Diagnosis of infection is dependent on many factors like right specimen, right container, right quantity, right timing, right transportation, right storage if required preprocessing, since the result is a true representation of the quality of the sample received.
Depending upon the size of the microrganisms there are different modality of diagnosing infections.
Bacteria, Staphylococcus, Escherichia coli, Pseudomonas are big enough to be visualized under light microscope and easy to culture. Different type of staining method are there like Gram stain to differentiate between gram positive, gram negative bacteria, Zeihl Neelsen stain for acid fast bacilli Culture of the bacteria is done on media poured in plastic petridishes which can be Blood agar, Chocolate agar , Mac conkey agar . Growth seen on these plates are utilized to identify different bacteria like hemolytic / non hemolytic on blood agar, Fastidious or difficult to grow bacteria on chocolate agar, Lactose and non lactose fermenting in Mac conkey agar. There are other selective media like Xylose lysine deoxycolate agar, Salmonella shigella agar for growth of specific bacteria. Anaerobic growth can be detected by Brain heart infusion broth and grown in anaerobic jar.
Bacteria grown on the media can further be identified and susceptibility can be done by automated method like VITEK 2.
Virus are smaller in size as compared to bacteria so they can be detected by Electron microscopy, serological method.
Electron microscopy uses a beam of electrons and wave like characteristics to magnify an objects image of virus or bacteria or any other biological specimen also .There are three type of electron microscopes TEM (Transmission electron microscope ) SEM (Scanning electron microscope ) REM ( Reflection electron microscope )
Serological diagnosis is either detection of the viral antigen or detection of the antibody formed against the virus in human body in patient serum sample. Different principals like Enhanced chemilumiscence (ECLIA), Enzyme linked Fluorscent antibody test (ELFA), Rapid serologic diagnosis by strip-based card lateral flow immunochromatography method. All these tests are semiquantitative assay. Quantitative assay is ELISA (Enzyme linked immunofluorescent assay) which uses color tagged anti-human antibody to attach to antigen antibody complex and further quantitates the amount of antigen or antibody.
Fungal diagnosis can be made by plating on special media SDA (Sabourd,s dextrose agar ) and identified by seeing the gross morphology of color of the growth . Microscopy method of LPCB (Lacto phenol cotton ball) preparation also shows the arrangement of the fungus in the slide and helps to identify the mycelial growth. Fungus like candida can be identified and susceptibility to drugs can be done by automated method like VITEK 2 .
Parasites can be identified by Gross examination and microscopy for ova /cyst /of parasites using iodine or normal saline.
Different type of organisms (Bacteria , virus , fungus and parasites ) can be identified by molecular methods like PCR (Polymerase Chain reaction). In this method the DNA/RNA of the organism is broken down in the sample provided and than the products are amplified by multiplication. These are further tagged to fluorophores and the quantity of these products are expressed as detected / not detected qualitatively or copies/mL
New modality like MALDITOF (Matrix associated laser desorption/ionization) is an ionization technique that uses a laser energy absorbing matrix to create ions from large molecules with minimal fragmentation. Mass spectra of expressed proteins generated and are analyzed by dedicated software and compared with stored profiles for species determination called biotyping. it can diagnose bacteria, virus, fungus and parasite also as well diagnose sensitivity pattern like methicillin resistance, carbapenem resistance.
One of the other new testing modality is the FILMARRAY test, which integrates sample preparation, amplification, detection and analysis of the RNA/DNA present of microorganisms. It requires just a few minutes of hands-on time and turn out time is just one hour, giving faster results and better patient care.
