Microbiology Fundamentals: Tools of the Laboratory

The Five I’s of Microbiology

• Inoculation
  • Culture: Growing microorganisms.
  • Medium (plural, media): Nutrients for microbial growth.
  • Inoculum: Small sample of microbes.
  • Inoculation: Introducing an inoculum into media to culture microbes.
  • Clinical specimens from body fluids, discharges, anatomical sites, or diseased tissue.
• Incubation
  • Incubator: Temperature-controlled chamber for microbe multiplication.
  • Temperatures: 20 to 45°C.
  • Atmosphere: Oxygen or carbon dioxide may be required for certain microbes.
  • Microbes grow and multiply, producing visible growth in the media.
• Isolation
  • Process where an individual bacterial cell, when separated on a nutrient surface, forms a colony.
  • Colony: A macroscopic cluster of cells on a solid medium, originating from a single cell.
  • Requires:
  • A medium with a firm surface.
  • A Petri dish.
  • An inoculating loop (streak plate method).
• Inspection and Identification
  • Microbes identified through:
  • Microscopic appearance.
  • Cellular metabolism characterization.
  • Nutrient requirements, growth by-products, enzymes presence, and energy derivation mechanisms.
  • Genetic and immunologic characteristics.

Media

• Culture media may be contained in:
  • Test tubes.
  • Flasks.
  • Petri dishes.
• Media may be inoculated with:
  • Loops.
  • Needles.
  • Pipettes.
  • Swabs.
• Sterile technique is necessary.

Physical States of Media

• Three physical states:
  • Liquid.
  • Semisolid.
  • Solid (that can be converted to liquid).
  • Solid (that cannot be liquefied).
• Agar:
  • Complex polysaccharide isolated from Gelidium.
  • Solid at room temperature.
  • Liquefies at 100°C; solidifies at 42°C.
  • Flexible and moldable.
  • Not a digestible nutrient for most microorganisms.

Chemical Content of Media

• Defined or synthetic:
  • Composition is precisely chemically defined.
  • Contain pure organic and inorganic compounds.
  • Molecular content specified by an exact formula.
• Complex:
  • One or more components is not chemically defined.
  • Contains extracts of animals, plants, or yeasts.
  • Examples: Blood, serum, meat extracts or infusions, milk, yeast extract, soybean digests, and peptone.

Media For Different Purposes

• General-purpose media:
  • Grow a broad spectrum of microbes.
  • Generally complex.
• Enriched media:
  • Contains complex organic substances such as blood, serum, hemoglobin, or special growth factors for the growth of fastidious microbes.
  • Used in the clinical laboratory to encourage growth of pathogens present in low numbers.

Selective Media

• Contains one or more agents that inhibit the growth of certain microbes.
• Important in the primary isolation of a specific microorganism from a sample containing many species.
• Speeds up isolation by suppressing unwanted background organisms and favoring the desired ones.

Differential Media

• Allow multiple types of organisms to grow but display visible differences in how they grow, such as:
  • Variations in colony size or color.
  • Media color changes.
  • Production of gas bubbles.
  • Variations are often from chemicals in the media with which microbes react.

Selective and Differential Media

• A medium can be both selective and differential.
• Example: MacConkey agar: suppresses the growth of some organisms while visually distinguishing the ones that do grow.
  • Dyes are used as differential agents because many are pH indicators that change color in response to the production of an acid or a base.

Miscellaneous Media

• Reducing medium:
  • Contains a substance that absorbs or slows the penetration of oxygen.
  • Important for growing anaerobic bacteria.
• Transport media:
  • Used to maintain and preserve specimens that have to be held for a period of time before clinical analysis.
• Carbohydrate fermentation media:
  • Contains sugars that can be fermented with a pH indicator to show this reaction.

Isolation

• Colony: a macroscopic cluster of cells appearing on a solid medium arising from the multiplication of a single cell.
• Isolation requires:
  • A medium with a firm surface
  • A Petri dish
  • An inoculating loop (streak plate method)
• Streak plate method, loop dilution (pour plate), and spread plate are different methods for isolating bacteria.

Inspection and Identification

• Microbes can be identified through:
  • Microscopic appearance
  • Characterization of cellular metabolism
  • Determination of nutrient requirements, products given off during growth, presence of enzymes, and mechanisms for deriving energy
  • Genetic and immunologic characteristics

Size of Macroscopic versus Microscopic Organisms

• Macroscopic organisms are measured in centimeters (cm) and meters (m).
• Microscopic organisms are measured in millimeters (mm), micrometers (\mum), and nanometers (nm).

Types of Microscopy

• Bright-Field:
  • Widely used; light transmitted through specimen.
  • Denser, more opaque specimens absorb light.
  • Can be used for live, unstained material and preserved, stained material.
• Dark-Field:
  • Modified bright-field microscope with a stop to block light from entering the objective lens.
  • Peripheral light reflected off specimen sides.
  • Brightly illuminated specimens surrounded by a dark field.
  • Used to visualize living cells distorted by drying or staining.
• Phase-Contrast:
  • Takes advantage of cell structure density differences.
  • Transforms subtle light wave changes into light intensity differences.
  • Useful for observing intracellular structures like endospores, granules, organelles, and locomotor structures.
• Fluorescence:
  • Uses ultraviolet (UV) radiation source.
  • Dyes (acridine, fluorescein) emit visible light when bombarded by UV rays.
  • Used in diagnosing infections and pinpointing particular cellular structures.
• Confocal:
  • Uses a laser beam to scan specimen depths and deliver a sharp image focusing on a single plane.
  • Used on fluorescently stained specimens or live unstained cells and tissues.
• Transmission Electron Microscope (TEM)
  • Viewing detailed structure of cells and viruses.
  • Electrons transmitted through thinly sliced (20–100 nm) specimens stained with metals.
  • Darker areas: thicker, denser parts; lighter areas: more transparent, less dense parts.
• Scanning Electron Microscope (SEM)
  • Creates a detailed three-dimensional view.
  • Surface of metal-coated specimen bombarded with electrons.
  • Shower of deflected electrons displayed as an image.

Preparing Specimens for the Microscope

• Mounting a sample on a glass slide.
• Preparation depends on:
  • Condition of the specimen: living or dead.
  • Aims of the examiner: observation, identification, or movement.
  • Type of microscopy available.

Fresh, Living Preparations

• Placed on wet mounts or in hanging drop mounts to observe as near to the natural state as possible
• Cells are suspended in water, broth, or saline to maintain viability and provide a medium for locomotion
• Wet mount:
  • Consists of a drop or two of culture placed on a slide and overlaid with a cover slip
• Hanging drop:
  • A drop of culture is placed in a concave (depression) slide, Vaseline adhesive or sealant, and cover slip are used to suspend the sample

Stains

• Staining is any procedure that applies colored chemicals (dyes) to specimens
  • Basic dyes have a positive charge
  • Acidic dyes have a negative charge
• Bacteria have numerous negatively charged substances and attract basic dyes
• Acidic dyes are repelled by cells

Negative versus Positive Staining

• Positive stain: dye sticks to the specimen and gives it color
• Negative stain: dye does not stick to the specimen but settles some distance from its outer boundary, forming a silhouette
  • Negatively charged cells repel the negatively charged dye and remain unstained
  • Smear is not heat fixed so the distortion and shrinkage of cells is reduced
  • Also used to accentuate a capsule
  • Nigrosin and India ink are used

Simple versus Differential Staining

• Simple stains: only require a single dye and an uncomplicated procedure
  • Cause all the cells in the smear to appear more or less the same color, regardless of type
  • Reveal shape, size, and arrangement
• Differential stains:
  • Use two differently colored dyes: the primary dye and the counterstain
  • Distinguish cell types or parts
  • More complex and require additional chemical reagents to produce the desired reaction

Differential Stains: The Gram Stain

• Developed in 1884 by Hans Christian Gram
• Consists of sequential applications of:
  • Crystal violet (the primary stain)
  • Gram’s iodine (the mordant)
  • An alcohol rinse (decolorizer)
  • Safranin (the counterstain)
• Different results in the Gram stain are due to differences in the structure of the cell wall and how it reacts to the series of reagents applied to the cells
• Remains the universal basis for bacterial classification and identification
• A practical aid in diagnosing infection and guiding drug treatment

Differential Stains: Acid-Fast Stain

• Differentiates acid-fast bacteria (pink) from non-acid-fast bacteria (blue)
• Originated as a method to detect Mycobacterium tuberculosis
  • These bacteria cell walls have a particularly impervious cell wall that holds fast (tightly or tenaciously) to the dye (carbol fuschin) when washed with an acid alcohol decolorizer
• Also used for other medically important bacteria, fungi, and protozoa

Differential Stains: Endospore Stain

• Similar to the acid-fast stain in that a dye is forced by heat into resistant bodies called endospores
• Stain distinguishes between endospores and vegetative cells
• Significant in identifying gram-positive, spore-forming members of the genus Bacillus and Clostridium