- •Saparbekova a.A., Aimenova Zh.E.
- •Composers: Saparbekova a.A., Aimenova Zh.E.
- •Content
- •Introduction
- •Safety measures in microbiological laboratory
- •Laboratory work №1 Methods of microscopic examination of microorganisms. Microscope components.
- •Electron microscopy
- •Scanning probe microscopy
- •Types of microscopes
- •Optical microscope
- •Lighting techniques
- •Optical configurations of microscope
- •Components of microscope
- •Laboratory work № 2 Nutrient media. Preparation of ware and media for sterilisation
- •Maintenance of Aseptic Environment
- •Laboratory work №3 Microorganisms morphology and methods of its study
- •Laboratory work № 4 Structure of bacteria and yeasts
- •In the history…
- •Blood agar plates (bap)
- •Chocolate agar (choc)
- •Sabouraud agar
- •Hay infusion agar
- •Potato dextrose agar
- •Inoculation of Culture Media
- •Importance of Using “Sterile Technique”
- •Inoculating the Agar Slant
- •Inoculating the nb
- •Inoculating the na butt
- •Laboratory work № 5 Cultivation of microorganisms
- •Laboratory work № 6
- •Isolation of accumulative pure cultures of bacteria
- •Common Methods of isolation of pure culture
- •Streak Plate Method
- •Various methods of streaking
- •Laboratory work № 7 Control for cultivation. Antimicrobial factors.
- •Laboratory work № 8 Microflora of microbial synthesis products
- •List of recommended literature
- •Saparbekova a.A., Aimenova Zh.E. Microbiology and virology
Lighting techniques
While basic microscope technology and optics have been available for over 400 years it is much more recently that techniques in sample illumination were developed to generate the high quality images seen today.
In August 1893 August Köhler developed Köhler illumination. This method of sample illumination gives rise to extremely even lighting and overcomes many limitations of older techniques of sample illumination. Before development of Köhler illumination the image of the light source, for example a lightbulb filament, was always visible in the image of the sample.
Köhler illumination is a method of specimen illumination used for transmitted and reflected light (trans- and epi-illuminated) optical microscopy. Köhler illumination acts to generate an extremely even illumination of the sample and ensures that an image of the illumination source (for example a halogen lamp filament) is not visible in the resulting image. Köhler illumination is the predominant technique for sample illumination in modern scientific light microscopy although it requires additional optics which less expensive and simpler light microscopes may not have.
The primary limitation of critical illumination is the formation of an image of the light source in the specimen image plane. Köhler illumination addresses this by ensuring the image of the light source is perfectly defocused in the sample plane and its conjugate image planes. In a ray diagram of the illumination light path this can be seen as the image-forming rays passing parallel through the sample.
Köhler illumination requires several optical components to function:
Collector lens and/or field lens
Field diaphragm
Condenser diaphragm
Condenser lens
These components lie in this order between the light source and the specimen and control the illumination of the specimen. The collector/field lenses act to collect light from the light source and focus it at the plane of the condenser diaphragm. The condenser lens acts to project this light, without focusing it, through the sample. This illumination scheme creates two sets of conjugate image planes, one with the light source image and one with the specimen. These two sets of image planes are found at the following points:
Light source image planes:
Lamp filament
Condenser diaphragm
Back focal plane of the objective
The eyepoint
Specimen image planes:
Field diaphragm
Specimen
Intermediate image plane (the eyepiece diaphragm)
The eye retina or camera sensor
The primary advantage of Köhler illumination is the extremely even illumination of the sample. This reduces image artifacts and provides high sample contrast. Even illumination of the sample is also critical for advanced illumination techniques such as phase contrast and differential interference contrast microscopy.
By adjustment of the field diaphragm the amount of light entering the sample can be freely adjusted without altering the wavelengths of light present, in contrast to reducing power to the light source with critical illumination. Adjusting the condenser diaphragm alters sample contrast. Furthermore altering the size of the condenser diaphragm allows adjustment of sample depth of field by altering the effective numerical aperture of the microscope. The role of the condenser diaphragm is analogous to the aperture in photography although the condenser diaphragm of a microscope functions by controlling illumination of the specimen, while the aperture of a camera functions by controlling illumination of the detector.
The optical microscope, often referred to as the "light microscope", is a type of microscope which uses visible light and a system of lenses to magnify images of small samples. Optical microscopes are the oldest design of microscope and were possibly designed in their present compound form in the 17th century. Basic optical microscopes can be very simple, although there are many complex designs which aim to improve resolution and sample contrast. Historically optical microscopes were easy to develop and are popular because they use visible light so that samples may be directly observed by eye.
The image from an optical microscope can be captured by normal light-sensitive cameras to generate a micrograph. Originally images were captured by photographic film but modern developments in CMOS and charge-coupled device (CCD) cameras allow the capture of digital images. Purely digital microscopes are now available which use a CCD camera to examine a sample, showing the resulting image directly on a computer screen without the need for eyepieces.
Alternatives to optical microscopy which do not use visible light include scanning electron microscopy and transmission electron microscopy.