Microbiology Tools of the Laboratory Inoculation and Isolation

Microbiology: Tools of the Laboratory

Inoculation and Isolation • Inoculation: producing a culture – Introduce a tiny sample (the inoculums) into a container of nutrient medium • Isolation: separating one species from another – Separating a single bacterial cell from other cells and providing it space on a nutrient surface will allow that cell to grow in to a mound of cells (a colony). – If formed from a single cell, the colony contains cells from just that species.

Figure 3. 2

Streak Plate Method • Streak plate method- small droplet of culture or sample spread over surface of the medium with an inoculating loop – Uses a pattern that thins out the sample and separates the cells Figure 3. 3 a, b

Loop Dilation Method • Loop dilation, or pour plate, method- sample inoculated serially in to a series of liquid agar tues to dilute the number of cells in each successive tubes – Tubes are then poured in to sterile Petri dishes and allowed to solidify Figure 3. 3 c, d

Spread Plate Method • Spread plate method- small volume of liquid, diluted sample pipette on to surface of the medium and spread around evenly by a sterile spreading tool Figure 3. 3 e, f

Media: Providing Nutrients in the Laboratory • At least 500 different types • Contained in test tubes, flasks, or Petri dishes • Inoculated by loops, needles, pipettes, and swabs • Sterile technique necessary • Classification of media – Physical state – Chemical composition – Functional type

Classification of Media by Chemical Content • Synthetic media- compositions are precisely chemically defined • Complex (nonsynthetic) media- if even just one component is not chemically definable

Classification of Media by Function • General purpose media- to grow as broad a spectrum of microbes as possible – Usually nonsynthetic – Contain a mixture of nutrients to support a variety of microbes – Examples: nutrient agar and broth, brain-heart infusion, trypticase soy agar (TSA).

Enriched Media • Enriched media- contain complex organic substances (for example blood, serum, growth factors) to support the growth of fastidious bacteria. Examples: blood agar, Thayer. Martin medium (chocolate agar)

Figure 3. 6

Selective and Differential Media • Selective media- contains one or more agents that inhibit the growth of certain microbes but not others. Example: Mannitol salt agar (MSA), Mac. Conkey agar, Hektoen enteric (HE) agar. • Differential media- allow multiple types of microorganisms to grow but display visible differences among those microorganisms. Mac. Conkey agar can be used as a differential medium as well.

Figure 3. 9

Miscellaneous Media • Reducing media- absorbs oxygen or slows its penetration in the medium; used for growing anaerobes or for determining oxygen requirements • Carbohydrate fermentation media- contain sugars that can be fermented and a p. H indicator; useful for identification of microorganisms • Transport media- used to maintain and preserve specimens that need to be held for a period of time • Assay media- used to test the effectiveness of antibiotics, disinfectants, antiseptics, etc. • Enumeration media- used to count the numbers of organisms in a sample.

Figure 3. 10

Incubation • Incubation: an inoculated sample is placed in an incubator to encourage growth. – Usually in laboratories, between 20° and 40°C. – Can control atmospheric gases as well. – Can visually recognize growth as cloudiness in liquid media and colonies on solid media. – Pure culture- growth of only a single known species (also called axenic) • Usually created by subculture – Mixed culture- holds two or more identified species – Contaminated culture- includes unwanted microorganisms of uncertain identity, or contaminants.

Inspection and Identification • Inspection and identification: Using appearance as well as metabolism (biochemical tests) and sometimes genetic analysis or immunologic testing to identify the organisms in a culture. • Cultures can be maintained using stock cultures • Once cultures are no longer being used, they must be sterilized and destroyed properly.

Figure 3. 2 Microscopes and Magnification. Unaided eye ≥ 200 m Light microscope 200 nm – 10 mm Tick Actual size Scanning electron microscope 10 nm – 1 mm Red blood cells Transmission electron microscope 10 pm – 100 m E. coli bacteria T-even bacteriophages (viruses) Atomic force microscope 0. 1 nm – 10 nm DNA double helix

Units of Measurement • 3 -1 List the metric units of measurement that are used for microorganisms.

Units of Measurement • • 1 µm = 10– 6 m = 10– 3 mm 1 nm = 10– 9 m = 10– 6 mm 1000 nm = 1 µm 0. 001 µm = 1 nm

Light Microscopy • The use of any kind of microscope that uses visible light to observe specimens • Types of light microscopy – Compound light microscopy – Darkfield microscopy – Phase-contrast microscopy – Differential interference contrast microscopy – Fluorescence microscopy – Confocal microscopy

Figure 3. 1 a The compound light microscope. Ocular lens (eyepiece) Remagnifies the image formed by the objective lens Fine focusing knob Coarse focusing knob Body tube Transmits the image from the objective lens to the ocular lens Arm Objective lenses Primary lenses that magnify the specimen Stage Holds the microscope slide in position Condenser Focuses light through specimen Diaphragm Controls the amount of light entering the condenser Illuminator Light source Base Principal parts and functions

Compound Light Microscopy • In a compound microscope, the image from the objective lens is magnified again by the ocular lens • Total magnification = objective lens ocular lens

Compound Light Microscopy • Resolution is the ability of the lenses to distinguish two points • A microscope with a resolving power of 0. 4 nm can distinguish between two points ≥ 0. 4 nm • Shorter wavelengths of light provide greater resolution

Figure 3. 3 Refraction in the compound microscope using an oil immersion objective lens. Oil immersion objective lens Unrefracted light Without immersion oil most light is refracted and lost Immersion oil Air Glass slide Condenser lenses Condenser Iris diaphragm Light source

Brightfield Illumination • Dark objects are visible against a bright background • Light reflected off the specimen does not enter the objective lens

Darkfield Illumination • Light objects are visible against a dark background • Light reflected off the specimen enters the objective lens

Phase-Contrast Microscopy • Accentuates diffraction of the light that passes through a specimen

Fluorescence Microscopy • Uses UV light • Fluorescent substances absorb UV light and emit visible light • Cells may be stained with fluorescent dyes (fluorochromes)

Figure 3. 6 b The principle of immunofluorescence.

Electron Microscopy • Uses electrons instead of light • The shorter wavelength of electrons gives greater resolution

Transmission Electron Microscopy (TEM) • Ultrathin sections of specimens • Light passes through specimen, then an electromagnetic lens, to a screen or film • Specimens may be stained with heavy-metal salts

Transmission Electron Microscopy (TEM) • 10, 000– 100, 000 ; resolution 2. 5 nm

Scanning Electron Microscopy (SEM) • An electron gun produces a beam of electrons that scans the surface of a whole specimen • Secondary electrons emitted from the specimen produce the image

Scanning Electron Microscopy (SEM) • 1, 000– 10, 000 ; resolution 20 nm

Figure 3. 22

Preparing Smears for Staining • Staining: coloring the microbe with a dye that emphasizes certain structures • Smear: a thin film of a solution of microbes on a slide • A smear is usually fixed to attach the microbes to the slide and to kill the microbes

Preparing Smears for Staining • Live or unstained cells have little contrast with the surrounding medium. Researchers do make discoveries about cell behavior by observing live specimens.

Simple Stains • Simple stain: use of a single basic dye • A mordant may be used to hold the stain or coat the specimen to enlarge it

Differential Stains • Used to distinguish between bacteria – Gram stain – Acid-fast stain

Gram Stain • Classifies bacteria into gram-positive or gram-negative – Gram-positive bacteria tend to be killed by penicillin and detergents – Gram-negative bacteria are more resistant to antibiotics

Gram Stain Color of Gram-Positive Cells Color of Gram-Negative Cells Primary Stain: Crystal Violet Purple Mordant: Iodine Purple Decolorizing Agent: Alcohol-Acetone Purple Colorless Counterstain: Safranin Purple Red

Figure 3. 12 b Gram staining. Rod (gram-negative) Cocci (gram-positive)

Special Stains • Used to distinguish parts of cells – Capsule stain – Endospore stain – Flagella stain

Endospore Staining • Primary stain: malachite green, usually with heat • Decolorize cells: water • Counterstain: safranin

Figure 3. 14 b Special staining. Endospore staining