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TOOLS OF THE LABORATORY

Chapter 3 Module 1

  • Images from McGraw-Hill unless otherwise linked to source.

HOW DO YOU STUDY SOMETHING YOU CAN'T SEE? FIG 3.1

  • Use the Six I's of Microbiology to investigate microorganisms.

1. IDENTIFICATION

  • Goal: Attach a name or identity to the microbe.

  • Process: Accomplished through the use of keys, charts, and computer programs that analyze the data to arrive at a final conclusion.

2. INFORMATION GATHERING

  • Purpose: Additional tests for microbial function and characteristics.

  • Methods:

    • Inoculations into specialized media to determine biochemical traits.

    • Immunological testing.

    • Genetic typing.

  • Results provide specific information unique to certain microbes.

3. INOCULATION

  • Definition: The sample is placed into a medium that supports its growth.

  • Types of Medium: May be solid or liquid, held in tubes, plates, flasks, or even eggs.

  • Delivery Tools: Typically a loop, needle, swab, or syringe.

    • Example: Streak plate methods.

4. SPECIMEN COLLECTION

  • Timing: Conducted before proceeding with the six I's.

  • What is Collected: Nearly any object of interest can be sampled, including common sources like:

    • Body fluids

    • Foods

    • Water

    • Soil

    • Plants and animals

    • Unique locations such as icebergs, volcanoes, and rocks.

  • Purpose: Collect samples that contain microbes of interest.

5. INCUBATION

  • Definition: Inoculated media placed in a controlled environment (incubator) promote growth.

  • Outcome: The microorganisms multiply, resulting in the development of a culture.

  • Purpose: Generates a culture to provide higher quantities for testing.

  • Tools Used: Incubator.

6. ISOLATION

  • Definition: Separating individual microbes.

  • Purpose: To create a pure culture.

  • Methods:

    • Specimen inoculated on solid media to create isolated colonies.

    • An isolated colony is then inoculated into fresh media.

ISOLATION TECHNIQUES

  • Streak Plate:

    • A small droplet of a sample is spread across four quadrants, gradually thinning out the sample.

  • Loop Dilution/Pour Plate:

    • Serial dilution using agar in test tubes; the sample is mixed and then poured onto plates to reduce the number of microbes present.

  • Spread Plate:

    • A small volume of diluted sample is transferred to a plate and evenly spread on the surface.

INSPECTION OF MICROORGANISM

  • Definition: Observing cultures visually for growth and examining them microscopically for the appearance of cells.

  • Purpose: To analyze initial physical characteristics of microbial cultures.

  • Tools Used: Microscope.

INFORMATION GATHERING OF MICROORGANISM

  • Definition: Testing of cultures to develop an overall profile of the microbes.

  • Purpose: To provide specific information unique to a certain microbe.

  • Methods:

    • Biochemical tests.

    • Immunological testing.

    • Drug sensitivity tests.

    • Genetic typing.

IDENTIFICATION OF MICROORGANISM

  • Definition: Analysis of collected data to identify the microbe(s) in the original sample.

  • Purpose: Lays the groundwork for future research and applications in other fields of microbiology.

  • Methods: Utilize physical characteristics, data from biochemical and immunological tests, and genetic information.

THE MICROSCOPE

Key Characteristics

  • Magnification: Ability to enlarge objects, typically using light and curved lenses.

  • Resolving Power: Ability to show detail by separating or distinguishing small or closely adjacent objects.

PARTS OF THE MICROSCOPE

  • Components:

    • Ocular lens

    • Revolving nosepieces

    • Objective lens

    • Stage

    • Stage clamps

    • Condenser

    • Diaphragm

    • Light source

    • Coarse focus

    • Fine focus

MAGNIFICATION IN TWO PHASES

  1. The objective lens forms a magnified real image.

  2. This real image is projected to the ocular lens where it is magnified again to form a virtual image.

TOTAL MAGNIFICATION

  • Equation: Total magnification of the final image is the product of the separate magnifying powers of the two lenses:
    exttotalmagnification=extobjectivepowerimesextocularpowerext{total magnification} = ext{objective power} imes ext{ocular power}

THE PURPOSE OF OIL

  • Function: Increases the resolving power of the microscope by replacing the air gap with a high refractive index oil to reduce light refraction, allowing more light to travel through the specimen.

FIELD OF VIEW

  • Concept: "Everything the light touches"

  • Observation: As you increase magnification, you decrease the field of view.

CONTRAST AND STAINING

  • Challenge: Microbes are colorless.

  • Solution: Specimens must be stained with a colored dye to visualize them.

  • Function of Staining: Increases contrast between the specimen and the slide background allowing better visualization of microorganisms.

  • Definition of Contrast: The ability to distinguish the specimen from the background.

STAINING EXAMPLES

  1. Simple Stains: One dye is used; reveals shape, size, and arrangement.

  2. Negative Stain: Stains the area around the microorganism.

  3. Acidic Dyes: Anionic, negatively charged chromophores used for staining. Negative staining occurs when the microbe repels the dye, and the dye stains the background.

  4. Differential Stains: Use a primary stain and a counterstain to distinguish cell types or parts (e.g., Gram stain, acid-fast stain, and endospore stain).

  5. Structural Stains: Reveal specific cell parts not displayed by conventional methods, such as capsule and flagellar stains.

STAINING EXAMPLES - VISUALS

  • Gram Stain:

    • Purple cells are gram-positive.

    • Red cells are gram-negative.

  • Acid-Fast Stain:

    • Red rods are acid-fast Mycobacterium.

VARIATIONS ON THE OPTICAL MICROSCOPE

  1. Bright-field: Most widely used; specimen appears darker than surrounding field. Used for live, unstained, and preserved/stained specimens.

  2. Dark-field: Brightly illuminated specimens surrounded by a dark field; suitable for live and unstained specimens.

  3. Phase-contrast: Transforms light waves changes into differences in light intensity, best for observing intracellular structures.

FLUORESCENCE MICROSCOPE

  • Description: Modified microscope with an ultraviolet radiation source and filter.

  • Dyes: Uses dyes that emit visible light when bombarded with shorter UV rays.

  • Application: Useful in diagnosing infections.

SCANNING CONFOCAL MICROSCOPE

  • Functionality: Uses a laser beam of light to scan the specimen.

  • Benefit: Integrates images to allow focus on multiple depths or planes.

SPECIMEN PREPARATION FOR OPTICAL MICROSCOPES

  1. Wet mounts and hanging drop mounts: Allow examination of live cell characteristics such as size, motility, shape, and arrangement.

  2. Fixed mounts: Made by drying and heating a film of the specimen, which is then stained to visualize cells or their parts.

ELECTRON MICROSCOPY

  • Definition: Forms an image with a beam of electrons that travel in wavelike patterns when accelerated to high speeds.

  • Comparison: Electron waves are 100,000 times shorter than visible light waves.

  • Power: Electrons have significant power to resolve minute structures due to their wavelength.

  • Magnification Range: Between 5,000x and 1,000,000x.

TYPES OF ELECTRON MICROSCOPES

  1. Transmission Electron Microscopes (TEM): Transmit electrons through the specimen; indicates specimen thickness with light and dark contrasts.

  2. Scanning Electron Microscopes (SEM): Provide a three-dimensional view and show the surface of specimens in extreme detail.

MEDIA: PROVIDING NUTRIENTS IN THE LABORATORY

  • Media can be classified based on three properties:

    • Physical state:

    • Liquid: Broth; does not solidify.

    • Semisolid: Contains solidifying agent.

    • Solid: Firm surface for colony formation.

    • Chemical composition:

    • Synthetic: Pure organic and inorganic compounds in an exact formula.

    • Complex: At least one ingredient that is not chemically definable.

    • Functional type:

    • General purpose, enriched, selective, differential, anaerobic, transport, assay, enumeration.

PHYSICAL STATES OF MEDIA

  1. Liquid: Broth; does not solidify.

  2. Semisolid: Contains a solidifying agent.

  3. Solid: Firm surface for colony formation; contains a solidifying agent. Can be liquefiable or non-liquefiable.

AGAR

  • Properties:

    • Most commonly used solidifying agent in microbiology.

    • Solid at room temperature but liquefies above 100°C, re-solidifying below 42°C.

    • Provides a framework for holding moisture and nutrients; most microbes do not digest it.

MOST COMMONLY USED MEDIA

  • Nutrient Broth: Liquid medium containing beef extract and peptone.

  • Nutrient Agar: Solid media containing beef extract, peptone, and agar.

CHEMICAL CONTENT OF MEDIA

  1. Synthetic: Contains pure organic and inorganic compounds in an exact chemical formula.

  2. Complex: Contains at least one ingredient that is not chemically definable.

  3. General Purpose Media: Grows a broad range of microbes, typically nonsynthetic.

  4. Enriched Media: Contains complex organic substances (e.g., blood, serum, hemoglobin) or special growth factors required by fastidious microbes.

SELECTIVE & DIFFERENTIAL MEDIA

  • Selective Media: Contains ingredients that inhibit growth of some microbes while promoting others.

  • Differential Media: Allows growth of several types of microbes with visible differences among them.

  • Transport Media: Used to maintain and preserve specimens before clinical analysis.

EXAMPLES OF SELECTIVE AND DIFFERENTIAL MEDIA

  • Triple Sugar Iron Agar (TSIA):

    1. Uninoculated.

    2. Acid production (red to yellow change).

    3. No reaction.

    4. Acid production only in the butt (yellow butt).

    5. Acid production throughout with hydrogen sulfide (black) precipitation in the butt.

    6. Alkaline reaction with hydrogen sulfide precipitation.

  • CHROMagar Orientation Media: Used to identify common urinary pathogens with color-forming reactions for at least seven species, facilitating rapid identification and treatment.

MISCELLANEOUS MEDIA

  • Carbohydrate Fermentation Medium: Contains fermentable sugars and a pH indicator to show acid production resulting from fermentation.