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Methods for Microbial Detection and Identification

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The detection and identification of microorganisms is a significant feature of microbiology research. It can be applied to many areas such as environment, industry, and medicine. The methods for microbial detection and identification have been developed over time, from traditional methods leaning heavily on culture, morphology, physiology, pathology, and biochemical testing that are comparatively time-consuming and labor-intensive, to modern mass spectrometry and genetic technologies that contributed to the soaring of microbiology research in recent years. Identification methods can be divided into two groups – phenotypic and genotypic: phenotypic methods clarify microorganisms according to their phenotypic characteristics, while genotypic methods are reliant upon the interpretation of genetic information.

Methods

Description

Phenotypic Methods

Morphological Methods

Microscopy

Microscopic microbial identification uses microscopes to view microorganisms that cannot be seen with the naked eye. There are several branches of microscopy including optical, electron, and scanning probe microscopy. Along with the microbiological knowledge of shapes, arrangements, motility, gram staining features, and so on, microscopic methods are practical and can identify and obtain morphological information of microorganisms quickly.

Macroscopic Morphology

Culturing microorganisms is still one of the most widely applied techniques in microbial studies. Different microbe species give rise to colonies that may be quite distinct to others, which makes it possible to preliminarily identify microorganisms by observing colonial morphology.

Physiological / Biochemical Characteristics

Culture Methods

Cultural characteristics of microorganisms refer to the growth characteristics and morphology in various kinds of culture media. Pure culture should be taken before further observation, which can be obtained under different cultural conditions such as aerobic or anaerobic environment, selective media, and physiological activities like substrate consumption and enzyme production. These biochemical characteristics provide valuable clues for microbial detection and identification.

Phage Typing

Phage typing is a method used for detecting single strains of bacteria. It is used to trace the source of outbreaks of infections. The viruses that infect bacteria are called bacteriophages and some of these can only infect a single strain of bacteria. These phages are used to identify different strains of bacteria within a single species.

Automated ID/AST Instrument

Microbiology instruments can provide rapid and accurate identification and susceptibility testing. They can automatically interpret biochemical bar after microbial culture, and provide reliable results by database matching. The automated ID/AST instrument largely reduces the time for culture and biochemical testing to as little as 5 to 8 hours.

Immunological Methods

Serological Tests

Serological tests can be used to identify similar species or to classify the same species, and to help clinical diagnosis by detecting microbial antigens and antibodies. The protocol involves using the antiserum from known strain, type, or strain for serological testing of specific serological reaction with target microorganism.

ELISA

Enzyme-linked immunosorbent assay (ELISA) is an analytical technique to detect the presence of an antigen or antibody in a given sample. It shows wider applications in clinical diagnosis, pathological studies, and quality control studies. The variations in ELISA allow us to detect either antigen or antibody, identifying the different strains of microbes at a time and also in characterization of the epitope distribution on the microbial surface.

Immunoaffinity Chromatography

Immunoaffinity chromatography (IAC) combines the use of liquid chromatography (LC) with the specific binding of antibodies or related agents, which can isolate certain particulates by coupling antibodies with a solid inert matrix, usually in a column, and allowing the culture to pass through, separating the antigen from other materials through a series of buffers.

Chemical Analysis

MALDI-TOF MS

Matrix-Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) is a proteomics-based approach for microbial identification and characterization, which has been widely used as an identification technique in microbiology laboratories. With significant advantages compared with other tools, such as biochemical identification and phenotypic tests, MALDI-TOF MS revolutionized the approach to the rapid and accurate identification of bacteria, shortens the period from days to one hour within.

HPLC

Identification of microorganisms by means of high-performance liquid chromatography (HPLC) has provided an alternative to diversified approaches, which can target multiple microbial components such as cell wall mycotic acid, nucleotides, and so on.

Molecular Methods

Whole-Genome Sequencing

Whole-genome sequencing provides a comprehensive understanding of the entire genome, which not only determines the likelihood that genetic material comes from an individual or group but also suggests additional information on genetic relationships, origin or susceptibility to specific diseases. The major processes of whole-genome sequencing workflow are alignment, variant calling, annotation and calculating related metrics.

Ribotyping

Ribotyping is a method for bacterial identification and characterization that employs rRNA based phylogenetic analysis. Given that rRNA genes (such as 16S rRNA) are highly conserved within a bacterial species, identifying 16S rRNA gene polymorphisms is a reflection of the evolutionary lineage of the bacterial species, and can shed light on bacterial classification, taxonomy, epidemiological investigation, and population biology.

Real-Time PCR

Real-time polymerase chain reaction (real-time PCR), also known as quantitative polymerase chain reaction (qPCR), is used to amplify and simultaneously monitor, and quantify or semi-quantify targeted DNA molecules, which is a conventional technology in biology labs to provide insights into gene expression and phenotypes of microorganisms on the genetic level.

DNA Fingerprinting

RAPD-PCR

Random amplification of polymorphic DNA (RAPD-PCR) employs short primers (8-12 nucleotides long) with arbitrary sequences that bind nonspecifically to template bacterial DNA, which results in amplification of random, repetitive regions of template DNA, thereby providing a unique profile for bacterial identification.

RFLP

Restriction fragment length polymorphism (RFLP) is a method for identifying bacterial strains using unique fingerprints, which relies on the presence of variations in homologous DNA sequences. This PCR-based method employs restriction enzymes, which can recognize and cut amplified DNA (PCR product) into DNA fragments of different lengths. Electrophoresis of the enzyme digests distinguishes individuals, populations, or species or to pinpoint the locations of genes within a sequence.

AFLP

Amplified fragment length polymorphism (AFLP) employs restriction enzymes (usually a pair) to fragment genomic DNA and then amplifies a subset of restriction fragments using ligated adaptors. This amplification is achieved by using primers that are complementary to the adaptor sequences but also have certain unique nucleotides. Therefore, only a small number of restriction fragments are selectively amplified.  AFLP fingerprints are then analyzed using gel electrophoresis, yielding a set of distinct DNA fragments from a single bacterial genomic DNA, which offers high specificity and discriminatory potential in the absence of any prior genome sequence knowledge.

Microarray

DNA microarray, also known as gene chip, is a well-established technology that can be used to measure gene expression levels. The whole process takes place on a special piece of glass or silicon chip where thousands of genes can be detected in a single reaction, allowing for the high-throughput identification of microorganisms in a fast and accurate manner.

Metagenomics

Metagenomics is a culture-independent technology that can provide insights into the collective genome of a microbial community, indicating microbial diversity and ecology in specific environments. Metagenomics is generally based on next-generation sequencing, which reads billions of DNA base pairs in a single run. With abundant and comprehensive genetic information, microbial metagenomics provides a solid basis for the design and analysis of the omics study of microbiome characteristics.

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