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Index >> Microscopy Methods in Microbiology >>Factors Affecting Staining

Microscopy Methods in Microbiology
Microroscopy Methods in Microbiology Part 2
Microroscopy Methods in Microbiology - Introduction
Observation of Microbes in the Living State
Observation of Microbes in Smears
Dyes and Staining
Useful pH Range and Colour Change of Some Indicator Dyes
Factors Affecting Staining
Microscopy and Micrometry
Microscopy and Micrometry - Introduction
Behaviour of Light
Light Theories
Resolving Power
Refraction and Reflection
Bright Field Microscopy
Use of Bright Field Microscope

Factors Affecting Staining

	Factors Affecting Staining: The result of staining procedure depends upon many factors like, the careful preparation and application of staining reagents, preparation and fixation of smear, chemical nature and the physical organization of the structure to be stained, application of mordant, specification of glassware and microscope etc. They are discussed in the relevant sections with detail. Modern microscopes are so designed that they require carefully specified thickness of slide and cover glass. For the best results we recommend slide having size of 75 X 25 mm and 1.0-1.2 mm thickness. Cover glass should be 18 mm square with 0.13-0.16 thickness. Not sticking to these specifications result into bad quality of image production.
This is very much true for the microbiological work since, it involves the use of high power objectives. Slides and cover glasses to be used for the bacterial preparations should be grease free. To achieve this, hold the new slide with forcep, make it free from grease by passing six to ten times through a blue Bunsen flame. Heating should be strong enough without resulting in the cracking of the slide. Cracking can be avoided by allowing the slide to cool before laying down. Further handling of these slides should be carried out though the edges only. They should be sufficiently protected from the laboratory contaminations. The cleaning and recirculating of the used slides is not a common practice in the developed countries.
It has been demonstrated that routine staining procedure do not kill some bacteria like spore former bacilli and acid-fast bacilli, such slides must be decontaminated by autoclaving and destroyed immediately since, members of these group are found to be pathogenic to man and animals. Though, the recycling of used slides is not appreciated, it can be done satisfactorily by giving the slides following treatment. Place the slides in 40% formaldehyde solution for 12 hours to sterilize them or wash the slides and clean them with cleaning solution or cleaning powder or 50% hot chromic acid.
Wash the slides thoroughly with water, rinse with distilled water and dry in oven. Cover glasses are also washed in the similar fashion and they are stored in the stoppered jar containing 50% ethyl alcohol. Before use they are dried with the soft cloth. For routine work cleaned cover slips are available. Recently some manufacturers have introduced plastic cover slips. They should not be used for a stained preparation, but they are found to be quite satisfactory for the wet mount preparations.
	The usefulness of slide or cover glass for the staining purpose can be checked by putting a drop of water over it. On a cleaned slide or cover glass a drop of water will spread evenly to form a thin film, while, on an uncleaned slide, water will collect into small drops and fiIm is not formed. Thus , microscopic observation of microbes either in living or in stained state need careful preparation of the specimen and then the observation under microscope. For both the steps special skill is required which can be mastered only by the sufficient practise. Thus, to get a successful result is an art rather than a science

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