are inoculated in polystyrene test tube which contained TSB and incubated at 24 h at 37°C. The sessile isolates of which biofilms formed on the walls of polystyrene test tube are stained with safranine for 1 h, after planktonic cells are discharged by rinsing twice with phosphate-buffered saline (PBS). Then, safranine-stained polystyrene test tube is rinsed twice with PBS to discharge stain. After air drying of test tube process, the occurrence of visible film lined the walls, and the bottom of the tube indicates biofilm production (Figure 6) [14].
Tube method. The first two polystyrene test tubes from the left indicate biofilm production. Other test tubes rather than the first two polystyrene test tubes from the left indicate lacking of biofilm production.
Microtiter plate (MtP) assay is a quantitative method to determine biofilm production by microplate reader. Bacterial suspension is prepared in MHB supplemented with 1% glucose and adjusted to 0.5 McFarland (1.108 cfu/ml). This bacterial suspension is 20-fold (1/20) diluted to reach 5.106 cfu/ml. Then 180 μl of Mueller-Hinton Broth (MHB) supplemented with 1% glucose [15] and 20 μl of bacterial suspensions are inoculated into 96-well flat-bottomed sterile polystyrene microplate to obtain 5.105 cfu/ml as a final concentration (tenfold dilution (1/10)). Microplates are incubated at 24 h at 37°C. The sessile isolates of which biofilms formed on the walls of wells of microplate are stained with only 150 μl of safranine for 15 min, after planktonic cells in wells of microplate are discharged by washing twice with phosphate-buffered saline (PBS) (pH 7.2) and wells are dried at 60°C for 1 h [14]. Before staining with safranine, fixation of biofilms can be done by either subjecting to 150 μl of methanol for 20 min or drying at 60°C for 1 h. Then safranine-stained wells of microplates are washed twice with PBS to discharge safranine stain. After air drying process of wells of microplate, dye of biofilms that lined the walls of the microplate is resolubilized by 150 μl of 95% ethanol or 33% glacial acetic acid or methanol. Then microplate is measured spectrophotometrically at 570 nm by a microplate reader [15, 16]. The studies are repeated in triplicates. Uninoculated wells containing sterile MHB supplemented with 1% glucose that are considered to be the negative controls are used as blanks. The blank absorbance values are used to identify whether biofilm formation of isolates exists or not. The wells of isolates of which OD values are higher than blank well are considered to be biofilm producers. Cut off value (ODc) can provide categorization of isolates as biofilm producer or not.
Negative value obtained from this formula and represented as zero indicates lack of biofilm production, whereas positive value indicates biofilm production (Figure 7).
Microtiter plate assay indicating biofilm production.
To interpret results, categorization can be done as no biofilm production (0), weak (+ or 1), moderate (++ or 2), and strong biofilm production (+++ or 3) by the calculation of cutoff value (ODc) shown below [15]:
OD ≤ ODc no biofilm production
ODc< OD ≤ 2 × ODc weak biofilm production
2 × ODc< OD ≤ 4 × ODc moderate biofilm production
4 × ODc< OD strong biofilm production.
PCR techniques is used for not only identification of pathogens by amplifying species-specific nucleic acid sequences but also detection of virulence factors by amplifying target virulence genes such as biofilm genes with the usage of gene-specific primers, even in the uncultured pathogen present in the sample.
Forward and reverse primers of biofilm-associated gene are designed. Firstly, multiple alignments were done in the NCBI to find oligonucleotide sequences specific to the species. Then primer pair of biofilm-associated gene is designed by using Primer3Plus verified by FASTA analysis checking specificity of primers for microbial sequences in the database [17, 18].
Genomic DNA of microorganism is extracted by extraction kits of which protocols can vary according to species and Gram-positivity or Gram-negativity of microorganisms. DNA of microorganism is measured spectrophotometrically by microplate spectrophotometry reader to determine the amount of DNA extracted as microgram per microliter.
Biofilm-associated gene is amplified by PCR such as qualitative real-time PCR, multiplex and conventional PCR that is used to detect whether biofilm-associated gene is present or not in microorganism. If conventional and multiplex PCR protocols are applied to detect biofilm gene, rather than qualitative real-time PCR, PCR product isolated is visualized on an agarose gel containing a DNA-intercalating dye such as ethidium bromide to confirm the presence of amplified gene (Figure 8). Only in qualitative real-time PCR, the amplicon is detected by fluorescence using a pair of specific hybridization probes labeled with fluorescence dye [11].
Image of
Microtiter plate (MtP) assay is a qualitative assay to detect efficacy of agent against biofilm production by microplate reader.
Bacterial suspension is prepared in MHB supplemented with 1% glucose and adjusted to 0.5 McFarland (1.108 cfu/ml). This bacterial suspension is 20-fold (1/20) diluted to reach 5.106 cfu/ml.
A 180 μl of agent doses and 20 μl of bacterial suspension are dispersed to each well of microplate to obtain 5.105 cfu/ml as a final concentration (tenfold dilution (1/10)). After incubation at 37°C for 24 h, ongoing processes are done according to MtP assay as mentioned previously for the determination of effect of agent against biofilm production [14, 16].
Biofilms remained after eradication by agent are measured by this technique. Biofilms of bacteria that line the walls of wells are formed according to MtP method.
After the content of microplate is discharged, 200 μl of each dose of agents is dispersed to each well of microplate of which the walls are lined with biofilm. A 200 μl of distilled water is added into a well of microplate of which the walls are lined with biofilm as a control. Then the effect of agent against mature biofilm is determined according to MtP assay as mentioned previously. Minimum concentration of agent eradicating mature biofilm that is named as minimum biofilm eradication concentration (MBEC) can be determined by this modified plate assay. MBEC50 and MBEC90 indicate the minimum concentrations of agents inhibiting 50 and 90% of mature biofilm formed.
In summary, biofilm formation process on abiotic surfaces by bacteria is done. Quantification of sessile biofilm-embedded bacteria lined on abiotic surface and sessile biofilm-embedded bacteria remained on abiotic surface after addition of agent on abiotic surface on which mature biofilms formed is determined by plate counting. Bacterial suspension is prepared and adjusted to 0.5 McFarland (1.108 cfu/mL) in Mueller-Hinton Broth (MHB) supplemented with 1% glucose [15]. This bacterial suspension is 200-fold (1/200) diluted to gain 5.105 cfu/mL. Kirschner wire orthopedic implants are placed into each test tube containing 5.105 cfu/mL isolate and incubated at 37°C for 24 h to lead bacteria to produce biofilm on Kirschner wire. After incubation, Kirschner wires on which biofilms are produced are discharged and rinsed with PBS (pH 7.2) and then transferred into each test tubes containing agent concentrations. After incubation at 37°C for 24 h, Kirschner wires are discharged and placed into test tubes containing 1 mL of sterile MHB and sonicated at 42 kHz for 2 min after vortexed for 5 min. Then 100 μl samples of each test tube sonicated and vortexed are inoculated on Mueller-Hinton agar (MHA) and incubated at 37°C for 24 h [19].
The lowest concentration of agent in which bacterial growth is below or equal to control is determined as biofilm minimum inhibitory concentration (bMIC) for biofilm. bMIC50 and bMIC90 indicate the minimum concentrations of agent inhibiting 50 and 90% of biofilm-embedded bacteria. After incubation, the lowest concentration of agent in which colonies of biofilm-embedded bacteria are not grown is determined as biofilm minimum bactericidal concentration (bMBC) of agent for biofilm [19].
Checkerboard assay is used for the determination of combination effects of two different agents. A 250 μl twofold dilutions of each agent from the stock solutions are dispersed to each row and column to obtain final varying concentrations by starting at fourfold of zero MIC for each isolate. So each well contains distinct combination of concentrations of two agents. First wells of rows and columns are left behind for sole treatments of each dose of agents. One well is used for bacterial control (Figure 9). Kirschner wires on which bacterial biofilm is produced are dispersed to each well. This microplate is incubated at 37°C for 24 h. After incubation, Kirschner wires are discharged and sonicated at 42 kHz for 2 min after vortexed for 5 min. The lowest concentration of agent in which bacterial growth that is not observed is determined as biofilm minimum inhibitory concentration (bMIC) of agent for biofilm. Then 100 μl samples of each test tube sonicated and vortexed are inoculated on MHA and incubated at 37°C for 24 h. After incubation, the lowest concentration of agent in which colonies of biofilm-embedded bacteria are not grown is determined as biofilm minimum bactericidal concentration (bMBC) of agent for biofilm. For the determination of whether the synergism is present between agents or not, fractional inhibitory concentrations (FICs) index that are calculated for each agent are summed up according to formula written below:
The schematization of checkerboard assay.
When ∑FIC is equal and lesser than 0.5, between 0.5 and 1, equal to 1, higher than 1 and equal and lesser than 4, and higher than 4, it is interpreted that the effect between agents in combination is synergistic, partial synergistic, additive, indifferent, and antagonistic, respectively [20].
The wells having the highest synergy rates of two agents that constitute the combinations are determined by taking the average and standard deviation of FIC indexes calculated of the wells with the lowest drug combination without bacterial growth in each row and column (Figure 9).
Measurements of biofilm genes repressed or induced by agent are done by quantitative real-time PCR (qPCR). So efficacy of agent against biofilm-associated genes can be detected by qPCR.
Complementary DNA (cDNA) is copied from RNA by enzyme reverse transcriptase. Gene expression in pathogen is monitored by qPCR copying cDNA from RNA of target gene. Amplified cDNA probed for identification. Fluorescent probes such as dye SYBR Green are used to indicate double-stranded DNA, consequently amplification. Accumulation of PCR amplicons labeled fluorescently is monitored through the qPCR processes (Figure 10). Visualization of amplicon on agarose gel is not needed to confirm amplification in qPCR [11].
Quantitative real-time polymerase chain reaction (qPCR) of
Total RNA of microorganism is isolated according to protocols of RNA isolation kits. Kit protocols can vary according to the species of microorganism. Total RNA of microorganism is measured spectrophotometrically by microplate spectrophotometry reader to determine the amount of total RNA isolated as microgram per microliter. Then cDNA is synthesized from total RNA with qPCR using primer pair of the biofilm-associated gene, which is designed using Primer3 and verified by FASTA analysis, which controls the specificity of the primers for microbial sequences in the data system, after multiple alignments were done in the NCBI to find oligonucleotide sequences specific to the species [17, 18].
Extracellular polymeric substances (EPS) not only contain polysaccharides but also contain proteins such as extracellular enzymes. These expressed proteins located in the matrix of EPS can be detected and characterized by mass spectrometry (MS) [1]. Large biologic molecules can be also detected and characterized in complex biologic structures such as EPS by MS. Chemicals involved in biofilm process are examined in detail by MS. Electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) are the types of MS [2]. In time-of-flight (TOF) mass spectrometer, mass is analyzed by ions desorbed in vacuum chamber. These two technics are combined and called MALDI-TOF.
Sample is ionized and vaporized by laser. Ions generated from sample by laser pass through the column of MALDI-TOF device toward TOF detector by an electric field. Depending on the mass/charge ratio of molecule, measurements are done by TOF. If this ratio is smaller, ions move faster (Figure 11).
MALDI-TOF mass spectrometry device [
Bacteria are identified, expression of bacterial proteins such as surface proteins and exoenzymes like β-lactamase in response to antimicrobials can be monitored, and growth of bacteria is measured by applications of MALDI. MS has high sensitivity and requires minimum amount of sample [2].
Biofilm-embedded bacteria can be estimated by biologic assays that is an indirect assay. Biological assays that measure production of microbial product give an opinion about estimation of the number of microorganism within biofilm. Amount of biologic product is correlated with biofilm-embedded microorganism producing the product by standardization of planktonic microorganism. Biologic products produced by planktonic microorganism are similar to biologic products produced by biofilm-embedded bacteria. Standardization curves of each microorganism tested need to be formed. Measurement of total protein at the absorbance is 550 or 950 nm; tryptophan fluorescence, endotoxin [2], ATP production via bioluminescence caused by luciferin and luciferase, urease production to estimate number of attached microorganism, and electron transport via the production of formazan are done by biological assays [3].
Biofilms cause resistance to many antimicrobial agents. The results of biofilm produced on indwelling medical devices are recurrent, untreatable infections and failure of medical device. To overcome chronic and recurrent infections, it is important to detect biofilms of microorganisms, determine antibiofilm activity of agents against biofilm, and determine antibacterial activity of agents against biofilm-embedded microorganism with the appropriate methods by clinical microbiologist and biofilm researcher microbiologist. Identification of genes involved in biofilm formation and measurement of gene expression as a result of antibiofilm and antibacterial activity of agents can be advantageous in biofilm studies.
The manufacture/fabrication of all men- made objects made up of metals/alloys involve the process of solidification at same stage. It processes of phase change, a liquid phase giving way to a solid phase. In metals and alloys, however, solidification involves the formation of crystals, a crystalise solid exhibiting regularity in atomic spacing over a considerable distance [1]. This is dissimilar from a process involving glasses and polymers. However, even in the case of metal/alloy, when crystals are deposited as a consequence of solidification from the melt, though, there exist certain extents of internal symmetry, there are certain irregular external forms and shapes. This tendency can be attributed to the uneven growth rates throughout the process of solidification and the constraints in the growth process during the last stages of freezing. In the ordinary general case, the solidified grain or cell representing the structure of the solid is a normal crystalline unit formed in the cast structure. However, under the specific case of eutectic freezing, the solidified cell consists of two separate crystal structures with the simultaneous growth of separate phases [2].
The most important practical applications of the process of solidification is found in the production of castings. Indeed, casting can be defined as liquid metal forming. The process consists of introducing the liquid metal of appropriate composition into the mould effecting its solidification under controlled conditions of cooling, pouring, etc. to obtain the desired cast structure [3, 4]. A molten metal has a viscosity which is about one-twentieth of the corresponding solid. Thus, instead of spending, high energy, overcoming the high flow of stresses of a solid to shape if by adopting bulk metal forming operations of forging, extrusion, rolling, etc. a liquid metal with essentially zero shear stress is required to be handle. A detailed study of the process of solidification, therefore, enables one to know and hence control the microstructure of the casting that decides is microstructural properties.
The process of freezing of a solid from its melt is accompanied by to very important phenomena which decides the intrinsic properties of the resultant casting. At the first instance, freezing is associated with volume contractions as a consequence of the development of a more closely packed solid [5]. At the same time a reduction in the molecular motion is experienced when the randomly moving molecules in the liquid phase generate the nuclei that finally grow into the solid phase. Latent heat of crystalization is liberated at the solid/liquid interface. This liberated heat energy markedly affects the rate and mode of crystal growth. The general fall of temperature to give way to freezing causes a lowering of the solubility of the alloying elements in the melt. Solute atoms are rejected at the solid–liquid interface. The solubility of the alloying elements is further affected as a result of the changed composition of the alloy grossly affecting the final structure of the solidified melt.
Where the casting process is the last stage of fabrication or it has to be followed by further mechanical working, solidification possesses pay an important role in deciding the microstructure of the product and hence its final structure related properties. In this respect, two distinct cases can be considered:
One of the major problems concerning the process of casting involves the local variation of the resultant microstructure leading to compositional variations. The above is illustrated in Figure 1.
Schematic presentation of alloy properties as influenced by its position in the melt.
As seen in Figure 1, the dendritic arm spacing (
Heavy reduction through mechanical working is not a very efficient method of modifying the edge cast structure. Any initial heterogeneously develop structure, due to the adoption of a faulty solidification process has some tendencies to persist. Therefore, it can be said with authority, any effective control of product quality must be exercised during the process of solidification itself.
The process of solidification comprises of successive stages of
Any phase change has to get initiated by the emergence of the new phase at some instant of time. Likewise, when a solid phase emerges out of the liquid metal/alloy, it begins with the appearance of Nuclei. These are the cluster of atoms which come together during their course of random motion in the melt and can be termed as the embryonic crystals. These embryonic crystals permit further sitting at atoms on their surfaces which causes the growth of the solid phase. However, many of the nuclei again disappear in the melt, the clustered atoms again moving randomly in the melt. Only those nuclei which are stable and meet the thermo-dynamic requirements, allow growth to take place on their surfaces.
The coming together of the randomly moving atoms, from within the melt to form the embryonic crystals, the Nuclei, is known as Homogeneous Nucleation. These are smaller zones of higher density, formed by the ordered cluster of atoms [6]. Mahata et al. have conducted experiments to understand homogeneous nucleation in solidification of aluminium by molecular dynamics simulation [7]. They are of the opinion that there are many methods like X-ray scattering [8] etc., to monitor solid to liquid transformation. However, these methods are limited by several factors that make it difficult to study Homogeneous Nucleation in pure metals.
The precipitation of the group of atoms as the embryonic fresh phase in the melt, is subjected to a change in the free energy. The total free energy change comprises of two components, VOLUME FREE ENERGY CHANGE and INTERFACE FREE ENERGY CHANGE. Thus, a thermodynamic set of conditions is set up for the formation of the Nuclei and for the Nuclei to be stable and not to dry–out prematurely, these thermodynamic conditions have to be met with.
Thermodynamically, when a solid comes out as a liquid, there is a negative free energy change in the system. This change of free energy is directly proportional to the new volume(solid) transformed. Thus, for a spherical solid particle formed in a liquid,
Thermodynamically, when a new interface is generated due to the emergence of a solid from a liquid, there is a gain of free energy at the interface created. This free energy gained is gained is proportional to the surface area of the solid particle created. For the same sphere, as considered above, with a radius of
The volume free energy change and the interfacial free energy change are both presented graphically in Figure 2. The Figure 2 also depicts the overall free energy change as a consequence of the two components when the solid volume is created in the melt.
Change of free energy (volume and interface) as a consequence of the creation of solid phase.
As seen in the Figure 2, for small values of
From the above it follows, homogeneous nucleation conditions are not favourable at the beginning for the stability of the nuclei as considerable undercooling is necessary for homogeneous nucleation to be effective. In the practical case of casting in a foundry, however, the melt need not be supercooled to make the homogeneous, stable nuclei form to start the solidification process. This is because, in the practical melt in the foundry, solidification processes are initiated by heterogeneous nucleation.
For heterogeneous nucleation, the initial interface for growth is provided by a foreign particle [9]. This foreign particle can be provided from outside or formed in the melt itself. The impurities, foreign particles or even the mould wall (the subtstate) can provide for a part of the surface energy required for nucleation. It is a known fact that less activation energy (free energy barrier) is required for nucleation. Therfore, the presence of the substate as mentioned above reduced the free energy barrier and can be very helpful in creating more growth capable nuclei. This is known as heterogeneous nucleation which need less activation energy than homogeneous nucleation [10]. This second phase to act as a nucleus, however, must be capable of being wetted by the melt forming low contact angles and also it must have some structural affinity with the crystalline solid to be formed on it. This second phase could be any one or any combination of the following:
impurities in the metal
the wall of the mould
deliberately added particles to encourage a particular mode of crystallisation
Once the heterogeneous nuclei meet the growth conditions, growth occurs on them. After a certain lapse of time, when the temperature of the melt is lowered, the homogeneous nuclei become stable, and more solid may get deposited on them. At the same time, fresh nucleation may occur generating further stable nuclei. These fresh nuclei may be of the same phase as the first nuclei or of a different phase.
The growth process is conceived as the sitting of further atoms on the stable nuclei which brings in the growth of individual crystal or a general growth in the mass of the solid as solidification proceeds
Nucleation does not take place randomly in the metal/alloy melt throughout the liquid because in the actual case of solidification, and uniform lowering of temperature throughout the melt cannot be obtained. There exist a thermal gradient between the cool mould wall surface exposed to the ambience and the interior of the solidifying melt that would eventually form the casting. Therefore, in the practical case, nucleation is initiated at the mould surface and the growth of the solid phase proceed being directed towards the centre of the casting. This growth takes place in a preferred crystallographic direction as dictated by the characteristic of the solidifying crystal. For an example, in a cubic crystal the preferred crystallographic direction is <001>. With the aid of the temperature gradient, the grains oriented favourably grow at a faster rate than the others.
With the lapse of time, depending on the no of effective nuclei and the initial growth rate setup by the initial temperature gradient, the growth of the crystals in the lateral direction gets obstructed. This is because the laterally growing crystals impinge into each other restricting growth of the neighbouring crystals. Also any growth of any crystal ahead of the others, into the high temperature melt is inhibited due to the unfavourable temperature conditions. Such a situation gives rise to planar or plane front growth where in a seemingly plane interface proceeds into the melt causing growth [11]. The interface is plane macroscopically whereas in actual, it is a terraced structure microscopically. This is a typical condition leading to the formation of columnar grains which is often observed in cast ingots. These columnar grains grow in a direction opposite to the direction of heat-flow. This is illustrated in Figure 3.
Schematic presentation of planar growth giving rise to columnar dendrite.
The occurrence of planar growth giving rise to a columnar structure involves thermal condition present in Figure 4.
Schematic presentation of the thermal condition for plane front growth.
It is assumed, a positive temperature gradient exists at the solid–liquid interface. Here, the liberated latent heat of crystallisation is not enough to reverse the temperature gradient due to the freezing; i.e., a situation is not created when some pockets in the inside of the melt, away from the interface, are at relatively lower temperature. This situation is favoured at slow cooling rates which ensure a stiff and positive temperature gradient. Under these conditions only, the interface assumes the shape of a seemingly plane surface a columnar grain-structure is favoured.
The thermal conditions get grossly distributed when sufficient accumulation of the liberated latent heat of crystallisation at the interface is experienced. The liberated heat now disturbs the thermal gradient. Though there is a positive thermal gradient due to the cold mould surface, the local evolution of latent heat produces a reverse temperature gradient at the interface. This is illustrated in Figure 5. Where a zone of thermal super cooling indicating pulls in the melt at the interface or adjacent to it at temperatures lower than the equilibrium temperature are witnessed. Obviously, growth does not occur due to the general advancement of the plane-front but by preferential growth processes in these undercooled pulls in the melt.
Schematic presentation of generation of thermal undercooling.
The planar growth pattern is disturbed as the minimum temperature in the liquid melt is not witnessed at the interface. Plane-front growth is hindered and growth occurs by other means. Depositions of further atoms on the surface of the nuclei may occur in regions of greater under cooling in preference to the interface. The thermal super cooling greatly influences the final structure of the solidified melt.
Constitutional supercooling in an alloy is best illustrated in Figure 6.
Phase diagram of a typical binary alloy.
The Figure 6 presents the solidification and hence the phase changes in a simple binary alloy of ‘A’ and ‘B’. Let us consider the alloy of Co. The initial alloy deposited from Co has a composition confirming to ‘C1’. Obviously, ‘C1’ has a composition pertaining to ‘B’ which is less than that of the original alloy ‘Co’. Therefore, as ‘C1’ is formed, the residual liquid gets slightly enriched in ‘B’. Thus, as solidification proceeds ‘B’ is continuously rejected into the liquid. This rejection occurs at the solid–liquid interface throughout the process of freezing. A constitutional gradient is, thus, created in the liquid, solute ‘B’ being continuously rejected at the interface. The concentration of ‘B’ is maximum at the interface and gradually diminishes as one goes towards the interior of the liquid melt. This compositional variation is presented in Figure 7(a). The change of composition brings in a corresponding change in the equilibrium freezing temperature of the alloy as presented in Figure 7(b). Each composition on the solute distribution curve has its corresponding equilibrium freezing temperature as it depends on the corresponding composition of an alloy.
Schematic presentation of rejection of solute at the interface (7(a)) and change of equilibrium temperature (7(b)) as a consequence of the solute accumulation.
The relationship between the actual (existing) temperature gradient in the melt and the equilibrium freezing temperature as a consequence of alterations in the composition of the alloy-melt, is illustrated in Figure 8.
Schematic presentation of constitutional supercooling as a consequence of solute rejection at the interface and the resultant alternation in the equilibrium freezing temperature.
Figure 8 clearly illustrates, before the actual (existing) temperature falls considerably for growth to occur, there, is a pool of melt where considerable supercooling can be witnessed at points farther within the melt. In this pool of supercooled liquid conditions are more favourable for freezing than at the interface. This condition is referred to as constitutional super-cooling.
Alloys having Eutectic composition or containing appreciable amounts of eutectic constituents, undergo eutectic freezing. The alloy of eutectic composition solidifies at a single temperature to precipitate a mixture of two phases
Alternate laminates of the two,
Rod-like or globular solids of apparently discontinuous phase in the matrix of the other phase.
It is the extent of undercooling and its relative location in the melt that immensely influence the mode of growth of the crystal in a solidifying melt. As suggested earlier, it can be a thermal undercooling or a constitutional undercooling. Different extents of undercooling may be found in a band of liquid adjoining the interface or even in the inside of the melt depending on the following:
Temperature gradient in the melt,
The equilibrium freezing temperature and
The nucleation temperature (which will also be dictated by heterogeneous nucleation)
Depending on the extent of under cooling growth can be Dendritic Growth, Cellular Growth or growth due to independent nucleation.
Dentric crystalline growth takes place on solidification of a metal/alloy melt when the liquid–solid interface moves into supper cooled liquid at a temperature lower than that of the interface. This is illustrated in Figures 5 and 6 wherein the thermal supercooling or the constitutional supercooling, as the case may be, generate pools in the liquid melt with temperature less than that at the interface. To understand dendritic growth it is important to realise that any protuberance on the solid face may tend to be stable and act as a centre for further growth in preference to other locations due to undercooling. The general advancement of the interface is retarded by the liberated lateral heat of crystallisation or by a solute barrier, but the local growth centres have the possibilities to grow into the zones of supercooling. This gives rise to dendritic growth. This is characterised by commercial alloys forming solid-solutions. It can be emphasised, under rapid solidification conditions non equilibrium condition of solid –liquid interface influence the dendritic characteristics to a great extent [12].
Primary axis of the dendrite is a result of preferred growth at the edge or corner of an existing crystallite. The projection develops into a needle, an then into a plate following the general direction of heat flow. This growth direction is usually associated with a particular crystallographic direction. Again, lateral growth of the primary crystal, needle or plate, is restricted by the liberation of latent heat of crystallisation or solute accumulations that had earlier restricted the growth of the original interface. However, the secondary or tertiary branches may grow by a similar mechanism that helped the formation of the primary stem. This is presented in Figure 9 which depicts the branch like dendritic growth.
Schematic diagram showing the growth of a typical dendritic arm.
This unidirectional dendritic growth produces columnar dendritic structure.
In a pure metal dendritic growth is detected by interrupted freezing and decantation (once a portion freezes, it is separated from the liquid, i.e., the liquid is decanted from the freezing crystal). On the other hand, in alloys dendritic growth is revealed by the characteristic cored structure. Coring is resulted from the differential freezing processes. The centre of the dendrites are deficient in solute which are rejected to the interdendritic zone, as explained earlier.
Dendritic growth may be associated by crystals growing independently on independently formed nuclei, elsewhere in the melt depending on the preventing thermal conditions. This independently growing crystal within the melt has an interface on its periphery. Thus, it is capable of growing in all directions generating an approximately equiaxial grain. With a less marked undercooling, when the undercooling is not enough to form dendrites, cellular growth may still take place. Thus, cellular growth precedes dendritic growth. The cellular substructure is produced as a cluster of hexagonal rods. These rods grow into the liquid and reject solute on their boundaries at the respective interfaces. After a certain level of undercooling is achieved by both thermal and the constitutional means, cellular growth gives way to dendritic growth. This proceeds by the preferential development of some of the cells. This intermediate, rod like structure is also referred to as
As shown in Figure 10 when the temperature gradient is very shallow or the rate of freezing is very rapid, the undercooling achieved may be sufficient to promote nucleations at points in the melt,
Schematic representation of the influence of undercooling on the growth pattern and grain morphology.
distant from the main interface. In such an eventuality, the nuclei are free to grow in all directions on their periphery. An equiaxial grain structure, is thus, produced by independent nucleations. Figure 10, thus, exhibits the effect of increased undercooling (with the creation of different temperature gradient) on the mode of growth. It also shows the growth pattern with this different temperature gradients existing in the melt in the growth direction away from the mould wall into the interior of the liquid-melt.
Three factors have major influence on the casting structure.
The alloy constitution (composition) decides whether the structure will be of a simple phase or eutectic grains or both. The alloy composition also indicates the tendency of the alloy to respond to constitutional supercooling. The extent of constitutional supercooling is certain to influence the growth pattern that decides the crystallographic morphology of the casting.
The thermal conditions to which the liquid melt/alloy is exposed during solidification, refer to both, the rate of cooling and the temperature distribution in the solidifying melt. This is also related to the thermal properties of the melt as well as that of the mould. Obviously, the above would influence the cast structure by dictating the mode of growth.
The inherent nucleation and growth conditions in the melt are decided by the presence of foreign particles as well as the solute present in the melt. These solute atoms could be present as trace impurities or may be due to deliberate additions to influence nucleation. Obviously, these will influence/modify the possibilities of nucleation and growth, influencing the cast structure.
To elaborate the above we take help of Figures 11 and 12.
Effect of temperature gradient variations on the extent of undercooling that influence the crystal structure.
Effect of liquidus temperature profile on extent of supercooling and crystal structure.
The alloys considered in these figures form a continuous range of solid solutions. The Figures 11 and 12 illustrate the mode of crystallisation and hence the structure of the casting, as governed by the interaction of temperature and compositional gradients in the liquid.
Figures 11 and 12 depict the effect of temperature gradient and that of liquidus temperature profile, respectively on the structure of the casting. Initially when the melt is at higher temperature the existing temperature gradient is stiff [Ti (in Figure 11)] planar growth is encouraged and columnar grain structure is favoured. This is assisted by a slow cooling rate. This continues till the temperature gradient is sufficiently shallow to generate considerable undercooling which disturbs planar growth and growth proceeds adopting other modes, as explained earlier. Figure 12 clearly indicates, with a given temperature gradient the alterations of equilibrium temperature profile, which could be due to the alterations in the solute concentration, undercooling is witnessed with liquidus profile TE (ii), with the liquidus profile TE (i) and the given temperature gradient ‘T’, undercooling is not witnessed and growth proceeds by plane-front growth giving rise to columnar grain structure. From the above, it can be concluded that columnar growth is promoted under stiff temperature gradients. Columnar growth is also favoured at slow cooling rates because of the following:
slow cooling rates establish low rate of nucleation in comparison to the growth rates, allowing growth to overtake nucleation.
As seen in Figure 13 when the cooling rates are slow the solid rejected at the interface get sufficient time to migrate into the melt interior, away from the interface. The equilibrium temperature is altered.
Change in equilibrium temperature profile as a consequence of solute concentration crystal variation.
It changes from TE (ii) to TE (i) (Figure 13). This is parallel to a situation as in Figure 12 when with TE (i) the extent of undercooling are negligible or absent. Such a situation promotes columnar growth.
In a foundry the various local factors like the extent of superheat, the extent of heterogeneous nucleation, the mould characteristics, etc. decide the variations in the thermal gradient (G) and the rate of cooling (R). Needless to say, the ratio G/R forms an important parameter to decide the mode of growth and the consequence of structure development.
Figure 14 illustrates that as the G/R ratio progressively changes from a high to a low volume the effect of undercooling becomes more and more pronounced. Columnar, plane-front growth gradually gives way to independent nucleation.
Schematic presentation of G/R ratio influencing the effect of undercooling and the resultant structure.
During freezing the thermal conditions prevailing in the melt continuously change. Thus separate structural zones as shown in Figure 15 are encountered in the solidifying melt.
Schematic presentation of critical changes in the G/R ratio during freezing and its influences on the different structural zones.
These zones are consequences of the continuously changing G/R ratio in the melt. Assuming of a lower value of the G/R ratio with the lapse of time results in the increasing extents of undercooling which are instrumental in the separable structural zones as preserved in Figure 15. The above can be made more clear with the aid of Figures 16 and 17.
Variation of undercooling with alteration in the thermal gradient showing different grain morphology.
Variation of undercooling with alternation in the thermal gradient showing different grain morphology.
Both Figures 16 and 17 provide for an explanation of the mixed structure in a solidifying casting on the basis of the prevailing thermal conditions. To start with, as presented in Figure 16, the temperature gradient is stiff. Solidification initially occurs under this marked thermal gradient. This is often sufficient to cause columnar dendritic growth in the outermost region and adjacent to the mould wall as shown in Figure 16 in this central zone (in some cases, throughout the entire solidifying melt) the temperature gradient is shallow (Figure 17).
This shallow gradient generates excessive undercooling. Here solidification proceeds by widespread nucleation, the rate of nucleation being very high. Independent nucleation occurs in the interior of the melt. These nuclei, without any barrier for growth across their periphery, grow into equiaxed grains. To be more specific, initially the temperature gradient is stiff. The rate of cooling is low, G/R assume high values. Initial solidification, thus, occur under a marked temperature gradient which is sufficient to cause columnar dendritic growth in the outermost layer. Gradually ‘G’ decreases, i.e., the temperature gradient becomes shallow and ‘R’ the rate of cooling increases as a consequence of increasing extents of undercooling. The shallow temperature gradient in the casting and the increasing extents of undercooling in the melt give rise to the formation of independent nucleation in the melt interior forming equiaxed grains, being free to grow on their unhindered periphery.
Schematically the practical, ideal cast structure can be presented as in Figure 18.
Schematic presentation of a theoretical grain structure of the casting.
Although, the above refers to alloys forming solid solutions, analogous changes occur in alloys subject to eutectic freezing. The Figure 18 shows small dendrites (equiaxed) in the outermost surface because of chilling effects at the cold mould wall.
Two further factors, other than the thermal and constitutional undercooling, also influence the cast structure particularly concerning the formation of equiaxed crystals. These are:
Crystal multiplication is a consequence of the fragmentation of dendritic arms in the columnar zone due to local factors like thermal fluctuations and change in growth rates, etc. Also the nuclei formed on the inside of the mould wall may get washed off when further metal is being poured into the mould. Some of these detached nuclei may vanish being unstable, while some may get transported to favourable sides in the liquid and grow into equiaxed grains.
Turbulence during pouring
Thermal convective currents between the hot central portion and the relatively cooler surface region,
Gravitational separation due to the difference in densities between the solid and the liquid. In general the fragmented dendrules in the melt tend to go to the bottom of the melt due to density differences between the solid dendrules and the surrounding liquid melt. The only exception is ‘Be’. Here the density of the liquid is higher than that of the solid.
To sum up the formation of the equiaxed zone consisting of equiaxed grains, is promoted by the following:
Heterogeneous nucleation in situ
Crystal multiplication and
Transportation of crystallites by gravity or by mass movement into the interior of the melt
Obviously, the above form certain factors which influence the crystallographic morphology of the casting.
For a casting to be produced, it is essential that appropriate technique to be adopted for the liquid metal to be fed into the mould cavity. It is an issue inviting special considerations since the viscosity of the melt increases with drop of temperature making its flow sluggish and time taking which may result in considerable solidification prior to the completion of the feeding process. Also, the metal/alloy shrink on solidification producing solidification shrinkages in the casting which are discontinuities in the casting. For a healthy casting production ample facilities must be made be made available for compensating for these shrinkage. On these considerations, we can take the case of a pure metal which solidifies at a constant temperature or even alloys having a narrow freezing range. In these cases, three are clearly defined interfaces between the solidified region and the ‘still-liquid’ region. Any solidification contraction has the liberty to be compensated by the ‘still-liquid’ melt adjacent to it. If sufficient liquid metal is available the process of compensation of the physical contraction continues by the general lowering of the free liquid surface resulting in the production of a sound casting with no solidification shrinkages. The supply of liquid metal is accomplished by the provision of liquid metal reservoir known as feeder head or riser.
However, in many cases the case is not as simple. A clearly defined solid/liquid interface does not exist. Solidification takes place through a zone simultaneously. Even in certain cases the solidification zone may extend throughout the melt entirely. In these zones crystals at different stages of growth can be seen with the residual low melting point liquid. The alloy is in a pesty zone or in a mushy stage. Contraction sites are dispersed in the casting making feeding of these contraction sites a very difficult and even impossible, for the production of sound casting.
Under these conditions, liquid metal is fed ink the contraction sites in three successive stages.
In the early stages, growing crystal bodies are suspended in the liquid. Free movement of the liquid across the crystals is possible. Thus, any contraction can be easily compensated by a feeder head with the general lowering of the free liquid surface.
Here, after some lapse of time, the grains grow to certain extent and form contact among themselves forming a network of solid. Liquid movement becomes confined to intergranular channels. These channels get diminished continuously. Frictional resistance to the liquid movement in these channels increases. It becomes considerably difficult for the liquid to reach the solidification sites that undergo contraction. Thus, feeding for compensating the contractions becomes progressively difficult.
Now the final stages of solidification have reached. The intergranular channels are completely blocked by the growing crystals. Thus, isolated pockets of liquid are generated which solidify independently. The resultant contractions cannot be fed from external sources. These are always with long freezing range and cool under shallow temperature gradients. The shrinkage defects in these alloys persist resulting in spatter porosities distributed in the entire castings, even extending to the casting surface.
An external feeder head, known as the riser, is employed to compensate for the solidification shrinkages so that a sound casting results. For successful functioning of the riser the principle of directional solidification is employed. It is also known as progressive solidification in which solidification starts farthest from the riser and proceeds into the riser so that any side of shrinkage has an unfailing supply of liquid metal. The successful functioning of the riser must ensure the following:
Riser should be the last one to solidify in the casting system. This means throughout the process of solidification the riser must have liquid metal for feeding during freezing.
Freezing must start farthest from riser and continue through the casting towards the riser.
There must be a continuous path of feeding the liquid metal from the riser to the solidifying site.
The cooling rate of the casting need to be controlled for this purpose. The cooling rate on the other hand can be controlled by controlling the pouring temperature of the metal, pouring rate, promoting differential cooling by use of chills, differential heating by addition of exothermic materials, use of padding, etc. The temperature gradient and cooling rate are very important consideration in a solidifying melt. It is opined [13, 14] by setting up an appropriate temperature gradient and cooling rate by selecting the necessary pouring rate and temperature the cast structure can be controlled and the casting upgraded. A fine grain structure can be, thus, obtained by proper selection of the pouring rate and temperature. These fine grained structure can enhance the ability of the casting to inhibit the slide of the dislocations. This can result in the increase of yield strength and the ultimate strength of casting. These measures stiffen the temperature gradient assisting the setting up of a path feeding from the riser to the contraction sites in the mould.
Though, no two alloys have identical feeding characteristics, on the basis of the major contrasts of solidification they can be put under three categories.
This group of alloys freeze with marked skin formation. These have of obvious short (narrow) freezing zone. These include Low carbon steels, Brasses, Aluminium Bronzes, Aluminium Copper, etc.
In these group of alloys progressive or directional solidification measures can be easily achieved. Sound castings can be obtained with proper feeding from the feeder head. However, the casting yield may suffer sometimes as the feeder head has to be finally discarded.
These are alloys with long feeding ranges. These include Medium and High carbon steels, Nickel based alloys, Gun metals, Mg alloys, Complex Al alloys, etc.
In these alloys solidification proceeds simultaneously in much of the casting, even in the entire casting. All the three stages of freezing can be clearly witnessed as explained earlier. In the third and last stage of freezing widespread porosity may occur. In such castings of alloys progressive solidification is very less unless heavy chilling is used to disturb the thermal gradient. The chilling induces very sharp temperature gradient and help formation of a sound casting by setting up of narrow freezing zones. However, the casting feeding in these alloys is not always based on directional solidification. Efforts are made to follow measures such that concentration of porosity is not localised but distributed in the casting. To achieve the above mentioned disperse porosity measures have to be adopted for equalisation of cooling rates over the entire zone instead of going for a sharp temperature gradient. These disperse micro pores could be more acceptable than concentrated porosities [15].
These are the alloys which show expansion on freezing. These include Grey Cast Iron. In the Gr III hypoeutectic Grey Cast Irons freezing is initiated with the growth of austenite dendrites. Contraction on freezing occurs much like the other alloys with considerable freezing range. Then the eutectic freezing begins. Graphite precipitates out of the solid. Interdendritic liquid gets enriched with carbon. The solidification of this austenite-graphite eutectic is accompanied by volume expansion. A positive pressure is caused. In a completely rigid mould this expansion makes it virtually self-feeding. In practice, however, the positive pressure tends to cause mould-wall-movement. This movement increases the mould dimensions and sets up a tendency for contraction giving rise to an increase in the internal porosity.
To sum up, in the Gr.I alloys feeding by risers is easy. In the Gr.2 alloys feeding by riser is helped by ensuring directionality in solidifications and in the Gr.III alloys, in the contraction stage all the three stages of bulk feeding and interdendritic feeding stages may be encountered.
As mentioned earlier the temperature gradient in a casting system can be made stiff from a shallow one by adopting several means. This is illustrated in Figures 19 and 20 [16].
Shallow temperature gradient showing extensive pasty zone.
Effect of stiff temperature gradient on pasty zone (narrow zone of crystallisation).
A stiff temperature gradient can reduce the extent of pasty zone with the associated advantages in setting of directionality in solidification. On the other hand, a shallow temperature gradient can be set up and extensive pasty zone resulting in simultaneous freezing in an extended zone in the melt and help distribution of micropores.
Casting of a metal/alloy is a manufacturing process in which the liquid metal/alloy Is poured into a pre-formed mould. The liquid solidifies in the mould and the solidified liquid, known as the casting, is finally retrieved from the mould. In the whole process of producing the casting, the solidification processes play a major role in deciding the cast structure which dictate the structure related properties of the casting and decide its end-use. Throughout the entire process of solidification the metal/alloy shrink, generating discontinuities in the casting in the form of shrinkage cavities. These have to the compensated for by the supply of liquid metal from a liquid-metal-reservoir, known as the feeder-head or the riser. For efficient functioning of the riser it is desirable to ensure directionality in solidification by shaping of the necessary thermal gradient ‘G’ and the rate of cooling ‘R’. Infact the ratio ‘G/R’ plays a very important role in deciding the cast structure when it changes from a high value to a low value with the lapse of time. It decides the mode of growth which can be planar, cellular dendritic or growth due to independent nucleation and dictates the consequent development of the cast structure. The extent of superheat in the melt, the extent of heterogeneous nucleation, the mould characteristics, etc. Setup the required ‘G/R’ ratio. External factors like the pouring rate and pouring temperature can be altered suitably to vary the ‘G/R’ ratio such that the desire cast structure can be obtained.
In our mission to support the dissemination of knowledge, we travel throughout the world to present our publications, support our Authors and Academic Editors at international symposia, conferences, and workshops, as well as to attend business meetings with science, academic and publishing professionals. We are always happy to meet our contributors in our offices to discuss further collaborations. Take a look at where we’ve been, who we’ve met and where we’re going.
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