Mb lab

• Proper use and care of the microscope

  1. Get your microscopes from the technician counter keeping the microscope in an upright position

  2. Hold the microscope arm with your right hand and support the base of the microscope with your left hand. Never hold the microscope like a bag or in any position other than upright to prevent accidental damage of loose parts

  3. Do not drop the microscope or remove any of its parts. All parts of a microscope are expensive and difficult to procure

  4. Lost or damage parts of the microscope should be reported immediately to your facilitators

  5. Wipe the microscope lenses and its mechanical parts with clean dry lens paper

  6. Gently clean the oil immersion lens (objective marked 100X) with clean lens paper. Xylene is the solvent of choice to clean hardened oil sticking from the Oil Immersion Objective (OIO)

  7. Do not use alcohol for cleaning as some lens adhesives are soluble in alcohol
• Condenser

  The lens located below the stage which collects light and delivers it to the objective
• Stage

  The platform under the objectives where you place the specimen to be viewed
• Arm

  The C-shaped part where the microscope is grasped when it is moved or transported
• Coarse adjustment

  The knob at the body tube used to focus the specimen viewed under the LPO
• Lamp

  A built-in light source for electric microscopes, controlled by an adjustment dial that regulates light intensity and a power switch
• The resolving power of the microscope describes the ability of a lens to gather fine details of the specimen being observed
• Limit of Resolution = 0.61 λ / η sin θ NA (Numerical Aperture)
• The limitations in both magnification and resolving power of the microscope lens prevent seeing very small microbes such as viruses and observing fine details of a cell
• An objective with a higher numerical aperture will have a shorter working distance
• Iris diaphragm

  Regulates the amount of light reaching the specimen, located above the condenser and below the stage
• θ (theta) is half the value of the angular aperture, which affects the working distance of the objective
• λ (lambda) is the wavelength of visible light illuminating the microscope, ranging from ~380 nm to ~700 nm
• Microscope Objectives

  • Low Power Objective (LPO) - marked 10X
  • High Power Objective (HPO) - marked 40X
  • Oil Immersion Objective (OIO) - marked 100X
• Translational control knobs

  Move the slide left or right, and forward or backward
• Fine adjustment

  The knob at the body tube used to focus specimen under the HPO and OIO
• Two tiny objects separated by a distance smaller than the limit of resolution cannot be resolved as two separate objects
• Microorganisms in their Fixed State: Stained Prepared Slides

  1. Secure prepared slides of bacteria, yeasts, molds, algae, and protozoa. Examine them under the microscope using the HPO for molds, yeasts, algae, and protozoa and the oil immersion objective (OIO) for the bacteria

  2. Mount the slide on the stage and check the illumination of the microscopic field by tilting the mirror and opening the iris diaphragm. Get the brightest microscopic field as this will get dimmer once the switch to the OIO is done

  3. Focusing under the OIO takes a little practice and care. With the nosepiece halfway between the HPO and the OIO (100X), apply a small drop of immersion oil
• Microorganisms in their Living State: Wet – Mount Preparation

  1. Place a drop of hay infusion / pond water / yeast cell suspension on a clean glass slide

  2. Carefully cover the drop with a cover slip

  3. Examine the preparation under LPO and HPO. Reduce the amount of light entering the microscope through the lamp control and iris diaphragm, as several live microorganisms are more readily seen in dim light

  4. Identify, illustrate and describe all the microorganisms that you see in the worksheet (Section I.B.)
• Keep fingers off optical surfaces and clean up lenses after a season of oil immersion work using lens paper
• Focusing under the OIO

  With the nosepiece halfway between the HPO and the OIO (100X), apply a small drop of immersion oil on the center of the specimen on the slide. Gently swing the OIO into position over the drop. Refocus by adjusting the fine knob
• Yeasts

  • Eukaryotic microbes that reproduce asexually through budding or fission. Example: Baker’s yeast (Saccharomyces cerevisiae)
• It takes a little practice and care to focus under the OIO
• Microbial Morphology

  • Common shapes and arrangements of bacteria
  • Yeasts
  • Molds
  • Protozoa
  • Algal cells and colonies
• Molds

  • Filamentous fungi composed of hyphae, reproduce asexually by forming spores. Example species: Aspergillus niger, Rhizopus stolonifer, Penicillium notatum
• Basic dyes are used for positive staining because they adhere to negatively-charged intracellular components
• Basic stains adhere to negatively-charged parts of the cell like nucleic acids
• Materials needed for the exercise

  • Cultures
  • Media and Reagents
  • Equipment
• Making smears

  Ensure clean and oil-free glass slides. Degrease slides with acetone. 2. Label the slide with scientific name of organism, staining method, your name, section, and group number. 3. Place water and bacteria on the slide. 4. Prepare bacterial smear
• Acidic dyes are used for negative staining because their negative charge is repelled by the cell’s negatively-charged outer surface thus preventing the dye from entering the cell
• Other common staining techniques include the endospore stain which tests for the presence of highly-resistant, intracellular structures and the acid-fast stain which is used to confirm the presence of tuberculosis and leprosy in clinical samples
• The difference in charge between acidic and basic dyes is used to either color the cells themselves (positive staining) or the background of the cells (negative staining)
• Simple staining increases visibility of cells and allows for the observation of cell morphology and arrangement
• If the slide with the prepared smear was overheated during the heat-fixing step, it might lead to undesired outcomes
• Bacterial smear preparation

  Place two loopfuls of distilled water at the center of the circle on a labeled glass slide. Aseptically touch a colony of the test bacteria and transfer it onto the drop of distilled water. Prepare a bacterial smear by mixing the bacteria with water and spreading it within the marked circular area on the slide. Air-dry the smear to form a translucent film. Heat-fix the smear by passing the slide over an open flame 3 to 5 times
• Air-dried smear should be heat-fixed before proceeding with the staining step
• Gram-positive organisms have thick peptidoglycan layers, while Gram-negative organisms have a relatively thin peptidoglycan layer but a thick lipopolysaccharide layer
• Simple staining

  Prepare a smear. Flood the slide with methylene blue. Drain off the dye after 1 minute and wash off the excess dye with water. Blot dry the slide. View the smear under oil immersion. Take note of the shape of the individual bacterial cells and their arrangement. Try using other stains like crystal violet and safranin
• If a smear contains two different-shaped bacteria after simple staining, it may indicate contamination or a mixed culture
• Bacteria have a relatively thin peptidoglycan layer but have a thick lipopolysaccharide layer