Lab 2

STAINING

Types of staining techniques Special stain Simple staining (use of a single stain) Differential staining Spore stain Capsule stain Identification For demonstrate morphological shape & arrangement. Gram stain Acid fast stain

Gram Staining

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.

Appears violet after Gram’s stain Gram Stain: It is the most important differential stain used in bacteriology because it classified bacteria into two major groups: b) Gram negative: Appears red after Gram’s stain Gram positive: Appears violet 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 Aceton-Alcohol. Therefore, cells will be counterstained with safranin and turned red.

Crystal violet ↓ Iodine Alcohol Safranin

Solutions used Gram positive Gram negative 1-Crystal violet Cells violet 2-Iodine Increase the affinity of attraction between the cell and stain C.V.I complex ,cells remain violet 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 violet 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 violet Cells red

Gram –ve E.coli Gram +ve S.aureus Step 1: Crystal Violet Step 2: Gram’s Iodine Step 3: Decolorization (Alcohol) Step 4: Safranin Red

Gram Stain procedure Cultures of bacteria Gram 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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