Instrumentation

• Light Path


  1. Light passes from the light source to condenser and specimen

  2. Through the specimen to the objective lens into the prism housed in the microscope tube

  3. The prism focuses the light through the oculars and into view

  
• Sterilization

  Process of destroying microorganisms using heat, chemicals, or radiation
• Sterilization by steam under pressure

  1. Autoclaving

  2. Temperatures > 100oC reached

  3. Condensation on cooler, wettable objects results in a rapid transfer of latent heat

  4. Achieved at lower temperatures than with dry heat

  5. Simplest autoclaves are versions of the pressure cooker

  6. Steam generated by applying heat to a small volume of water within a sealed apparatus

  7. All air must be displaced before the correct conditions of temperature and pressure are obtained - 121oC and 15 psi

  8. Air pockets trapped between the articles will act as hot air at low temperatures and fail to sterilize
• Demineralizers/ Deionizers/Ion Exchangers

  • Uses ion exchange resin and removes all the dissolved ions present in the raw water
  • Water is of high purity (more than distilled), with low electrical conductivity (less than 10 microS/cm at 25 oC)
• Uses of demineralized water

  • Laboratories and Research Institutes
  • Food and Diary Industries
  • Pharmaceutical Industries
  • Electronic and Storage Battery Industries
• Spectrophotometer

  Isolates wavelengths, by prisms, slits or monochromators and the entire spectrum is available
• Components of the Single Beam Spectrophotometer

  • Cuvettes (Absorption Cells)
• Care of Cuvettes

  1. Should be free of scratches

  2. Dichromate solutions should not be used
• Atomic Emission Spectroscopy

  • Metals excited in the flame of a Bunsen burner
  • Light intensity/wavelength is directly proportional to the number of atoms emitting energy and concentration of substance being tested
  • Increasing the thermal energy of the flame will increase the number of excited atoms and the emissions will be more intense
• Atomic Emission Spectroscopy - Flame Colours

  • Lithium - red
  • Sodium - yellow
  • Potassium - violet
  • Rubidium - red
  • Magnesium - blue
  • Copper - bright green to blue (depends on impurities)
• Atomic Emission Spectroscopy - Burners

  • Total consumption burner - the gasses such as Hydrogen and air are mixed with the sample in the flame
  • Premix burner - the gasses are mixed with the sample and atomized before being burnt
• Atomic Absorption (AA) Photometer - Non-Spectral Interference

  • Matrix interference - Sample is thicker than the standard resulting in differences in the rate of sample uptake and nebulization
  • Chemical interference - Phosphate interference when testing for calcium
  • Ionization interference - Dissociation does not stop but continues to excitation of electrons resulting in depletion of ground state atoms
• Fluorometry

  • Photoluminescence occurs when light energy stimulates the emission of a photon
  • Fluorescence is caused by photons exciting a molecule, raising it to an excited state
  • Process of absorbing light at one wavelength and emitting light at another for a period of 10-8 seconds
  • Electrons move to a higher wavelength due to the absorption of energy but emitted fluorescent energy is of a low energy and longer wavelength as some is loss as vibrational energy
• Single beam fluorometer

  • Uses a Hg vapour lamp with a filter but has a limited excitation spectrum
  • Sample cell has two transparent sides perpendicular to each other, rather than parallel
• Double beam fluorometer

  • Uses a Xenon arc lamp
  • Choppers (switches) alternates the beam between the reference and the sample
• Fluorometry Applications

  • Measurement of therapeutic drugs
  • Quantification of bilirubin and immunoglobulins
• Principles of Operation

  • Electrical Impedance - Conductivity difference between cells in a fluid and the fluid itself
  • Optical/ Light Scatter - Light hitting a particle and scattering it
  • Nephlometry/ turbidimetry
  • Osmometry - Measurement of the osmotic strength of a solution or compound
• Electrical Impedance Method

  1. Blood is diluted with an electrolyte (conducting) solution, the cells do not conduct

  2. It is aspirated and separated into two volumes - one mixed with diluents and delivered to the red cell bath, other to the white cell bath, where the white cells are mixed with a cytochemical – lytic agent

  3. Current flows between an external electrode, placed in the red cell solution and the internal electrode

  4. Movement of the suspension between the electrodes occurs

  5. The cells produce a resistance in the electrical path, which results in 'pulses' occurring

  6. The 'size' of each pulse is proportional to the size of the particle which produced it

  7. Abnormal cells are 'flagged'
• Light Scatter

  • Occurs when light passing through a solution encounters a molecule and collides with it, with the light being scattered in all directions
  • Angel and direction of light scatter is dependent on the size of the particle and the wavelength
  • Types of light scatter: Rayleigh, Mie, Rayleigh – Debye
• Flow Cytometry

  • Principle of hydrodynamic focusing, where individual cells are labelled with fluorescent markers and passed in single file through a fluidic stream
  • Cells are hit by a laser beam producing scattered visible light and fluorescence
  • Physical and chemical properties of cells/ particles are analysed
• The Flow Cytometer

  1. Sample is injected into the center of the sheath fluid (water or saline)

  2. The combined flow is reduced in diameter, forcing the cell into the center of the stream

  3. Allows the laser to hit one cell at a time and the scattered light is detected by filters, mirrors and lens

  4. Components: Light source, flow cell, optical components, electronics, computer

  
• Flow Cytometry: Cell Sorting – Liquid Suspension

  1. Cells pass through a chamber with a small hole forming a fluid jet

  2. A voltage is applied producing charged and uncharged particles (computer told what to look for)

  3. Chamber is vibrated, forces the jet stream into droplets

  4. The droplets then pass through an electrical field, which separates them into different collector tubes of further analysis
• Fluorescent dyes used in Flow Cytometry

  • Each has its own excitation and emission spectra
  • Dye used is dependent on the parameter being examined i.e., DNA/ RNA - Acridine Orange, Antibody: Antigen reactions - Rhodamine
• Applications of Flow Cytometry

  • Leukaemias and lymphomas
  • Immune disorders
  • Breast cancer
• Turbidimetry

  • Turbidity is the cloudiness or haziness due to suspended particles in a solution
  • Turbidity causes a decrease in the incident light as it passes through a particulate solution
  • May be caused by light scattering, reflectance or absorption
• Turbidimetry vs Nephelometry

  • Turbidimeter – measures the number of particles in solution by their ability to scatter light (cloudiness)
  • Turbidimetry – Detection of light scattered towards a detector in the direct path of the beam
  • Nephelometer – measures suspended particles in a liquid or gas colloid
  • Nephelometry –Detection of light scattered or reflected towards a detector that is not in the direct path of the beam, usually 90o
• Components of the turdibimeter and nephelometer

  • Consists of a light source, filters, lens, cuvet and detector
  • Light source - mercury arc, tungsten/ iodide lamps and helium – neon lasers
  • Filters are positioned near the detector, whose placement is dependent on the type of light measured
• Clinical Applications of Turbidimetry and Nephelometry

  • Measurement biological fluids, plasma proteins, drug monitoring and antibiotic sensitivities
• Electrochemistry

  • Requires the use of two electrodes or half-cells connected to a measuring device
  • The circuit is completed by a liquid junction or "salt bridge" (allows the passage of electrons from the electrodes)
  • To measure, one electrode must have a known potential (reference electrode), the other electrode has a varying potential (indicator electrode)
  • Electrochemical methods include Potentiometry, Amperometry, Coulometry
• Potentiometry

  • A potentiometer is an instrument, which measures the potential (voltage) in a circuit
  • An electrochemical cell can be created by placing metallic electrodes into an electrolyte where a chemical reaction either uses or generates an electric current
• Potentiometry - Electrochemical Reactions

  1. Metals have different electronegativities therefore in solutions of their salts zinc readily loses electrons to copper

  2. Zinc atoms losses electrons become positive ions and go into aqueous solution, decreasing zinc electrode mass

  3. Copper receives the two electrons which are converted into uncharged copper atoms, the deposit on the copper electrode, increasing its mass
• Types of Electrodes (pH meters)

  • An electrode or half-cell consists of
  • - Calomel Electrode, Silver: Silver Chloride Electrode
• Calomel Electrode

  1. A wire in contact with elemental Hg, covered with a thin film of Mercurous Chloride (Calomel)

  2. Immersed in a saturated Potassium Chloride (KCL) solution, which keeps the temperature and the Chloride activity constant
• Silver: Silver Chloride Electrode (AgCl: AgCl)

  1. More popular than the Calomel electrode, as it is not as sensitive to the effects of high temperatures

  2. Has an Ag or Platinum wire coated with Ag

  3. Wire is immersed in a solution of known Chloride ions(electrolyte).
• 2+(aq) + 2e- -> Cu(s)

  1. Zinc is oxidized and loses electrons; this occurs at the anode

  2. Copper is reduced as it gains electrons; this occurs at the cathode
• Electrode

  A single metallic conductor in contact with an electrolyte solution
• Types of Electrodes (pH meters)

  • Reference Electrodes
  • Ion – Selective Membrane Electrodes / Indicator Electrodes
• Calomel Electrode

  A wire in contact with elemental Hg, covered with a thin film of Mercurous Chloride (Calomel), immersed in a saturated Potassium Chloride (KCL) solution
• Calomel Electrode half cell reaction

  Hg2 Cl2 + 2 e- -> 2Hg + 2Cl-
• Silver: Silver Chloride Electrode (AgCl: AgCl)

  More popular than the Calomel electrode, as it is not as sensitive to the effects of high temperatures, has an Ag or Platinum wire coated with Ag, wire is immersed in a solution of known Chloride ions (electrolyte)