Simple and Gram's staining

First Year of Bachelor of science

1. Monochrome Staining (SIMPLE STAINING) & Diffrential Staining (GRAM’S STAINING) By : Jaynesh K Amb . Assistant Prof. Microbiology Shree Kidivav Science College Simar . Gir - Somnath

2. INTRODUCTION Simple staining is a method of staining in which bacteria are stained by using a single stain. Simple staining is also called as monochrome staining or positive staining. Examples of simple stain are Methylene blue, Safranin , Malachite green, Basic fuchsin and crystal violet etc. In simple staining procedure cell are uniformly stained . The simple stain can be used as a quick and easy way to determine cell shape, size and arrangements of bacteria. True to its name, the simple stain is a very simple staining procedure involving single solution of stain. Any basic dye such as methylene blue, safranin , or crystal violet can be used to color the bacterial cells. These stains will readily give up a hydroxide ion or accept a hydrogen ion, which leaves the stain positively charged.  Since the surface of most bacterial cells and cytoplasm is negatively charged, these positively charged stains adhere readily to the cell surface. After staining,   bacterial cell morphology  (shape and arrangements) can be appreciated. Monochrome Staining (SIMPLE STAINING)

3. A clean grease free slide is taken .A grease free slide is is made by first washing the slide with detergent wiping the excess water and the slide is passed through flame. On these grease free slide smear is made by using a sterile wireloop and cell suspension. These slide is allowed to air dry . After air drying these slide is rapidly passed through a flame for three to four times for heat fixation. After heat fixation the slide is placed on the staining rack and flooded with a particular stain and these stain is allowed to react for three minutes. Futher the slide is washed under running water. The slide is air dried and washed under oil immersion. Procedure of Simple Staining

4. Simple staining procedure stain bacteria easily and helps in observation under microscope. It is useful in preliminary studies of morphological characters of cell that is its size, shape and arrangement Application 

5. The End

6. Diffrential Staining (Gram’s Staining)

7. INTRODUCTION Differential Staining  is a staining process which uses more than one chemical  stain . Using multiple stains can better differentiate between different  microorganisms  or structures/cellular components of a single organism. One commonly recognizable use of differential staining is the  Gram stain . Gram staining uses two dyes:  Crystal violet  and Fuchsin or Safranin (the counterstain ) to differentiate between  Gram-positive   bacteria and  Gram-negative  bacteria. Acid-fast  Stains are also differential stains. Differetial Staining (Gram’s Staining)

8. Gram staining involves three processes : staining with a water-soluble dye called crystal violet, decolorization , and counterstaining, usually with safanin . Due to differences in the thickness of a peptidoglycan layer in the cell membrane between Gram positive and Gram negative bacteria, Gram positive bacteria (with a thicker peptidoglycan layer) retain crystal violet stain during the decolorization process, while Gram negative bacteria lose the crystal violet stain and are instead stained by the safranin in the final staining process. The process involves three steps : Cells are stained with crystal violet dye. Next, a Gram's iodine solution (iodine and potassium iodide) is added to form a complex between the crystal violet and iodine. This complex is a larger molecule than the original crystal violet stain and iodine and is insoluble in water . A decolorizer such as ethyl alcohol or acetone is added to the sample, which dehydrates the peptidoglycan layer, shrinking and tightening it. The large crystal violet-iodine complex is not able to penetrate this tightened peptidoglycan layer, and is thus trapped in the cell in Gram positive bacteria. Conversely, the the outer membrane of Gram negative bacteria is degraded and the thinner peptidoglycan layer of Gram negative cells is unable to retain the crystal violet-iodine complex and the color is lost . A counterstain , such as the weakly water soluble safranin , is added to the sample, staining it red. Since the safranin is lighter than crystal violet, it does not disrupt the purple coloration in Gram positive cells. However, the decolorized Gram negative cells are stained red.

9. Step 1:  A heat fixed smear   is covered with a basic violet dye, Example: Crystal violet. This stain imparts its colour to all cells. It is referred to as a primary stain, since it is applied first. Step 2:  After a short time, the slide is   washed off and the smear is covered with iodine, a mordant. At this stage both Gram positive and Gram negative bacteria appear dark violet. Step 3:  Next, the slide is decolurized with   alcohol or an acetone alcohol solution. This solution is a decolurizing agent, which removes the primary stain from the cells of some species but not from others. Step 4:  The slide is immediately washed after   decolurization and the slide is then counter stained with basic fuchsin or safranin , a basic red dye. The smear is washed again, blot dried and examined under microscope Procedure of Gram’s Staining

10. The Gram stain remains the most commonly used stain because it detects and differentiates a wide range of pathogens. Selection of suitable culture media based on Gram’s stain finding. Counting of bacteria. Appreciation of morphology & types of bacteria in clinical specimens. Application

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