Microscopy and Experiment Controls

Scientific Method Steps

• Ask a question.
• Develop a hypothesis based on observations and previous knowledge.
• Design an experiment to test the hypothesis.

Variables in Experiments

• Variables should be considered in each experiment.
• Dependent Variable: What is measured or observed in response to the independent variable; there can be more than one.
• Independent Variable: Limited to one experimental condition that is manipulated.
• Control Experiment: Used to see the difference in results when the independent variable is omitted or held constant, providing a baseline to evaluate results and confirm the process is working as expected.
• It is not possible to make any conclusions if a control does not work.
• It is best to have both positive and negative controls in every experiment, if at all possible.
• Positive Control: Similar to the experimental test but known to give a positive result, confirming the basic experimental conditions can produce a positive result, even if the samples don't.
• Negative Control: Known to give a negative result, demonstrating the baseline result when a test does not produce a measurable positive result; the value is often subtracted as a "background" value from test sample results.
• The control experiment shows that the experimental process is working as expected. Example:
  • Testing the effects of different doses of a drug (independent variable) on the symptoms and signs (dependent variable) of an illness.
  • Negative Control: Omit the drug to see the effects of the illness without the drug's influence.
  • Positive Control: Give the drug in a concentration previously shown to limit or modify the illness symptoms, giving the expected effect on the illness.
  • The independent variable means giving different doses of the drug (different than the positive control) to see the effect of the drug on the illness dependent on dosage.

Compound Light Microscope (Brightfield)

• Objects appear dark, the field (background) is light.
• Exhibits little contrast.
• Contains two lenses between the eye and the object.
• Requires a light source.
• Carried with one hand on the arm and one hand under the base.
• Base: The bottom part used to carry the microscope.
• Stage: Holds the slide.
• Arm: Used for carrying
• Body/Observation Tube: Transmits the magnified image.
• Light Source: Located in the base.
• Image Inversion: The image appears inverted.
• Condenser Lens: Focuses the light into a cone to concentrate light on the slide.
• Iris Diaphragm: Controls the angle and size of the cone of light.
• Objective Lenses: Rotated to vary magnification; primary lenses that magnify 4x, 10x, 40x, 100x, and 1000x
• Eyepiece/Ocular Lens: Magnifies the image 10x.
• Use the highest power objective for bacteria.
• Coarse Adjustment: Used to focus low power objectives (4x & 10x).
• Fine Adjustment: Used to focus high power and oil immersion lenses.
• Field of View: Area seen through the microscope.
• Scanning: 4x
• Low Power: 10x
• High-Dry: 40x
• Oil Immersion: 100x - requires the use of immersion oil to get adequate resolution.
• Total Magnification: Ocular (10x) x objective lens.
• Always start with the low power objective.
• Lenses are parfocal (the image should remain in focus when changing between objectives).
• Numerical Aperture: Measurement of the lens's ability to gather light and resolve fine detail.
• Resolving Power: Shorter wavelengths of light provide greater resolution, longer wavelengths and white light give lower resolution..
• Refractive Index: The amount the light bends between the objective lens and the slide (changes when using immersion oil).
• Light Refraction: Light bends when passing from glass to air; immersion oil minimizes light loss and enhances resolution.
• Immersion Oil: Reduces refraction; without it, light bends outward between the slide and objective lens.
• Using immersion oil prevents light loss because it is not bent (refracted) from hitting air between the slide and objective lens.
• The focal point is where a clear image is formed.
• Curved lenses create multiple focal points (spherical aberration).
• Adjusting the iris diaphragm reduces spherical aberration, eliminating light rays around the edge.

Phase Contrast Microscope

• Allows contrast between the specimen and the dark field to be seen.
• Used for living, unstained microorganisms, allowing observation of internal structures.

Microscopy Observations

• Brownian Movement: Movement caused by molecules in an aqueous environment.
• True Motility: Microbes actively move from one position to another and change direction, spin, or roll.

Eukaryotes vs. Prokaryotes

• Eukaryotes: Larger and possess membrane-bound nuclei, nucleoli, and organelles and may have complex appendages (arms, legs, tails, etc.); they often have uneven cell division.
• Prokaryotes: Smaller and visible under lower power; they both have cell division,. Bacteria usually undergoes even cell division (binary fission).

Wet Mounts

• Fast and does not require much equipment, but true motility is difficult to observe because they are smashed between the slide and the cover.
• Hanging Drop: Allows observation of microbes in their natural 3D environment and allows motile organisms to move freely.
• A petroleum jelly seal reduces liquid evaporation.

Aseptic Technique

• Practices that keep from contaminating media.
• Use: Test tubes and culture media are only open to introduce or remove something

Sterilization, Inoculation, and Broth Cultures

• Sterilized= Free of all life usually by autoclave; it kills but does not physically remove bacteria.
• Inoculate: Introduce bacteria into growth media.
• Broth Cultures: Allow large numbers of bacteria to grow in a small space, as broth is a liquid with nutrients that allows bacteria to grow.
• Broth is used to quickly grow bacteria in a small space - no competition.

Culture Characteristics

• Turbidity: Cloudiness of the liquid
• Flocculent: Floating chunks.
• Sediment: Cells that float to the bottom.
• Pellicle: Like a floating mat of bacteria or scum layer; may form if bacteria needs lots of oxygen and can protect the bacteria.

Culture Types and Oxygen Requirements

• Agar Slant: Test tube with solid agar at an angle; used for solid growth of bacteria on a surface, easy to store and transport, and used for pure cultures.
• Agar Deep: Test tube with solid agar at the bottom; used with inoculating needle to test bacterial oxygen requirements, and used to grow anaerobic bacteria (requiring less oxygen).
• Semisolid Agar Deep: More jello-like/watery than agar deep; used with an inoculating needle to determine bacterial motility.
• Motile bacteria will look like an upside down christmas tree; bacteria are able to swim away from the inoculation site and may be used to test oxygen requirements.

Transferring Bacteria

• Inoculating Loop: Used to transfer bacteria to agar plates, broths, and slants; always flame the needle using a Bunsen burner flame until it turns red.
• Inoculating Needle: Used to transfer bacteria to agar deep.

Cell Division and Colony Isolation

• Binary Fission: Even cell division.
• Streak Plate Technique: A technique used to grow isolated colonies.

Bacterial Terminology

• Bacterium: Single (one bacterial cell).
• Bacteria: Plural.

Examples of Bacteria Types

• Lactococcus lactis: Gram positive.
• Bacillus subtilis: Gram positive.
• Escherichia coli: Gram negative.

Staining Bacteria Techniques

• Staining bacteria enhances contrast so we can see better detail and resolution.
• Simple Stain: All bacteria are stained with one reagent.
• Chromophore: Colored ion; can be positive, negative, or neutral depending on the dye.
  • Basic Dye: Chromophore is a positive ion (cation).
  • Acidic Dye: Chromophore is a negative ion (anion).
• Direct Stain: Stains the bacteria.
• Negative Stain: Stains the background of the slide, leaving the bacteria in its original form.
• Differential Stains: Bacteria react differently to multiple reagents.
• Structural Stain: Used to identify specific parts.
• Smear: A thin film of bacterial cells on a slide.
• Fix: To attach bacteria to the slide.
• Heat Fix: Bacteria fixed to the slide by passing it through a flame.
• Procedure for Heat Fix & Direct Staining (Simple):
  • Spread a thin bacterial culture over the slide.
  • Dry in air.
  • Pass the slide through the flame to fix.
  • Flood the slide with stain and rinse.
  • Blot dry.
  • Place a drop of oil on the slide and view with the oil immersion lens.
• Chemical Fix: Bacteria are fixed to the slide with 95% methanol for 1 min.
• Autolysis: The rupture of the cell wall due to cellular enzymatic digestion.
• Denature: To unfold or change the conformation of proteins, rendering them inactive.
• Bacteria: With a slightly negatively charged cell wall, they would be attracted to dyes that have a chromophore with a positive ion (basic dye), creating a direct stain.

Gram Staining

• Used to classify and identify bacteria as gram-negative or gram-positive by the color they appear.
  • Pink or Red: Gram-negative
  • Blue or Purple: Gram-positive
• Gram Staining Procedure:
  • Apply primary stain (crystal violet) to the bacteria, which enters the cell and stains all bacteria purple. This pigment is only retained by gram-positive bacteria by the end of the procedure.
  • Apply mordant (gram's iodine), which forms a crystal-violet-iodine complex, iodine binds to the crystal violet chromophores, making a larger molecule that gets trapped inside of thick cell walls of gram positive bacteria.
  • Apply decolorizing agent (ethanol)- This ONLY decolorizes gram-negative bacteria - washes crystal violet iodine complex out of gram-negative bacteria.
  • Apply secondary/counterstain to stain the decolorized bacteria with red safranin. If this last step is omitted, gram-negative bacteria would appear clear.
• Gram Positive Bacteria: Has a THICK peptidoglycan
  • Crystal violet enters through the thick peptidoglycan cell wall.
  • Gram's iodine reacts with crystal violet to form crystal violet iodine complex in the cytoplasm.
  • Cell wall is dehydrated by the alcohol, and the crystal violet iodine stays inside of the cell.
  • Applying safranin ensures no contamination; contamination will be evident if there are two colors (purple-blue and pink-red).
• Gram Negative Bacteria: Has a thin layer of peptidoglycan and an outer membrane layer made from lipopolysaccharides, which this can get dissolved by the decolorizing agent.
  • Gram's iodine reacts with crystal violet to form the crystal violet iodine complex.
  • The decolorizing agent dissolves the outer lipopolysaccharide layer and washes put the crystal violet from the thin peptidoglycan layer.
• The counterstain safranin colors stain the bacteria red since crystal violet has been washed out.
  • Older, dead bacterial cells may not retain crystal violet-iodine-complex (primary stain) because the cells are degraded. If heat fix for too long you will burn bacteria and rupture the cell wall.
  • If the smear is too thick, the reagents may not reach all of the cells.
  • If the dye is left on for too long, you may stain the glass slide and inability to see the bacteria.
  • If you pick up agar, you may have a hard time seeing bacteria.

Microscopy Questions

• Penicillium, a a fungi, was viewed with the low-power objective lens; the total magnification is 100x.
• Streak Plate Method: The streak plate method is a technique used to grow isolated colonies by streaking bacteria on agar plates; this is used to dilute bacteria cells on the surface of the plate so they can grow in colonies.
• Each colony should grow from a single bacterial cell, making them pure cultures.
• The loop should be flamed in between streaks when inoculating a streak plate: true.
• Use of needle to inoculate a broth: false, the needle is used to inoculate agar deeps.
• Match the culture media to the correct function:
  • To determine the motility of bacteria: semisolid agar deep.
  • To store pure cultures: agar slant.
  • Allow a large number of bacteria to grow: broth culture.
  • To test bacterial oxygen requirements: agar deep.

Microscopy Usage

• When using the oil immersion lens, oil should be touching the oil immersion lens and the slide.
• The organism provided should have more than one type of growth visible on the agar slant: false, agar slants are for pure cultures.
• True statements to determining whether an organism is eukaryotic or prokaryotic under the microscope:
  • Eukaryotes have membrane-bound organelles that can be seen under low or high-dry powered objectives.
• Questions regarding hand-washing and bacterial colonies:
  • What type of bacteria was removed? Transient bacteria picked up by our hands and normal flora that is naturally on our bodies.
  • What type of bacteria would you most likely find in section 5? Normal flora.
  • The independence and dependence of bacterial colonies:
    • Hand-washing time and the use of soap (independent).
    • Number of bacterial colonies (dependent).
• How can you tell different bacterial colony types apart when they are growing on an agar plate? Form, margin, surface, color, size, color, margins (edges), and how translucent or opaque they are.
• A "Control" in an Experiment: A sample used as a standard of comparison in a scientific experiment.

Questions cover microscopy techniques, image resolution improvement, and the function of microscope components. Also includes experiment control types: positive, negative, and variable types.