General Microbiology Notes PDF

# General Microbiology ## Microbiology Definitions - Micro: Small, Tiny - Bio: Life - Logy: Science ## Classification of Kingdoms - **Monera:** Bacteria, Cyano Bacteria (blue-green algae) - **Protista:** Chlorophyta, Phaeophyta, Rhodophyta, Bacillariophyta, Xanthophyta - **Mycota:** Unicellula...

# General Microbiology ## Microbiology Definitions - Micro: Small, Tiny - Bio: Life - Logy: Science ## Classification of Kingdoms - **Monera:** Bacteria, Cyano Bacteria (blue-green algae) - **Protista:** Chlorophyta, Phaeophyta, Rhodophyta, Bacillariophyta, Xanthophyta - **Mycota:** Unicellular Fungi, Filamentous Fungi, Yeasts ## Microscope Parts - **Ocular Lenses (Eye Piece):** - Used to see specimens not visible to the naked eye. - Magnification power is (10x) - Removable - **Nose Piece:** - Movable circular structure that carries the objective lens. - Rotates between different objective lenses to obtain the desired magnification power (4x, 10x, 40x, 100x). - **Objective Lenses:** - 3 or 4 commonly found in microscopes. Each lens has its magnification power. - **4x:** Low power, red - **10x:** Medium power, yellow - **40x:** High power, blue - **100x:** Called "Oil Immersion Lens." White oil because with 100x lens, we use cedar oil, which gives a higher magnification. Immersion because the 100x lens is immersed in cedar oil. - **100x** used for Bacteria - **Arm:** - Connects the base and head. - Provides support to head. - Used in carrying of the microscope. - **Stage:** - The part on which the slide is placed. - **Stage Clips** - Used to fix the slide on the stage. - **Aperture:** - A hole in the stage from which light passes from the condenser and reaches the specimen - **Base:** - On/off switch. - Voltage regulator. - Illuminator lamp. - Mirror ## Transport Precautions When Using a Microscope - Use tissue or cotton swabs for lens care. ## Solvents - Xylene - Alcohol (70%) - Acetone ## Dust Protection - Dust Covers - Low Power Lens (4x) - Dry power lens (10x-50x) - Oil Immersion Lens (100x) - Used with cedar oil, especially for small bacteria. **Total Magnification = M power of ocular lens x M Power of objective lens** ## Microbiological Lab Equipments 1. Petri dishes (Isolation, Purification) 2. Isolation (loop, needle) 3. Pipette 4. Automatic Micro Pipette 5. Pipette Filler 6. Filter Paper 7. Cylinder 8. Conical Flasks 9. Falcon Tubes 10. Test tubes 11. Glass slides 12. Personal safety equipment ## Apparatus/Devices 1. Autoclave - Sterilization 2. Incubator- Incubation process device 3. Oven - For drying, especially glasses (160-200° C) (1-2 hours) 4. pH meter - At temperature 5. Water bath: Solids turn into liquids 6. Digital balance - Sensitive or digital scale # General Microbiology - Isolation of Bacteria and Fungi ## Isolation of Bacteria and Fungi from Natural Sources - Bacteria and Fungi: Consider the most common microorganisms present in water, soil, air, etc. ## To make isolation and purification, or any other microbe experiments, we must work under aseptic conditions. These aseptic conditions include 1. Disinfectant is used to clean and kill microbial cells in the laminar flow and on the surface or floor of the lab. 2. Close the fans and windows. 3. Clean our hands, wear coats, gloves, and goggles. ## Procedure of Isolation 1. Prepare nutrient agar and PDA media for bacteria and fungi, respectively. 2. Sterilize media in an autoclave at 121°C, 1.5 atm for 20 minutes. 3. Pour media into sterilized Petri dishes and leave them to solidify. 4. Spread 5mL water on to the surface of agar media. - In the case of soil, there are two methods of isolation that can occur: 1. Direct spreading of soil particles on the surface of agar media. 2. Make a serial dilution of soil. - Prepare a set of test tubes, each has 9 mL of distilled H2O or liquid medium (from the same kind which was used for isolation). - Make sterilization in autoclave 121° C, 1.5 atm for 20 minutes. - Put 1g of soil (this soil must be cleaned by removing sand, stones, any plant parts. Also, it must be dried if it was yet in the first tube) Shake well to help in separation of spores and vegetative cells which are attached with soil particles into the solution. Leave this tube for few minutes to allow the soil particles to settle down. - Transfer 1mL from the first tube to the second one and shaking well. After that, repeat this step between the successive tubes. - Inoculate 0.5 mL from each tube to the opposite plate. - Incubate the plates in an incubator at 28°C for 48 hours for Bacteria and 7 days for Fungi. ## Microbe Morphology 1. Quadrant 2. Radiant 3. Continuous 4. T-Shape # How You Can Obtain and Culture Microorganisms in the Lab? ## Media Preparation - Definition: A specific aqueous solution of nutrients required for microbial growth. ## Media Components: - **Carbon Source:** Carbohydrates, Proteins - Function: Provides energy - Building Structures (Skeleton) - **Nitrogen Source:** Amino Acids, Proteins - Function: 1. Structure Proteins 2. Functional proteins 3. Nucleic acids (Genetic Material) - **Minerals:** - **Macroelements (Trace):** Fe, S, Na, K - **Macroelements**: The organism needs in very high quantities. - **Microelements:** The organism needs in small quantities, but if it exceeds a certain concentration, it becomes toxic to microbial cells. - **The basic function element is Coenzymes. ** - **Water**: Distinguishes a living cell from a dead cell - The percentage of water in the cytoplasm ranges from 70 to 80% and helps the circulation of cytoplasm and makes the organelles move and perform their function ## Transport Precautions When Using a Microsocope - Use tissue or cotton swabs for lens care. ## Obtaining the Sample - **Tap water:** Contains ions and microorganisms.  - **Distilled water:** Contains ions and microorganisms. - **Sterilized distilled water:** Does not contain ions and microorganisms.  This method is used because it doesn't affect the compounds that were prepared, and it doesn't interact with them, preventing the sample from being transformed into another compound. It also prevents the sample from being negatively affected by passing through it. **Growth Factors** - Amino Acids - Growth Regulators **pH Degree** - Acid - Neutral - Basic **Important Note:** Anything other than the elements above is not providing food but has its own function. # Examination of Bacterial Cells - **Living Cells:** Motile - **Fixed Cells:** Dead - Using flame - Using chemicals ## Morphological Appearance - **Bacteria:** Small colony, Oily appearance - **Fungi:** Large colony, Cotton appearance - **Bacteria:** White, yellow - **Fungi:** Most colors ## Preparation of a Bacterial Film (Smear): 1. Groove (depression) 2. 1gmL of sterile distilled water 3. Bacterial Suspension (3 parts) # Gram Staining ## Aim: 1. Gram-Positive Bacteria (G+ve) 2. Gram-Negative Bacteria (G-ve) 3. Study Morphology ## Procedures 1. Prepare a bacterial film. 2. Add crystal violet to the bacteria, let it stand for 1 minute. 3. Wash with tap water, drop by drop, to remove the excess stain. 4. Add iodine solution, let it stand for 1 minute. 5. Wash with tap water, drop by drop, to remove the excess iodine solution. 6. Wash with ethanol (75%), drop by drop, to remove excess stains and iodine solution. 7. Wash with tap water to remove the excess ethanol. 8. Put safranin on the smear, leave it for 30 seconds. 9. Wash with tap water, drop by drop, to remove excess safranin. 10. Dry the slide using filter paper, then examine with the oil immersion lens. ## Observation - **G+ve Bacteria:** Have violet color of crystal violet. - **G-ve Bacteria:** Have red color of Safranin. ## Notes: - **Mordant:** Iodine. - **Primary stain:** Crystal violet. - **Counterstain:** Safranin. - **Alcohol**: Hydrolyzing (dehydrating) agent - **Iodine**: Decolorizing agent ## Comment: - **G+ve:**  - **Chemical Structure of the Cell Wall:** Mg ribonucleate - **Lipid Content of the Cell Wall:** Small - **G-ve:** - **Chemical Structure of the Cell Wall:** Complex ## The Structure of a Typical Bacteria - **Granule** - **Capsule** - **Cell Wall** - **Plasma Membrane** - **Cytoplasm** - **Flagella** - **Genetic Material** - **Pilli** - **Ribosome** ## Bacterial Cell Forms 1. **Cocci** - Spherical - **Monococci** - **Diplococci** - **Tetracocci** - **Streptococci** - **Sarcinae** - **Staphylococci** 2. **Bacilli** - Rod-shaped - **Monobacilli** - **Diplobacilli** - **Streptobacilli** 3. **Spiral** 4. **Comma Shaped** # Nostoc SP - Kingdom: Monera - Division: Cyanophyta - Class: Cyanophyceae - Order: Nostocaceae - Genus: Nostoc - Species: - **Nodules** - **Akinete (Spore Cell)** - **Gelatinous Sheath** - **Hormagonium** - **Vegetative Cell** - **Heterocyst** # Oscillatoria SP - Kingdom: Monera - Division: Cyanophyta - Class: Cyanophyceae - Order: Nostocales - Family: Oscillatoriaceae - Genus: Oscillatoria - Species: - **Apical Cell** - **Vegetative Cell** - **Hormogonium** - **Gelatinous Sheath** - **Biconcave Separating Disc** # Chlamydomonas SP - Kingdom: Protista - Division: Chlorophyta - Class: Chlorophyceae - Order: Volvocales - Family: Chlamydomonadaceae - Genus: Chlamydomonas - **Flagellum** - **Basal Granules** - **Contractile Vacuoles** - **Nucleus** - **Cup-shaped Chloroplast** - **Eye Spot (Stigma)** - **Cytoplasm** - **Pyrenoid** # Pandorina SP - Kingdom: Protista - Division: Chlorophyta - Class: Chlorophyceae - Order: Volvocales - Family: Volvocaceae - Genus: Pandorina ## Colony - **Chlamydomonal Cell** - **Gelatinous Sheath** - **Daughter Colony** - **Gelatinous Matrix** # Volvox SP - Kingdom: Protista - Division: Chlorophyta - Class: Chlorophyceae - Order: Volvocales - Family: Volvocaceae - Genus: Volvox - **Vegetative Cells** - **Flagella** - **Daughter Colony** - **Antheridium** - **Cytoplasm strands (orgonium)** - **Gelatinous Matrix** # Spirogyra SP - Kingdom: Protista - Division: Phaeophyta - Class: Chlorophyceae - Order: Zygnematales (Conjugales) - Family: Zygnemataceae - Genus: Spirogyra - **Thallus Structure** - **Receptacle** - **Conceptacles** - **Air Bladder (in Pairs)** - **Medribe** - **Frond Part (Blade)** - **Stipe** - **Rhizoids** # Female Conceptacle (9) - **Ostile** - **Paraphysis** - **Epidermis** - **Cortex** - **Reserved Food** - **Medulla Cell** - **Multilayered Wall** - **Oogonium (9)** - **Unbranched Non-Sepatated Stalk** - **Eggs** # Diatoms - Kingdom: Protista - Division: Bacillariophyta - Class: Bacillariophyceae - Order: Pennales/Centrales - Family: Diatomeaeae - **Sideview (Gridle View)** - **Epitheca (External)** - **Gridle** - **Nucleus** - **Hypotheca (Internal)** - **Tap View** - **Polar Nodule** - **Central Nodule** - **Raphe** - **Silica Striation**