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Author Topic: VISUALIZATION OF MICROORGANISMS- Staining and Identification Techniques  (Read 6061 times)
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Francis Umeoguaju
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« on: April 10, 2010, 08:22:36 am »


In addition to difference in sizes, shapes, texture and  colour or colony, the organism can specifically be identified by viewing it in the microscope. 
   
Ordinary, the organism cannot be easily visualized with the microscope since it has no particular colour differences from the surrounding.  Usually the organism or the background is made to absorb a particular dye so that it would be easily differentiated from each other.
   
There are several method of staining used with  organism. The methods may be classified as simple and differential. Simple stains will react with all microbe in identical fashion. Differential stains gives varying result depending on the organism  being treated.
   
Pretreatment of slide before staining 

Before a microbe is stained on the slide, the organism is made to stay on the slide so as to avoid being washed off on the course of the staining. This is achieved by heat-fixing. To prepare an heat-fixed smear of the bacteria on a microscopic slide,
   
A small drop of the suspension of the bacteria is spread thinly on a slide with the aid of another slide.
   
The smear is then air dried (waving it about  hastens  drying)
   
The air dried smear is “fixed” by gentle heating over a low flame. After heat fixing, the specific stain is then applied.


Methylene Blue Staining

Methylene Blue is usually the salt; methylene Blue chloride, which will dissociate in water into a positively  charged methylene blue ion which is blue in colour and a negatively charged chloride ion which is colorless.
   
The Basic stain is attracted and binds to the slight negatively charged  bacterial cell wall.
   
   
Procedure for methylene blue staining


The heat fixed slide is flooded with methylene blue  solution. The stain is left in contact  with the smear for 30 sec to 1 minute. The stained slide is then rinsed thoroughly with water and left in a slant position to dry. The slide is then examined under oil immersion.
   


Gram staining technique for detecting microorganism

The dye exploit the basic differences with the outer layer of Bacteria so that certain bacteria groups retain the initial stain of crystal violet solution while other readily lose this dye during discoloration process. Those that retains it appears dark-blue or violet and are termed Gram-positive bacteria.
   
   
Procedure for gram staining
   
   -Heat fix the organism on the slide.
   -Flood the slide with a 1% aqueous solution of methyl or crystal violet and allow to stand for 1 min.
   -Pour off the crystal violet stain, wash with water and flood the slide with gram’s iodine solution. Allow a contact time of I minute.
   -Gently rinse the slide with water from a squirt bottle.
   -Blot the slide dry to remove excess water but do not allow smear to become dry.
   -Decolorize with acetone-ether (3.1) (i.e. 90ml acetone + 30ml  ethanol) or alcohol until the decolorizer flow down the slide almost uncolored (usually 10 sec).
   -Wash with water, blot or air dry.
   -Counter  stain  using 2% solution of  safranin or basic fuschin .
   -Wash  briefly with water, blot and dry quickly (too  prolong washing removes the safranin).
   
   

Microscopic examination of stained microscope slides
   
The  microscope used to examine the stained slides was interface with the computer. Ordinary microscope can be used to view the image but interfacing it with a computer system enables one to store the microscopic images for future references.
   
Interfacing a microscope to a computer required an additional hardware attached to normal compound or simple microscope. Such accessories can be purchased from the market.
   
The microscope slide is clipped on the  mechanical stage of a compound microscope and is moved so that the area of the slide to be examined is directly over the lens of the light condenser.
   
Sometimes, (that  is for  oil immersion), a drop  of  oil is placed on the area of the slide of interest. The lens of choice is then swung into position. Once in position, the coarse adjustment knob is used to bring the mechanical stage close to the lens, while watching  the image on the computer screen.  When the image is seen, it is then adjusted finely with the fine adjustment knob until the image becomes focused on the screen. The mechanical stage is then used to move the slide around to obtain a complete view of  the slide.
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