The detection and identification of pathogens are essential in clinical microbiology and the food industry, as it allows doctors to provide proper treatments to patients and ensures the detection of any foodborne pathogens, so preventing illness.
Identifying pathogens by using phenotypic methods, such as morphological or biochemical characteristics, is one of the most traditional ways of detection. The morphological characteristics of pathogens can be microscopic or macroscopic and include cell shape, size, staining (both Gram and Acid-fast stain), and other structures unique to particular types of pathogens (such as endospores or capsules). With regards to staining, a ‘Gram’ stain is used to classify bacteria into two groups: Gram-positive or Gram-negative. This is based on their cell wall structure and components by using a crystal violet dye. Gram-positive bacteria possess an outer membrane surrounded by a thick layer of peptidoglycan. Peptidoglycan can retain the crystal violet dye, allowing for staining to occur. Gram-negative bacteria do not have this peptidoglycan on their outer membrane, so the dye is not absorbed. Acid-fast staining, on the other hand, is a method used to detect the presence of mycolic acid, also present in the cell wall. The macroscopic morphology of pathogen are characteristics that can easily be examined by the naked eye, usually with the appearance of colonies (such as texture, shape, growing speed).
Pathogens can also be identified by growing them in specific media. Blood agar is the conventional differential media and is made up of 5-10% mammalian blood from sheep or horse. Various microorganisms can lyse red blood cells (and thus degrade hemoglobin), and variations in this rate can determine the species. Pathogens can have different hemolytic activities including α, β, and γ-hemolysis. Β-Hemolysis refers to complete lysis and breakdown of red blood cells, leading to ‘clear’ zones around such colonies, whereas α - and γ-hemolysis refers to partial and absent of hemolytic activity.
Apart from differential media, selective media can also be used. Selective media contains ingredients which promotes the growth of a specific microorganism while inhibiting the growth of others. MacConkey agar is a commonly used selective media for detecting and isolating Gram-negative bacilli and enteric bacteria, testing their ability of lactose fermentation. MacConkey agar contains crystal violet and bile salts which give inhibitory effects to gram-positive bacteria, and therefore, selects for growth of gram-negative bacteria. Lactose monohydrate within MacConkey agar acts as a primary source of fermentable sugars for lactose-fermenting bacteria, which then results in the formation of acid, producing the color change necessary for detection.
One other method for detecting pathogens is through examining its biochemical properties. These determine the presence of specific types of enzymes within the pathogen such as catalase or oxidase. Some microorganisms contain catalase which helps break down hydrogen peroxide to protect themselves. To perform a catalase test; 3% hydrogen peroxide solution is added to a small volume of bacterial inoculum and gas bubbles will occur if the pathogen contains catalase. The oxidase test, on the other hand, is used to test for the presence of cytochrome c oxidase, which is a bacterial electron transport chain enzyme for regulating aerobic respiration. Such tests ucartse a reagent called tetramethyl-p-phenylenediamine, which, in the presence of cytochrome c oxidase, turns the solution purple (due to oxidation). Therefore the tested pathogens can be easily classified.
Lastly, various genetic methods like PCR and DNA sequencing can be used to detect the presence of bacteria and viruses. Before conducting a PCR assay, DNA or RNA extraction is required. If the target sequence is within the sample, amplification of DNA sequence will occur, revealing which specific pathogen is present. Due note, however, that PCR can amplify the sequence of dead cells, leading to false positive results. Real-time PCR has improved some of these drawbacks of traditional end-point PCR, as it allows identification and quantification of pathogens within the sample at the same time.
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