Instruments Techniques and principals in Microbiology

1. Instruments used in microbiology are not fully automated 2. Minimized human interference 3. To Aid in rapid diagnosis of infections 4. Minimized turnaround time 5. Safe and Hazard less enviornment

POLY STAINER 5300 automated system for Gram stain
• The Polystainer is a fully user programmable robotic stainer capable of delivering high quality stained slides for clinical and research Microbiology labs • The Polystainer will take over the tedious job of slide staining and free lab staff to get on with the work only skilled scientists are trained to do

It takes 5-10 minutes to stained the slides
Maximum 20 slides are stained

• Need for Automated blood culture is, • The detection of microorganisms in a patient’s blood has diagnostic and prognostic importance. Blood cultures are essential in the diagnosis and treatment of the etiologic agents of sepsis. • the expeditious detection and identification of blood borne bacterial pathogens is an important function of the diagnostic microbiology laboratory

• The BACTEC 9000 series of blood culture instruments are designed for the rapid detection of microorganisms in clinical specimens. • The sample to be tested is inoculated into the vial which is entered into the BACTEC instrument for incubation and periodic reading. • Each vial contains a sensor which responds to the concentration of CO2 produced by the metabolism of microorganisms or the consumption oxygen needed for the growth of microorganisms. • The sensor is monitored by the instrument every ten minutes for an increase in its fluorescence, which is proportional to the increasing amount of CO2 or the decreasing amount of O2 present in the vial. • A positive reading indicates the presumptive presence of viable microorganisms in the vial.

• Media used for this instruments is as under
 o o o o o  o o o o o   BACTEC PLUS Aerobic/F Culture Vial 25 mL Enriched Soybean-Casein Digest broth (TSB) 0.05% Sodium Polyanetholesulfonate (SPS) Cationic and Non-ionic Adsorbing Resins Carbon dioxide (CO2) and Oxygen (O2) Sensor for the detection of fluorescence BACTEC™ PLUS Anaerobic/F Culture Vial 25 mL pre-reduced enriched Soybean-Casein Digest broth. 0.05% SPS Resins CO2 and Nitrogen gas (N2) Sensor for the detection of fluorescence. Volume: 3 to 10 mL of blood in each vial in paeds vial is 0.5 to 5 mL of blood is acceptable.

 o o o o o  BACTEC PEDS PLUS/F Culture Vial 40 mL Enriched Soybean-Casein Digest broth 0.02% SPS Resins CO2 , O2 Sensor for the detection of fluorescence BACTEC MYCO/F LYTIC

• The VITEK 2 is an automated microbiology system utilizing growth-based technology. The system is available in three formats (VITEK 2 compact, VITEK 2, and VITEK 2 XL) that differ in increasing levels of capacity and automation. Figure 1 shows the VITEK 2 compact system. All three systems accommodate the same colorimetric reagent cards that are incubated and interpreted automatically. The reagent cards have 64 wells that can each contain an individual test substrate. Substrates measure various metabolic activities such as acidification, alkalinization, enzyme hydrolysis, and growth in the presence of inhibitory substances. An optically clear film present on both sides of the card allows for the appropriate level of oxygen transmission while maintaining a sealed vessel that prevents contact with the organism-substrate admixtures. Each card has a pre-inserted transfer tube used for inoculation (described below). Cards have bar codes that contain information on product type, lot number, expiration date, and a unique identifier that can be linked to the sample either before or after loading the card onto the system. Figure 2 shows the GN card.

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Anaerobic Chamber
• Anaerobiasis is maintained in a gastight box or chamber by a gas mixture containing or 80-90% Nitrogen(N2),5% Hydrogen (H2), and 5-10% CO2 and by using a palladium catalyst. Usually the system have a positive pressure inside the chamber. The catalyst reduces the oxygen to water, thus removing atmospheric oxygen from the chamber • Carbon di oxide is included because many anaerobes require it for growth • Add anaerobic indicator to check the system how works well

• The Clinitek 500 is a semi automated, bench top instrument that performs urinalysis • Testing using Siemens Multistix 10 SG reagent strips. • The analyzer is a reflectance photometer that analyzes the color and intensity of the light reflected from the reagent area and displays the results in clinically meaningful units. • No calculations are required. • The Siemens Multistix 10 SG reagent strip includes testing areas for glucose, bilirubin,ketones, specific gravity, pH, protein, urobilinogen, nitrite, hemoglobin and leukocyte esterase.

LabUMat- UriSed Urinalysis Analyzer
• • The LabUMat test strip analyzer and UriSed microscopic sediment analyzer both manufactured by 77 Elektronika Since all necessary measurements which have to be done on urine samples analyze completed by this integrated system in one process, combination of LabUMat and UriSed accelerates laboratory throughput and provides the most effective and reliable solution for complete and professional urine analysis. The LabUMat is an automated desktop instrument designed for high volume urine test strip reading Automated test tube handling and test strip dipping The URISED is a professional urine sediment analyzer detecting urine particles in urine sediment with high accuracy. This instrument performs sample preparation, takes several pictures of each sample through its built-in microscope and evaluates them using the UriSed Evaluation Module . which is a high quality image processing software

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• The BACTEC™ MGIT™ 960 System is designed for the rapid detection of mycobacteria in all types of clinical specimens except blood and urine. (Each tube contains Modified Middlebrook 7H9 Broth Base, Casein peptone ) • The system includes a liquid culture medium (BBL™ MGIT™ Mycobacteria Growth Indicator Tube), a growth supplement and an antibiotic mixture (BBL™ MGIT™ PANTA™ • The BACTEC™ MGIT™ Growth Supplement provides substances essential for the growth of mycobacteria. • BBL MGIT PANTA contains a mixture of antimicrobial agents used to suppress the growth of contaminating bacteria.

• A fluorescent compound is embedded in silicone on the bottom of each of the BBL MGIT broth tubes. This compound is sensitive to the presence of oxygen dissolved in the broth. • Initially, the large amount of dissolved oxygen quenches the emissions from the compound and little fluorescence can be detected. Later, actively respiring microorganisms consume the oxygen and allow the fluorescence to be detected. • The BACTEC™ MGIT™ 960 System monitors the tubes for increasing fluorescence. Analysis of the fluorescence is used to determine if the tube is instrument-positive; i.e., the test sample contains viable organisms. Culture tubes which remain negative for a minimum of 42 days (up to 56 days) and which show no visible signs of positivity are removed from the instrument as negatives.

Fluorescence microscope
• A fluorescence microscope is an optical microscope used to study properties of organic or inorganic substances using the phenomena of fluorescence instead of, or in addition to, reflection and absorption.[ • In fluorescence microscopy, the sample you want to study is itself the light source. The technique is used to study specimens, which can be made to fluoresce. • The fluorescence microscope is based on the phenomenon that certain material emits energy detectable as visible light when irradiated with the light of a specific wavelength.

Fluorescence microscope
• The sample can either be treated with fluorescing chemicals. • The Sample Gets Excited The basic task of the fluorescence microscope is to let excitation light radiate the specimen and then sort out the much weaker emitted light to make up the image. First, the microscope has a filter that only lets through radiation with the desired wavelength that matches your fluorescing material. The radiation collides with the atoms in your specimen and electrons are excited to a higher energy level. When they relax to a lower level, they emit light.

Fluorescence microscope
• To become visible, the emitted light is separated from the much brighter excitation light in a second filter. Here, the fact that the emitted light is of lower energy and has a longer wavelength is used. The fluorescing areas can be observed in the microscope and shine out against a dark background with high contrast. • Fluorescence microscopy is a rapid expanding technique, both in the medical and biological sciences. The technique has made it possible to identify cells and cellular components with a high degree of specificity.

Principle of Fluorescence 1. Energy is absorbed by the atom which becomes excited. 2. The electron jumps to a higher energy level. 3. Soon, the electron drops back to the ground state, emitting a photon (or a packet of light) the atom is fluorescing.

Biosafety cabinet
• A biosafety cabinet (BSC), biological safety cabinet, or microbiological safety cabinet is an enclosed, ventilated workspace for safely working with materials contaminated with (or potentially contaminated with) pathogens in the laboratory. Several different types exist, differentiated by the specifics of construction. • The primary purpose of a BSC is to serve as the primary means to protect the laboratory worker and the surrounding environment from pathogens. All exhaust air is HEPA-filtered as it exits the biosafety cabinet, removing harmful bacteria and viruses. This is in contrast to a laminar flow clean bench, which blows unfiltered exhaust air towards the user and is not safe for work with pathogenic agents.[

Biosafety cabinet
• The U.S. Centers for Disease Control and Prevention (CDC) classifies BSCs into three classes. These classes and the types of BSCs within them are distinguished in two ways: the level of personnel and environmental protection provided and the level of product protection provided. • Class I cabinets provide personnel and environmental protection but no product protection • Class II cabinets provide both kinds of protection (of the samples and of the environment) since makeup air is also HEPA-filtered.

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