* Lab 2
** Types of staining techniques
Simple staining (use of a single stain)Differential staining
For demonstrate morphological shape & arrangement.
Identification
Special stain
Gram stain
Acid fast stain
Spore stain
Capsule stain
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* GRAM STAINING
Gram's Stain is a widely used method of staining bacteria as an aid to their identification. It was originally devised by Hans Christian Gram, a Danish doctor.* Gram Stain:
It is the most important differential stain used in bacteriology because it classified bacteria into two major groups:Gram positive: Appears purple after Gram’s stain b) Gram negative: Appears red after Gram’s stain
* Gram’s +ve Bacteria Gram’s -ve Bacteria
* Gram’s +ve Bacteria Gram’s -ve Bacteria* Gram-positive bacteria Have a thick peptidoglycan layer surrounds the cell. The stain gets trapped into this layer and the bacteria turned purple. Retain the color of the primary stain (crystal violet) after decolorization with alcohol Gram-negative bacteria have a thin peptidoglycan layer that does not retain crystal violet stain. Instead, it has a thick lipid layer which dissolved easily upon decoulorization with Alcohol. Therefore, cells will be counterstained with safranin and turned red.
* Crystal violet↓Iodine↓Alcohol↓Safranin
* Solutions usedGram positive
Gram negative
1-Crystal violet
Cells violet
Cells violet
2-Iodine Increase the affinity of attraction between the cell and stain
C.V.I complex ,cells become purple color
C.V.I complex ,cells become purple color
3-Alcohol- decolorizing agent to remove the stain from the cells
Cell wall dehydrate-pores shrinks- permeability of cell wall and membrane decreases –C.V.I. complex cannot pas out of cell wall, cells remain purple Lipid extracred from the cell wall – pores enlarged -C.V.I. complex removed from cells and cells become colorless 4-Safranin To give different color of the intial stain
Cells remain purple
Cells red
* Gram +ve S.aureus
Gram –ve E.coli Step 1: Crystal VioletStep 2: Gram’s Iodine Step 3: Decolorization (Alcohol)
Step 4: Safranin Red
* Gram Stain procedure
Cultures of bacteriaGram stain solutions: Crystal violet (primary stain) Gram’s iodine (mordant) Alcohol (decolorizing agent) Safranin (Secondary stain)or (counter stain)* The Gram staining method
1. A small sample of a bacterial culture is removed from a culture. In this example it is being taken from a broth culture of the pure microbe but it could be removed from a culture on solid medium .* The Gram staining method
2. The bacterial suspension is smeared onto a clean glass slide. If the bacteria have been removed from a culture on solid media it will have to be mixed with a drop of distilled water.* The Gram staining method
3. The bacterial dried slowly at first and then, when dry, heated for a few seconds for fixation* The Gram staining method
4. Cover the surface of the slide with Crystal Violet stain and let sit for ( one minute ).* The Gram staining method
5. Washing the slide with water.
* The Gram staining method
6..Cover the slide with Gram’s Iodine and time for ( one minute).Then Washing with water!* The Gram staining method
7. Cover the slide with Gram’s decolorizer and time for 10-20 seconds.And wash with water* The Gram staining method
8. Cover the slide with the counterstain, Safranin, and let sit for 30-60 seconds* The Gram staining method
9. washing with water* The Gram staining method
10.drying the slide and read with the oil immersion lens of the microscope. Look for Gram-negative and Gram-positive bacteria.* The Gram staining method
Typical Gram-positive bacteria* The Gram staining method
Typical Gram-negative bacteria* Gram Positive may be appear as Gram Negative: Using old culture. 2.Wrong in Technique Steps. a. If not treated with iodine. b. If alcohol stay on slide more time. c. Acidic PH of medium.
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