The document provides an overview of the Gram stain method. Some key points:
1) The Gram stain was discovered in 1884 by Hans Christian Gram and differentiates bacteria based on their cell wall composition into Gram-positive or Gram-negative.
2) It is a widely used initial method in microbiology to characterize and classify bacteria under a microscope.
3) Gram-positive bacteria retain the primary crystal violet stain due to their thick peptidoglycan layer, while Gram-negative bacteria are decolorized by the ethanol and take up the counterstain due to their thin peptidoglycan layer.
The document provides an overview of the Gram stain method. Some key points:
1) The Gram stain was discovered in 1884 by Hans Christian Gram and differentiates bacteria based on their cell wall composition into Gram-positive or Gram-negative.
2) It is a widely used initial method in microbiology to characterize and classify bacteria under a microscope.
3) Gram-positive bacteria retain the primary crystal violet stain due to their thick peptidoglycan layer, while Gram-negative bacteria are decolorized by the ethanol and take up the counterstain due to their thin peptidoglycan layer.
The document provides an overview of the Gram stain method. Some key points:
1) The Gram stain was discovered in 1884 by Hans Christian Gram and differentiates bacteria based on their cell wall composition into Gram-positive or Gram-negative.
2) It is a widely used initial method in microbiology to characterize and classify bacteria under a microscope.
3) Gram-positive bacteria retain the primary crystal violet stain due to their thick peptidoglycan layer, while Gram-negative bacteria are decolorized by the ethanol and take up the counterstain due to their thin peptidoglycan layer.
The document provides an overview of the Gram stain method. Some key points:
1) The Gram stain was discovered in 1884 by Hans Christian Gram and differentiates bacteria based on their cell wall composition into Gram-positive or Gram-negative.
2) It is a widely used initial method in microbiology to characterize and classify bacteria under a microscope.
3) Gram-positive bacteria retain the primary crystal violet stain due to their thick peptidoglycan layer, while Gram-negative bacteria are decolorized by the ethanol and take up the counterstain due to their thin peptidoglycan layer.
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Gram stain methods
Made by : DR ( Rasha Bennour)
Supervised by : Associate professor ( Omra O. Bugrein, MD ) Overview : • The most widely used staining procedure in microbiology is the Gram stain, discovered by the Danish scientist and physician Hans Christian Joachim Gram in 1884.to differentiate Pneumococci from Klebsiella Pneumonia. It is also known as “ Differential staining”, as it divides micro-organisms into two maingroups i.e. Gram’s +ve and Hans Christian Joachim Gram Importance of a Gram Stain: • The Gram stain is a very important step in the initial characterization and classification of bacteria. It is also a key procedure in the identification of bacteria based on staining characteristics, enabling the bacteria to be examined using a light microscope. The bacteria present in an unstained smear are invisible when viewed using a light microscope. Once stained, the morphology and arrangement of the bacteria may be observed as well. Furthermore, it is also an important step in the screening of infectious •This differential staining procedure separates most bacteria into two groups on the basis of cell wall composition: 1- Gram positive bacteria (thick layer of peptidoglycan-90% of cell wall)- stains purple. 2- Gram negative bacteria (thin layer of peptidoglycan-10% of cell wall and high lipid content) –stains red/pink. PRINCIPLE OF GRAM STAINING • The structure of the organism’s cell wall determines whether the organism is gram positive or negative. When stained with a primary stain and fixed by a mordant, some bacteria are able to retain the primary stain by resisting declorization while others get decolorized by a decolorizer. Those bacteria which retain the primary stain are called Gram positive and those bacteria which gets decolorized and then get counterstained are called Gram negative. REQUIREMENTS AND PREPARATION OF REAGENTS: • 1-Primary Stain : Crystal violet Solution A : • 1_Crystal violet = 2 gm • 2_Ethyl alcohol= 20 ml • Solution B : • 1_Ammonium oxalate = 0.8 gm • 2_Distilled water = 80 ml • Mix solution A and B. Keep for 24 hours and filter. Store in an amber colored bottle. • 2-Mordant : Gram’s Iodine – Iodine = 1 gm – Potassium iodide = 2 gm – Distilled water = to 100 ml • Mix and Store in an amber colored bottle.
• 3-Decolorizer : 95% Ethanol or 1:1 acetone with ethanol
– Acetone = 50 ml – Ethanol (95%) = 50ml • 4-Counterstain: safranin – Safranin O = 0.34 gm – Absolute alcohol = 10ml – Distilled water = 90ml
• Mix, filter and store in ambered
• colored bottle. Crystal violet (CV) dissociates into CV+ and Cl– ions in aqueous solutions. These ions penetrate through the cell wall and cell membrane of both Gram-positive and Gram-negative cells. The CV+ ion interacts with negatively charged components of bacterial cells and stains the cells purple.
Iodine (I), used as mordant interacts with CV+ and
forms large complexes of crystal violet and iodine (CV–I) within the inner and outer layers of the cell.
When a decolorizer such as alcohol or acetone is
added, it interacts with the lipids of the cell membrane. Since Gram negative organism have thin peptidoglycan layer(1-2 layers) and have additional lipopolysaccharide layer which gets so gram negative organism fails to retain the complex and gets decolorized as the complex is washed away In contrast, a Gram-positive cell becomes dehydrated from an ethanol treatment. This closes the pores in the cell wall and prevents the stain from exiting the cell. The large CV–I complexes become trapped within the Gram- positive cell also due to the thick and multilayered (40 layers) nature of its peptidoglycan.
After decolorization, the Gram-positive cell remains
purple and the Gram-negative cell loses its purple color. Counterstain, which is usually positively- charged safranin or basic fuchsin, is applied last to give decolorized Gram-negative bacteria a pink or red color. PROCEDURE OF GRAM STAINING • Smear preparation : 1-Take a grease free dry slide. 2-Sterilize the inoculating loop on a flame. 3-Transfer a loopful of culture (or the specimen) by sterile loop and make a smear at the center. Smear should not be very thin or very thick. 4-Allow the smeat to dry in the air. 5-Fix the dry smear by passing the slide 3-4 times through the flame quickly with the smear side facing up. Gram Staining : 1-Place the slides on the staining rods. 2-Cover the smear with crystal violet stain and leave for 1 minute. 3-Wash carefully under running tap water. 4-Flood the smear with Gram’s iodine solution and leave for 1 minute.
5-Drain off the iodine Wash the slide for the
again in a gentle stream of tap water.
6-Flood the slide with the decolorizing agent
then wait for 20-30 seconds. This can also be done by adding a drop by drop to the slide until the decolorizing agent running from the slides runs clear. .
7-Gently wash the slide under running tap
water and drain completely.
8-Counterstain with safranin for and and
wait for about 30 seconds to 1 minute.
9-Wash slide in a gentile and indirect
stream of tap water until no color appears in the effluent and then blot dry with absorbent paper. Bacterial Morphology: MCQs • 1. What is the correct order of staining reagents in Gram-Staining? a) Crystal violet, alcohol, iodine solution, safranin. b) Crystal violet, iodine solution, alcohol, safranin. c) Crystal violet, safranin, alcohol, iodine solution. d) Iodine solution, crystal violet, alcohol, safranin. Answer: b
Explanation: Gram staining is a
type of differential staining. In this process the fixed bacterial smear is subjected to the following staining reagents in the order listed: crystal violet, iodine solution, alcohol (decolorizing agent), and safranin. • 2-Which of the following are true for Gram- negative bacteria?
a) upon alcohol treatment, the permeability of
the cell wall increases.
b) crystal violet-iodine (CV-I) complex is
extracted.
c) pore size decreases and the CV-I complex
cannot be extracted.
d) alcohol treatment increases the permeability
of the cell wall and the CV-I complex can be extracted. • Answer: d
Explanation: Experimental evidence
suggests that during staining of Gram- negative bacteria the alcohol treatment extracts the lipid, which results in increased permeability of the cell wall. Thus the crystal violet-iodine (CV-I) complex can be extracted and the Gram- negative organism is decolorized. These cells subsequently take on the color of the safranin counterstain. • 3. In Gram-staining, iodine is used as a______________
a) fixative b) mordant c) solublizer d) stain • Answer: b
Explanation: In Gram-staining, iodine acts
as a mordant i.e. it combines with the dye or stain and thereby fixes it on the material. It increases the interaction between stain solution and the bacterial cell. THANK YOU FOR YOU ATTETION