GRAM STAIN:
Principle of gram staining technique:
Most bacteria can be differentiated by Gram's reaction due to
differences in their cell wall composition. Gram-positive bacteria have a large
amount of peptidoglycan in their cell wall, while Gram-negative bacteria have a
thin cell wall with less peptidoglycan.
Organisms that retain a purple color with crystal violet and iodine
complexes that are not stained by acetone or ethanol are called Gram-positive
bacteria. Organisms are termed gram-negative that lose their color when treated
with acetone or ethanol after being stained with crystal violet and
counterstained with neutral red or safranin. The iodine solution used in the
technique acts as a mordant.
Required:
1. Crystal violet stain
2. Lugol's iodine
3. Acetone-alcohol decolorizer
4. Neutral red
Method:
1. Correct the dry smear.
2. Cover the fixed smear crystal violet stain for 30-60 seconds.
3. Rinse the stain quickly with clean water.
4. Discard all water, and cover the smear with Lugol's iodine for 30-60 seconds.
5. wash out the iodine with clean water.
6. Decolorize rapidly (within seconds) with acetone alcohol. Rinse immediately with clean water.
7. cowl the smear with neutral red stain for two minutes
8. Rinse the stain with clean water.
9. Clean the back of the slide, and place the smear in a draining rack to air dry.
10. First examine the stain with a 40x objective and examine the stain
Dispersing the material, and then reporting the bacteria and cells with the oil immersion objective.
Results:
Gram-positive bacteria................... dark purple
Yeast cells .................................dark purple
Gram negative bacteria........pink to dark red
Center of pus cells. ...................Red
Epithelial cells ................................Pale red
Reporting:
Gram smear from clinical specimen
The report should include
the following:
Information:
*Number of bacteria present,
whether high, moderate, low, or low.
* Gram reaction of bacteria, whether gram positive or gram negative.
*Morphology of bacteria,
whether cocci, diplococci, rods, or coccobacilli.
* Whether organisms are
intracellular.
* Presence and number of pus cells per high power field.
* Presence of yeast cells and animal tissue cells
ZIEHL-NEELSEN- STAIN
Principle:
Principles:
The Ziehl-Nielsen (Zn) technique is used to stain Mycobacterium
species including M. tuberculosis, M. ulcerans, and M. leprae. Mycobacteria,
unlike some other bacteria, do not stain well with the Gram technique. However,
they can be stained with carbol fuchsia phenol. The stain binds to mycolic
acid, wax D, and long-chain fatty acids in the mycobacterial cell wall.
After staining, an acid staining solution is applied. It removes
the background red color in smears from tissue, cells, and normal flora of the
mouth except for mycobacteria that retain the color (fasten) and are therefore
called acid-fast bacilli, or simply AFB. . After staining, the smear is stained
with malachite green or methylene blue which stains the background material,
creating a color contrast against which the red AFB can be seen.
Sample:
1. Wholesale.
2. Cerebrospinal fluid.
3. Peritoneal fluid.
4. Pleural fluid.
5. Pus.
Required:
● Carbol Fuchsin stain....a primary dye.
● Acid alcohol (3% HCL in
isopropyl alcohol).
● Malachite green or methylene blue sample:
Method:
1. Heat fixation or alcohol fixation: This is recommended when a smear has not been prepared from sodium hypochlorite (bleach)-treated saliva and is not immediately stained. M. tuberculosis is killed by bleach during the staining process.. Heat-fixing untreated saliva will not kill M. tuberculosis while alcohol fixation is antiseptic.
2 )Cover smear carbol fouchon stain
3)Heat the stain until evaporation begins (ie about 60 °C). Do not overheat. Allow the hot stain to remain on the slide for 5 minutes. Heating the stain: Great care must be taken when heating carbol fuchsin, especially if the stain has been applied to a rack. Only a small flame should be placed under the slides.
4 Wash the stain with clean water.
5 Cover the smear with 3% acid alcohol (decolorizer) for 5 minutes
or until the smear is substantially decolorized, i.e., pale pink. Caution:
Acidic alcohol is flammable, so use it carefully away from open flames.
6 Rinse thoroughly with clean water.
7 Cover the smear malachite green stain for 1-2 minutes.
8 Wash the stain with clean water.
9. Clean the back of the slide, and place it in a draining rack to
dry (do not dry).
10. Using a 100x oil immersion objective, examine the smear microscopically. Scan the smear systematically
Results:
• AFB
Red, straight or
slightly curved rods, occur.
Can appear alone
or in small groups, with pearls.
Sales ............. Blue
• Background
material.........blue
Reporting of salivary smears:
A sputum sample must contain more than 10,000 bacilli/ml of sputum to be detected by a Zn smear.
More than 10 AFB per HPF
------------- +++
1-10 AFB per
HPF---------------++
10-100 AFB per 100
HPF--------- +
1-9 AFB per 100 HPF--------- Report correct number
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