Practical General Bacteriology Lab 1: The Microscope PDF

# Practical General Bacteriology ## Lab-1- The Microscope - There are many types of microscopes. The most common (and the first to be invented) is the optical microscope, which uses light to image the sample. Other major types of microscopes are the electron microscope, the ultra microscope, and...

# Practical General Bacteriology ## Lab-1- The Microscope - There are many types of microscopes. The most common (and the first to be invented) is the optical microscope, which uses light to image the sample. Other major types of microscopes are the electron microscope, the ultra microscope, and the various types of scanning probe microscope. - **Compound light microscope:** It's widely used in microbiology laboratories. The limit of magnification is about 1000X. It consists of two systems: - **The lens system:** which consists of: - **Eyepiece or Ocular lenses:** The lens the viewer looks through to see the specimen. The eyepiece usually contains a 10X or 15X power lens. - **Objective lenses:** One of the most important parts of a compound microscope, as they are the lenses closest to the specimen. A standard microscope has three, four, or five objective lenses: 4X, 10X (Low), 40X(high dry) and 100X(Oil). - **Illumination system:** - **Illumination:** The light source for a microscope. Older microscopes used mirrors to reflect light from an external source up through the bottom of the stage; however, most microscopes now use a low-voltage bulb. - **Iris diaphragm:** Adjusts the amount of light that reaches the specimen. - **Condenser:** Gathers and focuses light from the illuminator onto the specimen being viewed. - **Adjustment parts** - **Stage height adjustment (Stage Control):** These knobs move the stage left and right or up and down. - **Coarse adjustment:** Brings the specimen into general focus. - **Fine adjustment:** Fine tunes the focus and increases the detail of the specimen. - **Other parts:** - **Nosepiece:** The viewer spins the nosepiece to select different objective lenses. - **Specimen or slide:** The specimen is the object being examined. Most specimens are mounted on slides, flat rectangles of thin glass. - **Stage:** The flat platform where the slide is placed. - **Stage clips:** Metal clips that hold the slide in place. - **On/off switch:** This switch on the base of the microscope turns the illuminator off and on. - **Base:** The base supports the microscope and it's where the illuminator is located. - **Arm:** The arm connects the body tube to the base of the microscope. - **Tube:** Connects the eyepiece to the objective lenses. ## How To Use A Microscope 1. Turn microscope ON. 2. Rotate low power objective into place. 3. Place slide onto stage - center the specimen over the hole in the stage. Secure the slide with stage clips. 4. Look through the eyepiece and turn the coarse adjustment knob until the specimen comes clearly into view. Adjust slide if necessary. 5. Diagram what you see in the field of view. 6. Rotate medium power objective into place and repeat steps 4-5. 7. Rotate high power objective into place and repeat steps 4-5 using the fine adjustment knob only. ### When converting to 100x power, note the following - Don't convert to 100x power unless put a drop of oil on slide in the point of light density. - Don't move the slide from its place to put the drop of oil. - Don't move the mechanical stage by using coarse adjustment to put the drop of oil. - Only use the fine adjustment to demonstrate the field (using course adjustment leads to breaking the slide). ### What are benefits (purpose) of using immersion oil with the oil lenses? - **To avoid the mechanical contact between the slide and lenses.** - **The diffraction factor of oil approximately equal to the diffraction factor of glass so the light hadn't refract and led to increase the resolving power of the microscope.** ## Tools and Equipment ### Laboratory equipment | Equipment | Description | |---------------------------------|----------------------------------------------------------------------------------------------------| | Burette (US also buret) | Used to measure and dispense liquids, particularly in titration experiments. | | Gauze mat | A mesh material used to support glassware over a flame. | | Tripod | A three-legged stand that provides a stable base for supporting various laboratory equipment. | | Flame | A source of heat used for sterilization. | | Rubber tubing | A flexible tube used for connecting different pieces of equipment, like a Bunsen burner to a gas source. | | Bunsen burner | A device that burns fuel, usually gas, to create a safe flame for heating and sterilization. | | Microscope cover | A thin glass or plastic sheet that covers the specimen on a microscope slide to protect it from dust. | | Pestle | Used to grind substances in a mortar. | | Evaporating dish | Used for evaporating liquids. | | Petri dish | A shallow, circular dish used to grow bacteria or other microorganisms in culture. | | Crucible | A ceramic container used to heat and melt solids at high temperatures. | | Mortar | A bowl used for grinding solids into a fine powder. | | Graduated cylinder | Used to measure the volume of liquids with precision. | | Glass rod | Used to stir and transfer liquids. | | Magnet | Used to handle ferrous materials. | | Dropper | Used to dispense small quantities of liquids, often for adding reagents. | | Spatula | A tool for transferring or scraping dry powder or solid substances. | | Pipette | Used to aspirate and dispense liquid. | | Retort | A type of flask used for distillation or heating liquids. | | Beaker | A cylindrical container used for holding, mixing, and heating liquids. | | Eyepiece | The lens that magnifies the image for the viewer. | | Flask | A container used for holding and mixing liquids. | | Funnel | A cone-shaped device used for transferring liquids from one container to another. | | Test tube | A cylindrical container with a rounded bottom used to hold and culture samples of liquids. | | Stopper | Used to seal the top of a test tube or flask. | | Lamp | A source of light used for illuminating samples or work areas. | | Stand | A base used to support various laboratory equipment. | | Test tube rack | Used to hold and organize test tubes. | ## Lab-2- Culture Media - **Growth medium or culture medium** is a combination of substances designed to support the growth of microorganisms or cells. Different types of media are used for growing different types of cells. - **How many types of growth media are there?** - There two major types of growth media: - **Cell culture**: Which use specific cell types derived from plants or animals. - **Microbiological culture**: Which are used for growing microorganisms, such as bacteria or yeast. - **Pure culture:** Culture medium containing the growth of a single species of bacteria. It can be preserved by: - Cooling - Freezing - Lyophilization (Freeze drying) - **Mixed culture:** Culture medium containing the growth of two or more species of bacteria. ### Kinds of culture media Culture media can be divided according to their consistency: - **Solid media:** 2% agar - **Semisolid media:** 1% agar - **Liquid media:** 0% agar #### Agar - A complex carbohydrate extracted from sea algae called Gelidium, used in preparing culture media as a solidifying agent because of its characteristics which are: - Its melting properties: melt at 90-100C° and solidifies at 42C°. - It has no nutritive value for the majority of bacteria. ### Their uses and contents - **Natural media (non-synthetic):** Media contain natural material rich with vitamins and their structure and concentration are not defined such as milk and blood - **Defined media (synthetic media):** Medium contain chemical materials their structure and concentration exactly defined - **Semi-synthetic media:** Media contain natural material as well as chemical materials - **Living media:** Medium contain living tissue used to culturing viruses and cancer cell ### Routine laboratory media - **Basal media:** Basal media are those that may be used for growth (culture) of bacteria that do not need enrichment of the media. Examples: Nutrient broth, nutrient agar and peptone water. Staphylococcus and Enterobacteriaceae grow in these media. - **Enriched media:** The media are enriched usually by adding blood, serum or egg. Examples: Enriched media are blood agar and Lowenstein-Jensen media. Streptococci grow in blood agar media. - **Selective media:** These media favor the growth of a particular bacterium by inhibiting the growth of undesired bacteria and allowing growth of desirable bacteria. Examples: MacConkey agar, contain crystal violate that inhibit G+ve. - **Differential media (Indicator):** An indicator is included in the medium. A particular organism causes change in the indicator, e.g. MacConkey agar are differential media (contain lactose sugar and neutral red). - **Transport media:** These media are used when cannot be cultured soon after collection. Examples: Cary-Blair medium, Amies medium, Stuart medium. - **Storage media:** Media used for storing the bacteria for a long period of time. Examples: Egg saline medium, chalk cooked meat broth. - **Assay medium:** Medium used to assay the production amount of some material in bacteria - **Enumeration media:** That used to calculate the number of bacteria in water, soil, and food sample - **Characterization media:** That used to characterize and recognize the type of bacteria ## Preparation Of Culture Media 1. **Weighting the medium ingredients:** According to the direction written on its container. 2. **Dissolve with little amount of D.W.:** Then complete the volume to the volume you want and may be need using heating and stirrer for complete dissolving. 3. **Check pH.** 4. **Dispensing the medium into test tube by pipette.** 5. **Sterilization by autoclave.** 6. **Dispensed agar medium into petri dish when the heat reach to 45.** **Example:** Prepare 500ml of N.A. medium if the direction on the container wrote 8gm/liter | gm | ml | |---|---| | 8 | 1000 | | x | 500 | x = 8 * 500/1000 = 4 gm of media dissolve in a little amount of D.W. then complete the volume to 500 ml, then autoclaved and poured in plates ## Method Of Pouring the Media In Plate - The sterile plates should be on the table near the burner then: - Cool the solid medium to 45C° to avoid solidify it and to avoid forming of a drop on the cover of plates. - Remove the cover (or cotton plug) and sterile the upper part by the burner. - Remove the cover of the plate near the burner and pour the medium and close the cover of the plate. - Move the plate on the table 5 times in two directions to distribute the media equally in the plate. ## Sterility Test This test means putting the flasks, tubes, and plates which contain sterile media before using in an incubator at 37C for 24 hr. to ensure that there is no contamination while preparing and pouring the media. ## Shapes of Bacteria | Shape | Description | Examples | |---|---|---| | **Cocci** | Spherical | * **Diplococci:** Streptocococcus pneumoniae * **Tetrad** * **Staphylococci:** Staphylococcus aureus | | **Bacilli** | Rod-shaped | * **Streptococci:** Streptococcus pyogenes * **Chain of bacilli:** Bacillus anthracis | | **Others** | | * **Vibrios:** Vibrio cholerae * **Spirilla:** Helicobacter pylori * **Spirochaetes:** Treponema pallidum | ## Microbiology Lab - 2018 ### Lab Report Template | Field | | |------------------------|---| | Date | | | Student(s) Name(s) / | 1. | | | 2. | | Method Name | | | **Materials** | | | Material | | |----------------|---| | Detergent | □ | | Sponge | □ | | Burner | □ | | Lighter | □ | | Marker | □ | | Loop | □ | | Slides | □ | | Agar Medium | □ | | Broth Medium | □ | | Stain Set | □ |