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sampling exam chapter 3

sampling exam chapter 3

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Published by khaled
sampling exam chapter 3
sampling exam chapter 3

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Published by: khaled on Jun 20, 2008
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Staining method & culture media
Staining method and culture media
Staining
Staining is of primary importance for the recognition of bacteria since their clear protoplasm is so feeblyrefractile that it is difficult to see them in the unstained condition unless dark-ground illumination isemployed or, alternatively, use of phase-contrast techniques.
Smear preparation
Smears are making usually from lesions, exudates, blood, tissues and colony suspension of theisolated bacteria.
Smear to be stained must be firmly fixed to a glass slide before the various dye solutions areapplied.
Slides must be cleaned by soaking in 95% ethyl alcohol, then wiping dry with clean gauze,otherwise the material (smear material) may wash off.
Make a direct thin smear or film of clinical material.
Bacteria from solid media are transferred to a drop of distilled water or saline on a slide.
A loopful of broth culture is placed directly on the slide. Slides must be cleaned by soaking in95% ethyl alcohol, then wiping dry with clean gauze, otherwise the material (smear material) maywash off.
Make a direct thin smear or film of clinical material.
Bacteria from solid media are transferred to a drop of distilled water or saline on a slide
.
A loopful of broth culture is placed directly on the slide.
Allow the film to dry in the air. Drying is accelerated in the incubator.
Fix the film by quickly passing the slide through the Bunsen flame several times. (Proper fixingalso prevents washing off).
Types of stains
1.Simple stains.2.Differential stains.3.Special stains.4.General purpose stains.
1. Simple stains.LOEFFLER'S METHYLENE BLUE STAIN
It is the most valuable stain for staining bacteria.
It is excellent for the genus Corynebacterium where beading, barring, and granules may bedemonstrated.
In sporing bacilli stained with this stain the spore appear as unstained bodies within blue cells
.Staining Procedure
1- Stain for 1 min.
2- Rinse with water.
3- Drain or blot to dry.
By dr.khaled fujairah municipality1
 
Staining method & culture media
Micrococcus luteus methylene blue stain (x 1000(
2. DIFFERENTIAL STAINSA. GRAM’S STAIN
This is the most commonly employed and important of all the diagnostic staining techniques.
By this method bacteria can be recognised as Gram-positive (blue-black) if they retain theprimary dye complex of methyl violet and iodine in the face of attempted decolourization or asGram-negative (red) if decolourisation occurs as shown by the cell accepting the counter stain
.
Gram +ve cocci
Gram +ve cocci
By dr.khaled fujairah municipality2
 
Staining method & culture media
Gram
 –
ve bacilliGram +ve bacilli
GRAM’S STAINING METHOD (HUCKER’S MODIFICATIN
)
Procedure
Gram’s stain is best performed on young cultures because older cultures often decolourize tooreadily.
The slide is flooded with crystal violet stain, for ½ - 1 min.
Pour off the stain, wash in water.
Apply Lugol’s iodine solution, for ½ - 1 min.
Tip off iodine but do not wash.
Decolourize with a few drops of acetone, for not more than 4 seconds.
Wash thoroughly in water.
Counter stain with Safranin for ½ min.
Wash and stand on end to drain, or blot to dry.
B. ZEIHL-NEELSEN’S METHOD (Acid-Fast Stain
)
It is used for examination of Mycobacterium.
The principle of staining is depending on the resistance of this type of bacteria to decolarizationby acid alcohol, because the cell wall contains waxy material (mycolic acid) which prevents theremoval of carbol fuchsin from the cell.
Staining Procedure
Flood the slide with strong carbol fuchsin and heat until steam rises (but do not boil).
After 3-4 min apply more heat until rises again; do not let the stain dry on the slide.
About 5-7 min after the first application of heat washes the slide thoroughly under running water.
Decolourize in acid-alcohol until all traces of red have disappeared from the film. Decolourizationshould not be attempted in one stage; there should be intermittent washings in water and re-application of acid-alcohol.
Wash well with water and counter stain with Methylene blue for 1 min.
Wash and stand end to drain.
Acid-fast organisms arered, other organisms areblue
By dr.khaled fujairah municipality3

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