Lab 1 Microbiology Lab Manual PDF

Lab 1 Bacterial diagnosis ( VIETIC System) & BACT automatedSystem Vitek 2 The automated system is computer-assisted and has sophisticated software to analyses the growth rate and determine the antibiotic susceptibility report. Principle  Detect growth in...

Lab 1 Bacterial diagnosis ( VIETIC System) & BACT automatedSystem Vitek 2 The automated system is computer-assisted and has sophisticated software to analyses the growth rate and determine the antibiotic susceptibility report. Principle  Detect growth in microvolumes of broth with various dilutions of antimicrobials  Detection via photometric, turbidimetric or fluorometric methods. Advantages  increased reproducibility  decrease labor costs  rapid results  software  detects multidrug resistances  ESBL  correlates bacterial ID with sensitivity Disadvantage costs Type of Automated system 1. Vitek 1 and 2 2. Pheonix 3. MicroScan Walk Away system 4. MALDI- TOF ( Matrix – Assisted Laser Desorption \Ioniztion – Time Of Flight ) There are 4 reagent cards available for the identification of different organism classes as follows: 1. GN : gram negative fermenting and non-fermenting bacilli 2. GP: gram positive spore-forming and non-spore-forming bacilli 3. YST: yeast and yeast –like organisms. 4. BCL: gram positive spore-forming bacilli. Other cards a. Antibiotic susceptibility testing (AST) cards. b. Gram positive antimicrobial susceptibility testing (AST) cards. c. Gram negative antimicrobial susceptibility testing (AST) cards. d. Yeast antimicrobial susceptibility testing (AST) cards.  Chromatography terms The analyte : is the substance to be separated during chromatography. The eluent: is the solvent that will carry the analyte. The mobile phase: consists of the sample being separated and the solvent that moves the sample through the column. The stationary phase : is that interacts with the sample (mobile phase). Immunochromatography By The retention time : is time that takes analyte to pass through the Lec Dr monther adel system Assi Lec. Ali Alsaeedi 1. Types : Gel filtration (permeation) chromatography : are separated protein according their size. Principle: immunochromatography is a testing method for detecting a Smaller molecules are able to penetrate the pores of gel, disease by dropping the sample containing an analyte onto a test therefore, the smaller molecules move through the column strip. This is a speedy and simple technique that produces more slowly. Whereas larger molecules are unable penetrate diagnostic results in 10 to 15 minutes after dropping the sample the pores of gel, therefore, pass through the column quickly 2. Ion exchange chromatography what is immunochromatographic procedure for blood? A process that allows the separation of ions and polar Immunochromatography (IC) combines two basic techniques: molecules based on charged that carry it. (i) the separation of molecules based on their ability to migrate on By exchanging ions between the article that you want to separated solid supports by capillary flow. and the surface ions (media) that happens exchange process (ii) the identification of the target molecules based on the antigen– Principle: antibody reaction It is exchange the sample components that passed in column with ionic components (stationary phase) leading to  separated. Negatively charge exchangers bind with The stationary phase is microscope layer of liquid that coated positively charge ions (cations). glass or metal coil tubing called column. The instrument used to Similarity positively charged bind with negatively charged perform gas chromatography is called a gas chromatograph or ions (anion). aerograph, gas separate 3. Affinity chromatograph Principle is a method of separating biochemical mixtures based on a Principle: The gas compounds being analyzed interact with highly specific interaction such as that between antigen and wall column, which is coated with different stationary antibody, enzyme and substrate, or receptor and ligand. phases. This causes each compound to elute at different Important: time, known as the retention time of compound. The a. Purify and concentrate a substance from a mixture into a comparison of retention times is which gives gas buffering solution chromatography its analytical usefulness. b. Reduce the amount of a substance in a mixture 4. HPLC (High liquid chromatography): Is chromatographic technique is used in biochemistry an analytic chemistry to identify quantify and purify the individual components of the mixture. The stationary phase consist a pump that moves mobile phase and detector that provides a characteristic retention time and also information's about UV data. Principle In HPLC a pump (rather than gravity) provides the higher pressure required to propel the mobile phase and analytic through the density packed column 5. Gas chromatography: Is a common type of chromatography used in analytic chemistry for separated and analyzing compounds that can be vaporized without composition. In gas chromatography, the mobile phase is a carrier gas, such as helium or nitrogen.   The ELISA test, or the enzyme immunoassay (EIA), was the first screening test commonly employed for HIV. It has a high sensitivity (99%).. Basic Principle of ELISA 1- Use an enzyme to detect the binding of antigen (Ag) antibody (Ab). 2- The enzyme converts a colorless substrate (chromogen) to a colored product, indicating the presence of Ag:Ab ELISABy binding 3- An ELISA can be used to detect either the presence of Assi Lec. Ali Alsaeedi Antigens or antibodies in a sample depending how the test is designed. — Assi lec. Haider ali Khudair 4- ELISA was developed in 1970 and became rapidly accepted Introduction to ELISA Materials needed for ELISA 1. Testing sample — ELISA, or Enzyme-Linked Immuno Sorbent Assay, are 2. Antibody (1st, 2nd)/Antigen quantitative immunological procedures in which the Ag-Ab 3. Polystyrene microtiter plate reaction is monitored by enzyme measurements. The enzyme 4. Enzyme linkage or labeling allows to follow the target protein and if 5. Washing buffer present (qualify) and at what amounts (quantify). 6. Substrate 7. Blocking buffer (stop solution)  An enzyme conjugate is an enzyme bound or joined with an Antigen (Ag) antibody which binds with the target protein. Any molecule that induces production of antibodies when  This enzyme labeling is a safe and effective way to track the introduced in the body of an animal is called antigen. antibody  The term ELISA was first used by Engvall & Perlma in 1971.  4. False positives/negatives possible, especially with mutated/altered antigen. — OR — Any “thing” foreign to the immune system. e.g. bacteria, viruses, (or their parts), Protein molecule, Carbohydrate molecule, Allergens, etc - Antibody ab ; proteins produced by the immune system which help defend against antigens Specimen sample for ELISA Serum , CSF, sputum, urine, semen, supernatant of culture, stool Advantage of ELISA 1. Reagents are relatively cheap & have a long shelf life 2. ELISA is highly specific and sensitive 3. No radiation hazards occur during labeling or disposal of waste. 4. Qualitative & Quantitative 5. Suitable for automation 6. Easy to perform and quick procedures 7. Equipment can be inexpensive and widely available. 8. Huge capacity through standard 96-microwell pattern: miniaturization and automation. Disadvantages of ELISA 1. Measurement of enzyme activity can be more complex than measurement of activity of some type of radioisotopes. 2. Enzyme activity may be affected by plasma constituents. 3. Very specific to a particular antigen. Won’t recognize any other antigen  Principle of Immunofluorescence Assay Immunofluorescence assay is based on specific antibodies for detecting and visualizing particular proteins or antigens in biological samples using fluorescence microscopy. The immunofluorescence assay principle relies on the antibodies’ specificity for their target proteins. This specificity helps selectively label and visualize the location and distribution of specific proteins within cells, tissues, or other biological samples. Immunofluorescences & Radioimmunoassay Immunofluorescence can be helpful in clinical diagnostics for Assi Lec. Ali Alsaeedi detecting specific antigens or markers associated with diseases or conditions Assi lec. Haider Ali Khudair Introduction Different diagnostic tests require other immunological processes to detect antibodies and antigens. Immunofluorescence assay is one of the most commonly used immunological tests. Immunofluorescence is the combination of the two words immuno and fluorescence. Immune means immune or immunity; fluorescence implies fluorescent molecules that produce visible or invisible radiation. The essential components of the immunofluorescence technique Immunofluorescence assay is a significant technique are antibodies and fluorescent labels commonly used in immunology or molecular biology for 1. Antibodies: The antibodies are proteins the immune system detecting the presence and distribution of specific proteins or produces that specifically bind to antigens. Likewise, the antigens. This process uses antibodies labeled with fluorescent immune system recognizes antigens as foreign or non-self. molecules that bind to the target protein or antigen of inters In the immunofluorescence assay, specific antibodies target 1  and attach to the protein or antigen of interest within a to primary antibodies. The added step makes biological sample. this process more sensitive. 2. Fluorescent Labels: Fluorophores, or fluorescent labels, are attached to the antibodies. These labels emit fluorescent light when exposed to specific wavelengths of light. Each fluorophore emits light at a unique wavelength, which helps identify and differentiate different targets in the sample Types of Immunofluorescence Assay There are different types of immunofluorescence assays with slight variations in the use and types of antibodies used. 1. Direct Immunofluorescence or direct fluorescent antibody (DFA) test is a simple method requiring only primary antibody. The Lab3 primary antibody conjugates to a fluorophore Counter immunoelectrophoresis (CIE) (fluorescent dye), which directly binds to the Counter immunoelectrophoresis (CIE) is a modification of antigen of interest in the sample. the Ouchterlony method that speeds up the migration of an antigen and antibody by applying an electrical current. 2. Indirect Immunofluorescence or indirect fluorescent antibody (IFA) test is the most common immunofluorescence technique. Firstly, application of a primary antibody specific to the targeted antigen occurs. Then, there is the use of a secondary antibody labeled with a fluorescent dye particular to the primary antibody. Here, the secondary antibody binds  flow cytometry used for flow cytometry studies are used to identify and quantify immune cells and characterize hematological malignancies. They can measure: cell size. cell granularity basic principle of flow cytometry? flow cytometry Assi Lec. Ali Alsaeedi Assi lec.ali hamz The basic principle of flow cytometry is the passage of cells in single file in front of a laser so they can be detected, counted and Assi lec. yahya jalal sorted. Cell components are fluorescently labelled and then excited by the laser to emit light at varying wavelengths. detected Assi lec. Haider ali Khudair by the detectors Assi lec. intesar jabbar steps in flow cytometry Introduction flow cytometry is a laser-based lab test that can detect chemical and physical differences of cells or particles. There are four steps in most flow cytometry protocols: Healthcare providers commonly use it to evaluate 1. Sample Preparation. bone marrow, peripheral blood and other fluids in 2. Blocking. 3. Antibody Incubation. your body. Flow cytometry can help diagnose The three main components of a flow cytometer are the fluidics, several conditions, including infectious diseases optics, and electronics working of cytometry Flow cytometry runs on the principles of light scattering, excitation and emission. Fluorescently tagged cell components get excited when they pass through a laser beam, producing lights of different wavelengths. The fluorescence is used to analyze cellular properties advantages of flow cytometry It allows researchers to evaluate thousands of cells per second in a rapidly flowing single-cell suspension. This tool is used across the life sciences to statistically characterize cells is flow cytometry qualitative or quantitative? Flow cytometry (FC) is defined as a method for the qualitative and quantitative measurement of biological and physical properties of cells and other particles suspended within a high- velocity fluid stream and passing through a laser beam in a single file methods of cytometry Cytometry is a technique that is used primarily for the measurement of cells, although cytometers can also be used to characterize viruses and abiotic particles. There are two basic variations of the technique, flow cytometry and solid phase cytometry