Microorganism Shapes, Staining, Gram Staining, and Dispersal PDF

Basics Shapes of the Microorganisms  I. Shapes of the Bacterial Cells 1. Spherical Cells (cocci): They are found in different arrangements: 2. Short Rods: They are found as single cells.  3. Long Rods (Bacilli): They are found as: Non-sporulat...

Basics Shapes of the Microorganisms  I. Shapes of the Bacterial Cells 1. Spherical Cells (cocci): They are found in different arrangements: 2. Short Rods: They are found as single cells.  3. Long Rods (Bacilli): They are found as: Non-sporulated Sporulated long long rods, found rods in chains. 4. Spiral Shape 5. Vibrio (Spirillum) Shape 6. Actinomycetes: Long, thin, branched and aseptate hyphae, reproduce by conidia.  II. Shapes of the Fungi 1. Molds: Large, 2. Yeasts: Large oval thick, branched and cells, found as single septate hyphae, cells or in non-uniform reproduce by groups, reproduce conidia. by budding. Staining Methods  Chemical Makeup of Stains Benzene = organic compound Chromophore = color Auxochrome = ionization properties Benzene + Chromophore = Chromogen – Chromogen is a colored compound only Auxochrome with Chromogen allows the dye to form salt compounds that adhere to cells.  Basic Dyes Work best in basic pH The most used in bacteriological preparations Ionizes (Cl-, SO4-) Creates (+) Cationic chromogen Attracted to (-) acidic cell components [DNA, proteins] Examples – Methylene Blue CF – Crystal Violet – Carbol Fuchsin  Acidic Dyes Works best in acidic pH Ionizes (Na+, K+, Ca+ +) Creates Anionic (-) chromogen Attracted to (+) cell components [AA] Examples – Picric Acid Nigrosin – Nigrosin – 1. Simple Staining: in which the cells are stained using 1. Simple Staining: one stain (as staining with methylene blue or fuchsein). The simple staining is used to study: The morphological features of the microbial cells. The cell arrangement system. 2. Complex Staining: 2. Complex Staining: in which the cells are stained using 2 stains. The first stain is called primary stain and the second stain is called counter stain.  Simple Staining Fuchsein red Methylene blue stain stain It is a strong acidic It is a weak alkaline stain. stain. The duration time is The duration time is ranged from 0.5 – ranged from 3.0 – 1.0 minute. 5.0 minutes. It isn’t used to stain It is used to stain the bacteria present the bacteria present in a material can be in a material can be  Preparation of Smear for Staining Smears of microorganisms from solid cultures are prepared on clean glass slides as follows: 1 2 3 4 5 6 7 8 9 10  Heat fix (usually) – Kill organism – Heat fixing denatures bacterial enzymes, preventing them from digesting cell parts, which causes the cell to break, a process called autolysis – Adhere to slide – Accepts dye Problems – Too thick – Wash off specimen  Smear Staining OR Flooding the Flooding the smear with smear with methylene blue / fuchsein red / 1 Pouring off the stain. Washing the slide with water. Drying the slide using Bunsen flame.  Practical Spots 1. Fungi (Saccharomyces cerevisiae):  Practical Spots 2. Fungi (Saccharomyces cerevisiae):  Negative Stain Acid Dye (-) chromogen Repelled by (-) cell wall Cells – Colorless – Seen against dark background Gram Staining as a Type of Complex Staining (Differential Staining)  Procedure of Gram Staining 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  6. Pouring off the iodine solution. 5 7 Flooding the smear Washing the smear with the iodine with ethanol, then solution for 1 min. with water.  9. Pouring off the fuchsein red. 10. Washing the slide with water. 8 Flooding the smear with fuchsein red for 1 min. 11. Drying the slide. Gram Positive Gram Negative Bacteria Bacteria Decolorization with ethanol Fuchsein red Why Gram method is a differential staining method? because, Gram staining divides bacteria into 2 classes They do not Gram- decolorize with ethanol, positive (G+) thus appearing purple. bacteria They decolorize with Gram- ethanol, thus they accept fuchsein red and negative appear with red color. (G-) bacteria G – negative G + positive bacteria bacteria  Importance of Gram Staining  Gram staining has a taxonomic importance because it divides bacteria into 2 classes: G+ and G- bacteria.  Gram staining is used to confirm the purity of the bacterial cultures.  Gram staining is used in the diagnosis of some diseases. Bacillus subtilis Micrococcus spp Escherichia coli  Microbial Dispersal in Nature Spread of bacteria in nature is widespread and is due to: 1. Small size of the cell so it is easy to carry in the air. 2. Speed ​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 a few places free of microbes, such as: 1. Deep soil layers. 2. Uninfected internal tissues of living organisms. 3. Inside active volcanoes.  Microbial Dispersal in Nature (Air - Skin) Materials Required: 2 tubes of sterile deep nutrient agar. 2 sterile Petri plates. Water bath. Thermometer.  Gradual cooli ng 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 Ai one r plate. - Opening the plate away from the flame for 15 minutes. - Writing a word of skin on the lid - Closing of the the plate. Ski other plate. n - Opening the plate in the sterile area of the flame. - Moving the finger on the surface of 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.