Biochemical Tests Part 2 PDF

**BIOCHEMICAL TESTS PART 2** **RAPID RESULT TESTING** Miniaturized or automated testing for identification and or susceptibility testing of organisms. Some use dehydrated substrates in wells that detect pre-formed enzymes Some are Serological tests, to detect antibodies or antigens directly from...

**BIOCHEMICAL TESTS PART 2** **RAPID RESULT TESTING** Miniaturized or automated testing for identification and or susceptibility testing of organisms. Some use dehydrated substrates in wells that detect pre-formed enzymes Some are Serological tests, to detect antibodies or antigens directly from a specimen or from a growing organism **Chromogenic media** Rapid testing by Automation Rapid testing by Molecular techniques All these allow for increased accuracy and faster patient results -- usually within 4-6 hours **HOW ARE RAPID RESULTS DETECTED** **Agglutination:** Antigen/antibody reaction = clumping. **Fluorescence:** Non-fluorescent to start, becomes fluorescent if enzyme breaks down substrate. **Chromogenic reaction:** Dye colorless to start if enzyme present becomes colored. **Colorimetric:** Color produced due to acid or alkaline pH or a chemical reaction (enzyme breaks down substrate). **Turbidity:** Increased cloudiness if bacteria present. - Colorimetric or Turbidimetric methods can be qualitative or quantitative measurements. HOW TURBIDITY IS MEASURED - Light-rays are scattered and absorbed by bacteria in the solution rather than transmitted in straight lines through the sample. - If measure scattered light = **nephelometer** - If measures transmittance = **turbidimeter** AUTOMATED DETECTION SYSTEMS Automated systems use instruments that measure light change to detect positive or negative results **Photometer:** measures the **intensity of light**--converts light into an electric current that changes as the amount of light absorbed by particles in the liquid changes. **Spectrophotometer/Colorimeter:** measures the **concentration of a substance** or the **intensity of a color of a solution** by how much light of specific colors is absorbed/transmitted by the particles in the solution. **Fluorometer**- detects increasing levels of fluorescence SPECTROSCOPY/ SPECTROPHOTOMETER - Measures the amount of light that is absorbed or transmitted by a sample -quantitative measure of the concentration of matter in the sample -- i.e. amount of bacteria - Consists of two devices; a **spectrometer** and a **photometer**. **Spectrometer:** **collimator, monochromator & slit** - The collimator (lens) transmits a straight beam of light (photons) - This light passes through a monochromator (prism) that splits this light into several wavelengths - Then the slit selects & transmits only the desired wavelength you want to measure & this wavelength of light is shined onto a sample **Photometer:** - Detects the number of photons or the intensity of light that is transmitted or absorbed after the desired wavelength of light passes through the sample in cuvette -- converts this to an electrical signal which is digital display **VITEK 2** - Automated system first designed for the space program - Identifies bacteria (aerobic, anaerobic, some fastidious, facultative gram pos & neg) and yeasts - Cards have 64 wells that each contain a test substrate - Measure metabolic activities: acidification, alkalization, enzyme production, and growth in the presence of inhibitory substances - Uses a photometric system to detect changes in light like turbidity or the colored end products of substrate metabolism - Each test reaction is read every 15 minutes - Results can take from 4-6 hrs, some longer 13 -16 hrs. - Biocode is generated & compared to a database of known organism biocodes - Has cards to perform susceptibility testing of non-fastidious, aerobic or facultative bacteria - Antibiotic susceptibility testing (AST) determines the susceptibility of a bacterial strain to a panel of antibiotics. - The organism is placed in contact with different concentrations of different antibiotics. - In general, if the microorganism grows in a certain concentration of the antibiotic it is resistant to it & if it can't grow in a certain concentration it is susceptible. - The lowest concentration of an antibiotic which prevents visible growth of bacteria is called it's MIC. - This is compared to known organism MIC in Vitek database and reported as S (susceptible) or R (resistant) PCR - Polymerase chain reaction (PCR), is a technique where many copies of a specific target DNA region are made in in a test tube rather than an organism. **Target (Template) DNA :** - **The DNA extracted from the specimen** (may or may not have the specific target sequence you are looking for) - If target sequence is present, it will be used as a template on which the new DNA will be built. **DNA polymerase:** - Enzyme in cells that use existing single strands of DNA as a template to make new double strands of DNA - Reads the template and adds nucleotides one at a time - In PCR use **Taq polymerase** because it does not denature at the high temperatures required in each PCR cycle **dNTPs (deoxyribonucleoside triphosphates):** - Four dNTPs (adenosine triphosphate, guanosine triphosphate, thymidine triphosphate and cytosine triphosphate) - The "building blocks" added to ss of DNA by Taq polymerase to make new ds of DNA **Primers (Forward & Reverse):** - 2 short sequences of complementary DNA or RNA (20 base pairs) - Forward & reverse bind to either end of specific sequences on target DNA strand. - Taq polymerase can only add new nucleotides to an existing double stranded DNA- provides the starting point to add on to. - **Primers that are labeled are called probes** **PCR Buffer:** - Is necessary to create optimal conditions for activity of Taq DNA polymerase. - It is usually sold with Taq polymerase or you can make your own - Buffer maintains the pH between 8 & 9.5 - Includes salts like **Magnesium chloride** (MgCl~2~) & **KCl** that help the polymerase enzyme perform properly. **Next Steps:** - The first step in a PCR reaction is to extract and purify the DNA in your sample -- this is your template DNA which may or may not have the target DNA you are looking for - This DNA is placed a small tube with the polymerase enzyme, dNTPs, buffer & primers. - This tube is placed in an instrument called a thermocyler **Pcr thermocyler** - A thermal block with holes to hold the tubes with the PCR reaction mixtures - A cycler then raises and lowers the temperature of the block to complete a number of PCR cycles. - In a PCR cycle the DNA in the sample goes through repeating steps of denaturation, extension & annealing. Before a PCR cycle actually starts there may be a initial stage called a **Hot Start** where the temperature of the block is raised to 95^0^C for 1-10 min - This is only done if you are using a DNA polymerase that needs to be activated by heat to start working -- after this the actual PCR cycle starts (denaturation, annealing, extension) ANALYSIS OF PCR PRODUCT Easiest way to visualize PCR product is by agarose gel electrophoresis: - Nucleic acids or proteins separated based on size, charge and shape by an electric charge (from -- to +) - Visualized by staining with dye (fluorescence) - Compared to a ladder (size standard ruler) included on one lane of the gel VISUALIZING OUR SAME PRODUCT BY REAL TIME PCR (qPCR) - **qPCR combines PCR amplification and detection into a single step.** - PCR amplification products are assayed as they accumulate rather than at the end of the procedure - Positive result can be observed while assay is still running - Uses a **fluorescent reporter dye** or a **probe labelled with a fluorescent dye** - The Thermocyler used will also have a UV light source to excite the reporter & a camera to detect increasing fluorescence at each cycle of amplification **Two kinds of reporter dyes can be used** 1. **SYBR Green -- what we used** 2. **Taqman** **Advantages to qPCR:** is faster, does not require laborious post PCR methods for visualization of PCR products and allows for quantitation of amplified product. qPCR-SYBR Green - Fluorescent dye that binds to **any double strand of DNA** - DNA bound SYBR green has a **stronger fluorescent signal** than unbound -- increased fluorescence is measured - **Less specific** than Taqman- will bind to any amplified double stranded DNA product target & non target qPCR-Taqman Probe **Taqman probe** is a short sequence of DNA specific to a section of the target DNA. It has a **fluorescent dye on its 5' end** and **quencher on its 3'** end - While these are close together there will be no fluorescence emitted - During PCR the Forward & reverse primers attach to ss of target DNA And the Taqman probe attaches to the homologous region on the target DNA - DNA polymerase will start to extend the primers but then comes into contact with the probe. The probe cannot be extended. The polymerase has exonuclease activity -eats away the probe. This causes the fluorescent dye to come apart from the quencher and now emit a fluorescent signal. PHASES OF A qPCR CURVE **qPCR results are commonly displayed on an amplification curve that has 3 main phases:** **1. Baseline phase:** initial cycles of PCR 3 to 15 where there is little change in fluorescent signal. Or presence of low levels of background fluorescence not due to production of amplicon. **2. Exponential phase:** occurs around cycles 16-25 & represents the PCR product beginning to amplify and double at each cycle **3. Stationary or Plateau phase:** occurs late in the reaction, cycles 26-38. Components get used up, no increases in amplicon production OTHER INDICATORS ON A qPCR AMPLIFICATION CURVE **Threshold line:** - The cycle\# when the fluorescence of a PCR product is detected above the background signal - It is set by instrument **Ct (cycle threshold):** - Is the cycle\# where fluorescence in a sample crosses the threshold line. - Is a measure of the concentration of target DNA in the PCR reaction.  - It is inversely related to the starting amount of target. - The more target DNA present in the sample the lower the Ct cycle number **Absolute quantification:** - Samples of known quantity are serially diluted and then amplified to generate a standard curve - Unknown samples are then quantified by comparison to this curve **Maldi-Tof MASS SPECTROMETRY** - Introduced in 2009 in Microbiology - Very rapid identification of microorganisms -bacteria, fungus and mycobacteria **MALDI-TOF mass spectrometry involves:** - Using a laser to vaporize and ionize protein molecules - Measuring the molecules' mass-to-charge ratios - Generating a mass spectrum using time of flight mass spectrometry - Mass spectrum produced from an unknown organism is then compared to a database of mass spectra generated from known microorganism - Unknown strain is identified based on the closest match - Once a colony chosen for ID by MALDI it is placed in a well of the microplate - Overlay with 1uL of organic matrix (cinnamic acid) - Let air-dry before inserting plate in analyser **Once the slide has been placed in the analyzer...** - An ultraviolet laser beam hits the test spot on the slide - Matrix absorbs laser energy and also protects the sample - Excitation of the matrix causes vaporization -- then, microorganism proteins and matrix molecules are ejected from the target surface =**desorption** - Protons are transferred from the matrix to the proteins =**ionization** - Results in positively charged proteins in a gas phase - Electrostatic field and high voltage accelerate these ions into the flight tube (a mass spectrophotometric vacuum chamber) - Ion particles migrate to the detector at a speed that depends on their mass - Smaller ions reach detector faster than larger ones -- each ions time-of-flight is measured - A protein spectra is produced & compared to main database **MALDI-Tof LIMITATIONS** - Agar media ingredients if transferred may interfere with ID - Bacteria with mucoid capsules (*K. pneumoniae*) or spores (Clostridium) are difficult to ID - Can get a poor ID can be due to incomplete database, poor inoculum - Species that are too closely related may not identify correctly **Common Examples (there are more): *E.coli & Shigella, S. pneumoniae & S. mitis*** - Maldi-tof does not perform susceptibility testing at this time **Maldi-Tof ADVANTAGES** - Accurate, reproducible and very rapid, ID in minutes = more effective antimicrobial use and reduced patient care costs - Reduces the need to use a reference laboratory - Identifies bacteria, fungus & mycobacteria - sometimes at the sub-species and strains levels, allowing the detection of epidemic lineages - Can be used to ID anaerobic and fastidious or slow growing microorganisms - Low sample size, only 1 colony - After initial investment very low reagent costs **KIESTRA/WASP** - LAB OF THE FUTURE - Modular system (pick and choose what fits) - Process any type of specimen or container - High throughput: 250-400 inoculation per hour Decisions are made on images from computer screen rather than on actual plate Screening tests are quick tests performed in order to begin differentiating bacterial genus that have similar gram stain results **Example:** *Staph & Strep* are both gram positive cocci *Staph* are catalase +, *Strep* are catalase - - Help decide which identification & confirmatory tests to proceed with **BACTERIAL SCREENING TESTS** **Common screen Tests at Michener** **Catalase** Differentiates *Staphylococcus & Streptococcus* **Oxidase** Differentiates Gram negative organisms **Pbile** Differentiates *S. pneumoniae & Viridans streptococcus* **Spot indole** Differentiates Gram negative organisms **PYR** Helps differentiate some of the *Streptococci & some Staph* **Dnase** Differentiates Gram negative organisms & some *Staph* **CATALASE TEST** - **Tests whether an organism produces the catalase enzyme** - The catalase enzyme functions at the end of the ETC in oxidative metabolism of glucose - Once an organism produces superoxide dismutase to get rid of superoxide a less toxic by product, hydrogen peroxide (H2O2) is formed. - If organism produces catalase, it converts H2O2 to water and oxygen = protection from harmful effects of H2O2 - Add **hydrogen peroxide** to a colony on a glass slide and look for **immediate bubbles -- indicates catalase present** **OXIDASE TEST** **Tests whether an organism can produce the cytochrome C oxidase enzyme** **Oxidase reagent = 6% tetramethyl phenylenediamine dihydrochloride** Reagent is an aromatic amine that is colorless when in a reduced state -- which is how it starts off If organism produces the enzyme **cytochrome oxidase** the oxidase reagent becomes oxidized to a colored compound called **indophenol** and **turns a deep purple blue** **SPOT INDOLE TEST** **Tests whether an organism can produce the tryptophanase enzyme needed to breakdown tryptophan** - Indole detected by addition of **spot indole reagent p-dimethylaminocinnamaldehyde** Tryptophan Indole + pyruvic acid + ammonia Indole + p-dimethylaminocinnamaldehyde → turquoise color - Rapid indole results within seconds to up to 3 min - Detects indole production on actual colonies - **Green/Turquoise color is positive** No color is negative **DEOXYRIBONUCLEASE (DNase) TEST** **Tests whether an organism can produce the Dnase enzyme in order to breakdown DNA as a source of carbon and energy for growth.** - The test medium contains DNA, peptides as a nutrient source, and methyl green dye as an indicator - The methyl green dye and DNA form a complex that gives the agar a blue-green color at pH 7.5 - If DNAse is secreted, DNA in the medium hydrolyses into smaller particles which are no longer bound to the methyl green dye - Results in a clear zone in the agar around the bacterial growth. **PYR** **Test for the organisms ability to product the enzyme L-pyroglutamyl-aminopeptidase (PYRase)** - Some organisms produce the PYRase enzyme which hydrolyses L-pyrorolidonyl-B-napthylamide to B-naphthylamine - PYR is on a disk, if enzyme produced, a **deep pink violet color** (like lid on color developer bottle) develops with the addition of **p-dimethylaminocinnamaldehyde (spot indole reagent)** **BILE SOLUBILITY TEST** - Differentiates *S. pneumoniae* from other alpha hemolytic strep - Bile salts (sodium desoxycholate and sodium taurocholate) trigger catalytic enzymes in *Streptococcus pneumoniae* that promote the autolysis of cells - Bile salts lower the surface tension at the medium-membrane interface & *S. pneumoniae* will disintegrate (disappear) or lyse within 30 minutes in the presence of 10% sodium desoxycholate, a surfactant **OPTICHIN DISK TEST** Determine if an organism is susceptible to the chemical optochin (or ethyl hydrocupreine) - Differentiates *S. pneumoniae* from other alpha hemolytic strep - A colony of the test organism are streaked for confluent growth onto a BAP - An optochin disk is applied to the inoculum and the plate is incubated 35 ^0^C in CO2 - Zones of inhibition with a diameter ≥ 14 mm are interpreted as optochin-susceptible and isolate is S. pneumoniae - No zone is consistent with an alpha-haemolytic streptococcus other than S. pneumoniae