Gram Staining, Dispersal, and Motility PDF

1. Preparation of fixed smear. 3. Pouring off the crystal violet. 4. Washing the slide with water. 2 Flooding the smear with crystal violet for 1 min. ...

1. Preparation of fixed smear. 3. Pouring off the crystal violet. 4. Washing the slide with water. 2 Flooding the smear with crystal violet for 1 min.  6. Pouring off the iodine solution. 5 7 Flooding the smear with Washing the smear with the iodine solution for 1 ethanol. min.  9. Pouring off the fuchsin red. 10. Washing the slide with water. 8 Flooding the smear with fuchsin red for 1 min. 11. Drying the slide.  They do not decolorize Gram-positive with ethanol, thus (G+) bacteria appearing purple. They decolorize with Gram- negative ethanol, thus they (G-) bacteria accept fuchsin red and appear with red color. Gram Positive Bacteria Gram Negative Bacteria Decolorization with ethanol Fuchsein red G – negative bacteria G + positive bacteria q Gram staining has a taxonomic importance because it divides bacteria into 2 classes: ¨ G+ and G- bacteria. q Gram staining is used to confirm the purity of the bacterial cultures. q Gram staining is used in the diagnosis of some diseases.  Bacteria are widespread in nature and this is due to: 1. Small size of the cell so it is easy to carry in the air. 2. Speedy cell reproduction so the cells are found in large counts. 3. Diversity of their food needs so the cells are found in different ecosystems. ¨Despite this, there are few places free of microbes, such as: ¨. ¨1. Deep soil layers. ¨2. Uninfected internal tissues of living organisms. ¨ ¨3. Inside active volcanoes. Materials Required: 2 tubes of sterile deep nutrient agar. 2 sterile Petri plates. Water bath. Thermometer.  Gradual cooling to 50°c. Deep nutrient agar Melting the nutrient agar at 100°c. - Circular movement of the plates. - Allowing the plates to set. Pouring the nutrient agar into the plates  - Writing a word of air on the lid of one Air plate. - Opening the plate away from the flame for 15 minutes. - Closing the plate. - Writing a word of skin on the lid of the other plate. Skin - Opening the plate in the sterile area of the flame. - Moving the finger on the surface of medium to form straight lines. - Closing the plate. Incubating the plates in the inverted position at 30 °C / 24- 48h. After incubation, the microbial colonies appear and can be seen with the naked eye.  What is the microbial colony? The colony is defined as “the population of individual cell that is grouped together into a mass large enough to be seen on the surface of the solid medium”. Motility of Bacteria  Types of Motility Brownian movement True Motility (False Motility) Random, vibratory movement of the movement of the cells microbial cells that is caused by the from one position molecules, suspended in to another using the liquid, striking Flagella. bacteria.  Arrangement of flagella Monotrichous cell Lophotrichous cell Amphitrichous cell Peritrichous cell Testing the motility of bacteria using Wet mount Preparation  How to prepare the wet mount? I. Materials: qGlass slide. q Cover. q Inoculating loop. q Broth culture 24 h. old. q Bunsen flame.  II. Steps of preparing the wet mount: 1- Cleaning the slide and the cover. 2- Placing the cover horizontally. 3- Shaking the broth culture. 4- Sterilizing the inoculating loop. 5- Transferring one loop from the culture to the center of the slide. Liquid culture 6- Placing the cover on the slide in contact with the culture.  Microscopic examination of the wet mount Adding one drop of cider oil on the cover. Racking up the condenser of the microscope. Opening the iris diaphragm. Placing the oil immersion lens (100x) in its position and adjusting the lighting. Using the fine adjustment knob to focus on the bacterial cells.  Bacillus subtilis Micrococcus spp G+ long-rods with central G+ spherical cells, found spores, found in chains singly or in non-uniform groups. Listeria monocytogenes G+ short-rods, found singly or in short chains. Non spore-forming bacteria.  Escherichia coli Actinomycetes G- single short rods. Non Long, thin, branched, spore-forming bacteria aseptate hyphae, reproduce by conidia Mixed smear from Bacillus subtilis and E. coli Mixed smear from Micrococcus spp. and E. coli