Gram Stain Lab 3: University of Sulemani PDF

University of Sulemani College of Agricultural Engineering Sciences Food Science & Quality control General Microbiology Laboratory-lab 3 Gram Stain Learning Objectives  After completing this exercise, students should be able to: 1. Explain two important differ...

University of Sulemani College of Agricultural Engineering Sciences Food Science & Quality control General Microbiology Laboratory-lab 3 Gram Stain Learning Objectives  After completing this exercise, students should be able to: 1. Explain two important differences between the cell walls of gram-positive and gram-negative bacteria. 2. Recognize why the Gram stain is important in clinical diagnosis. 3. Identify which step of the Gram-stain procedure could be left out and the reason why. 4. Explain why antibiotics such as penicillin that work by preventing the crosslinking of glycan molecules in peptidoglycan are more effective against gram-positive than gram-negative bacteria. 5. State the advantage of the Gram stain over a simple stain Gram Stain The Gram stain is a differential stain. Four different reagents are used and the results are based on differences in the cell wall of bacteria. Gram Stain  Using two dyes in sequence, each of a different color, bacteria fall into two groups. 1. The first group retains the color of the primary dye: crystal violet (these are called gram positive). 2. The second group loses the first dye when washed in a decolorizing solution but then takes on the color of the second dye, a counter stain, such as safranin or carbolfuchsin (these are called gram negative). An iodine solution is used as a mordant (a chemical that fixes a dye in or on a substance by combining with the dye to form an insoluble compound) for the first (crystal violet) stain. https://www.youtube.com/shorts/VzL4K9nbTUY?feature=share  Differences in the Gram-stain reactions are related to differences in the biochemical composition of bacterial cell walls.  Gram-positive cell walls are composed of thick, strongly linked peptidoglycans (protein-sugar) complexes in which the crystal violet–iodine complex becomes trapped, thereby enabling the cells to resist decolorization.  In contrast, gram-negative bacterial walls have a high concentration of lipids (fats) in their outer membranes and a thinner layer of peptidoglycan.  The lipids dissolve in the decolorizer (alcohol, acetone, or a mixture of these) and are washed away along with the crystal violet–iodine complex.  After decolorization, the now colorless gram-negative organisms take up the red counter stain and appear pink. The gram-positive organisms, which have retained the crystal violet–iodine complex, appear purple after decolorization and counterstaining.. The Gram stain is one of the most useful tools in the microbiology laboratory and is used generally. In the analytical laboratory, it is used not only to study microorganisms in cultures, but also to stain and examine smears made directly from clinical specimens. Direct, Gram stained smears are read quickly to determine the relative numbers and morphology of bacteria in the sample.  The numerous modifications of Gram’s original method are based on the concentration of the dyes, length of staining time for each dye, and composition of the decolorizer.  The choice of decolorizing. agent depends on the speed wanted to accomplish this step. The slowest agent, 95% ethyl alcohol, is used in this exercise to permit the student to gain experience with decolorization.  Acetone is the fastest decolorizer, while an equal mixture of 95% ethyl alcohol and acetone acts with intermediate speed. The acetone-alcohol combination is probably the most popular in diagnostic laboratories. https://youtu.be/UktBAN_OIQM  An organism’s appropriate Gram-stain reaction depends on the presence of an intact cell wall.  As bacterial cultures age and the wall begins to disintegrate, gram-positive cells may not fully retain the crystal violet– iodine complex and will appear falsely gram-negative or as a mixture of gram-positive and gram-negative cells.  In this latter case, they are referred to as gram variable. For this reason, cultures that have incubated longer than 24 or 48 hours or patient specimens that are not examined within a few hours of collection may give false readings when Gram stained.  Smears of bacterial colonies or organisms in patient specimens that are decolorized for too long (known as over decolorization) may also give a false Gram-stain reading.  In addition, some antimicrobial agents that act on the bacterial cell wall may change the morphology and Gram- stain reaction of the bacterial cells  Procedures  Stain a smear by the following procedures : 1. Cover slide with crystal violet solution. Allow to stand for 1 minute (check with instructor; time varies with different batches of stain). 2. Wash off with tap water. 3. Cover slide with Gram’s iodine (a mordant) solution. Leave for 1 minute. 4. Wash off with tap water. 5. Hold the slide at an angle over the staining rack and decolorize with ethyl alcohol (95%) drop wise until no more color washes off (usually 10–20 seconds). This is a most critical step. Be careful not to over decolorize, as many gram-positive organisms may lose the violet stain easily and thus appear to be gram negative after they are counterstained. 6. Wash off with tap water. 7. Apply safranin (the counter stain) for 1 minute. 8. Wash off with tap water. 9. Drain and blot gently with bibulous paper. Air dry the slide thoroughly before you examine the preparation under the microscope. 10. When slides are dry, Examine all slides under oil with the oil-immersion objective. RESULT:  Colour:  Shape:  Gram’s reaction:  Name of microorganism: