Lecture 4 Lab - Microbiology Techniques PDF

Hanging Drop Technique Lab - 4-  Objectives Learn how to perform the motility test. Differentiate between Brownian movement and true motility.  Motile bacteria move about with structures called flagella (a few exceptional bacteria move with the help of axial...

Hanging Drop Technique Lab - 4-  Objectives Learn how to perform the motility test. Differentiate between Brownian movement and true motility.  Motile bacteria move about with structures called flagella (a few exceptional bacteria move with the help of axial filaments, which cannot be seen in the microscope). Nonmotile bacteria without flagella are called atrichous.  Looking at living bacteria are not as easy as one would think. First of all, living bacteria have no color, and they are small: therefore, they are really difficult to see, even with the oil immersion lens. Second, all bacteria have some vibrational movement, even nonmotile ones. The keys to a good hanging drop slide are 1) use a small drop of bacterial suspension, but do not let it dry out, and 2) use a young culture of bacteria.  Brownian movement is caused by water molecules bouncing around in the solution, knocking up against each other and the microorganisms. Kinetic energy inherent to all molecules causes this kind of movement. On the other hand, those bacteria with flagella will be very apparently moving about the field of vision, although perhaps not all of the bacteria will be moving. Some cells will "run" straight across the field, others will "tumble" across the field in a slower motion.  MATERIALS NEEDED fresh cultures (broth medium less than 24 hours old is optimal) of various bacteria (assigned by instructor) depression slides cover slips petroleum jelly  THE PROCEDURE Place a drop of the bacterial culture (optimally from a young broth culture) in the middle of a cover slip. Place a thin line of petroleum jelly around the edge of the cover slide, or at the 4 corners of cover slip. Turn the depression slide upside-down (depressed area facing down) and gently touch the cover slide. The jelly holds the cover slip to the slide and also keeps the suspension from drying out. Now flip the entire microscope slide/cover slip combination over. It should look like the diagram below. cover glass Bacterial suspension petroleum Hollow ground slide concave well  Site of flagella Peritrichous (E.coli) Monotrichous ( Vibrio cholerae)  Site of flagella Lophotrichous (pseudomonas) amphitrichous (Spirillum volutans) 3- Hanging drop technique.  Practical lecture 3 Acid Fast Stain  Acid - Fast Stain Differential Stain that divides bacteria into 2 groups  Acid - Fast  Non Acid - Fast Used to identify organisms in the Genera Mycobacterium (high lipid and wax content in cell wall)  Acid - Fast Stain 1. Gather all equipment and supplies 2. Label the slide with the patient’s initials or the specimen number. 3.Preparing and Fixing Smears  Acid - Fast Stain 4.Flood the slide with strong carbol-fuchsin (basic dye) for 5-10 min then heat intermittently , do not allow the stain to boil or dry 5.Rinse the slide with distilled water  Acid - Fast Stain 6.Decolorize with a mixture of acid/alcohol 7. Rinse the slide with distilled water. 8.Add counter stain (Methylene blue) for 2 min 9. Rinse with water 10. Air-dry Results of Acid - Fast Stain Acid-fast Non Acid-fast Fixation Primary stain Carbol fuchsin All cells are stained red. Decolorization Acid/alcohol Acid-fast cells keep their color Counter stain Methylene blue Any other cell type takes the blue color  Results of Acid - Fast Stain Acid - Fast Cells Red Non Acid - Fast Blue  Spore stain Bacterial endospores contain numerous protective layers which cannot be penetrated easily by a simple or Gram- stain. Excessive physical treatment (Steaming/ heating) is required to allow the dye to penetrate. Normal (vegetative) cells are initially stained with the dye as the spores, then decolorized and counterstained with another dye.  Spore stain 1- Steaming with a solution of Malachite green All cells and spores are stained green. 1 2-Decolorization with water Spores keep their color, cell loses its color 2 3-Counter stain with Safranin, cell stains red, spores remains green. 3  Other stain Capsule Stain Flagella Stain Thank you