Bacteriology - High-Magnification Microscopy with Immersion Oil

Several points should be considered when a specimen is being examined under a microscope. Bacteriological investigations focus primarily on the structural and cellular details of the specimen.

When the specimen is being examined for specific details, the maximum magnification that can be used with a dry objective is 40x. For greater magnification than this (63x or 100x) an immersion objective is needed.

These immersion objectives are designed so that the gap between the object and the objective is filled with a liquid which largely eliminates diffraction and enables a sharp image to be obtained, so improving the performance of the objective. The immersion fluid must satisfy various criteria. Its refractive index should be 1.515 (the same as that of glass) to render it invisible under the microscope. It must also have a viscous consistency to stop it migrating over the objective and dripping from it. Viscosity and density are important parameters to be considered when using the immersion fluid,so immersion fluids are usually in the form of an oil.

Bacteriological specimens are normally not mounted for routine applications; i.e. they are examined dry or directly under immersion oil. The immersion oil must be inert and not attack or change the stain. Immersion oils should also have little, if any, intrinsic fluorescence so as to avoid causing interference during fluorescence investigations.

Merck supplies a range of immersion oils:

• Classical - Oil of cedar wood for microscopy
  Natural raw materials are turned into oil of cedar wood meeting the requirements and specifications
  Cat. No. 106965 Oil of cedar wood for microscopy
  Refractive index (20 °C) 1.510 - 1.52
  Density (d20°C/4°C) 0.980 - 0.990 g/cm3
  Viscosity (20°C) 2000 - 3500 mPas
  Fluorescence 1L = 0.98 kg
  


• Synthetic - Immersion oil for microscopy
  Defined synthetic raw materials are used to produce this immersion oil
  Cat. No. 104699 Immersion oil for microscopy
  Refractive index (20 °C) 1.5150 - 1.5170
  Density (20°C/4°C) 1.0245 - 1.0265 g/cm3
  Viscosity (20°C) 100 - 120 mPas
  Fluorescence 1L = 1.02 kg
  



• Conforming to ISO standards - Immersion oil modified acc. to ISO standards for microscopy
  Synthetic raw materials with defined optically relevant properties form the basis of the immersion oil
  Cat. No. 115577 Immersion oil acc. to ISO 8036 modified for microscopy
  Refractive index (23°C) 1.5175 - 1.5185
  Density (20°C/4°C) 1.300 - 1.315 g/cm3
  Viscosity (23°C) 150 - 1500 mm2/s

Application
Under the microscope, first locate the part of the dry specimen to be examined. Swing the lens holder away, place a drop of immersion oil on the specimen at the point to be observed and return the lens to its original position. When finished, clean the lens and the specimen with ethanol.