Laboratory Tools in Microbiology LECTURES IN MICROBIOLOGY Laboratory Tools in Microbiology LESSON 3 Sofronio Agustin Professor

Topics Covered Microscopy Staining Techniques Methods of Culturing Microbes

Units of Measurement 1 µm = 10-6 m = 10-3 mm 1 nm = 10-9 m = 10-6 mm

Simple Microscope A simple microscope has only one lens

Parts of a student laboratory microscope Compound Microscope A compound microscope has two sets of lenses and is typically used in teaching and research laboratories. Parts of a student laboratory microscope

Optics : Magnification A specimen is magnified as light passes through the objective and ocular lenses. Total Magnification = objective lens X ocular lens (magnifications) The pathway of light and two stages of magnification of a compound microscope.

Resolution distinguishes magnified objects clearly. Optics: Resolution Resolution is the ability of the lenses to distinguish two points. A microscope with a resolving power of 0.2 um can distinguish between two points > 0.2 um. Resolution distinguishes magnified objects clearly.

Optics: Resolution Resolution can be increased by using immersion oil and shorter wavelengths of light.

Optics: Refraction Refractive index is the light-bending ability of a medium. The light may bend in air so much that it misses the small high-magnification lens. Immersion oil is used to keep light from bending.

Brightfield Microscopy Dark objects are visible against light background. Used to observe stained or unstained specimens. Most commonly used in laboratories.

Darkfield Microscopy Light objects are visible against a dark background. Light reflected off the specimen enters the objective lens. Used to screen for syphilis agent, Treponema pallidum.

Phase-contrast Microscopy Accentuates diffraction of light that passes through the specimen. Used to observe internal cellular detail of live specimens.

Differential Interference Contrast (DIC) Microscopy Accentuates diffraction of light that passes through the specimen. Uses two beams of light to produce more pronounced contrast.

Fluorescence Microscopy Cells are stained with fluorescent dyes called fluorochromes. Uses UV light as energy source. Fluorochromes absorb UV light and emit visible light Fluorescent staining of fresh sample of cheek scrapings

Confocal Microscopy Uses fluorochromes and laser light. The laser illuminates each plane in a specimen to produce a 3-D image.

Electron Microscopy Very high magnification of up to 100,000 X. Transmission electron microscope (TEM) View internal structures of cells Scanning electron microscope (SEM) Three-dimensional images

Transmission Electron Microscopy Transmission Electron Micrograph of (a) a virus and (b) a protozoan.

Scanning Electron Microscopy False-color Scanning Electron Micrograph of Paramecium sp.

Summary of Microscope Types

Optical and Electron Microscopy: A Comparison

TEM and SEM Compared Transmission Electron Microscope Scanning Electron Microscope

Scanning Probe Microscopy Scanning tunneling microscopy uses a metal probe to scan a specimen. Resolution 1/100 of an atom.

Scanning Probe Microscopy Atomic force microscopy uses a metal and diamond probe inserted into the specimen. Produces 3-D images.

Preparation of Specimens for Light Microscopy A thin film of a solution of microbes on a slide is a smear. A smear is usually fixed to attach the microbes to the slide and to kill the microbes.

Stains Positive stains Dye binds to the specimen Negative stains Dye does not bind to the specimen, but rather around the specimen.

Positive and Negative Stains Positive stains are basic dyes (cationic) that bind to negatively charged cells. Negative stains are acidic dyes (anionic) that bind the background.

Positive Stains Simple - One dye Differential Two-different colored dyes Ex. Gram stain Special Emphasize certain cell parts Ex. Capsule stain

Bacterial Stain Types

Simple Stain Use of a single basic dye is called a simple stain. A mordant may be used to hold the stain or coat the specimen to enlarge it.

Differential Stain: Gram Stain The Gram stain classifies bacteria into gram-positive and gram-negative. Gram-positive bacteria tend to be killed by penicillin and detergents. Gram-negative bacteria are more resistant to antibiotics.

Differential Stain: Gram Stain Color of Gram + cells Gram – cells Primary stain: Crystal violet Purple Mordant: Iodine Decolorizing agent: Alcohol-acetone Colorless Counterstain: Safranin Red

Differential Stain: Gram Stain

Differential Stain: Acid Fast Stain Cells that retain a basic stain in the presence of acid-alcohol are called acid-fast. Non–acid-fast cells lose the basic stain when rinsed with acid-alcohol, and are usually counterstained (with a different color basic stain) to see them.

Culture of Microbes Five basic techniques Media Microbial growth

Five Basic Techniques 1. Inoculate 2. Incubate 3. Isolation 4. Inspection 5. Identification

Summary of Laboratory Techniques

Isolation Technique A single visible colony represents a pure culture or single type of bacterium isolated from a mixed culture.

Isolation Methods

Culture Media Classified according to three properties Physical state Chemical composition Functional types

Culture Media: Physical State Liquid media Semi-solid media Solid media

Liquid Media Liquid media are water-based solutions that are generally termed broths, milks and infusions.

Semi-solid Media Semi-solid media contain a low percentage (<1%) of agar, which can be used for motility testing.

Solid Media Solid media contain a high percent (1-5%) of agar, which enables the formation of discrete colonies.

Culture Media: Chemical Composition Synthetic or chemically-defined media Nonsynthetic or complex media

Synthetic Media Synthetic media contain pure organic and inorganic compounds that are chemically defined (i.e. known molecular formula).

Complex Media Complex or enriched media contain ingredients that are not chemically defined or pure (i.e. animal extracts).

Culture Media: Functional Types Enriched media Selective media Differential media

Enriched Media Enriched media are used to grow fastidious bacteria.

Differential and Selective Media Selective media enables one type of bacteria to grow While differential media allows bacteria to show different reactions (i.e. colony color).

Differential Media

Selective Media Mannitol Salt Agar MacConkey Agar

Miscellaneous Media Examples of miscellaneous media are reducing, fermentation and transportation media.

Microbial Growth Incubation Inspection Identification Varied temperatures, atmospheric states Inspection Mixed culture Pure culture Identification Microscopic appearance Maintenance and disposal Stock cultures sterilization