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), MacConkey agar, Hektoen enteric (HE) agar.

• Differential media- allow multiple types of microorganisms to grow but display visible differences among those microorganisms. MacConkey 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 pH 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.

Tick Actual size

Red blood cells

E. coli bacteria

T-even bacteriophages(viruses)

DNA double helix

Unaided eye≥ 200 m

Light microscope200 nm – 10 mm

Scanningelectron

microscope10 nm – 1 mm

Transmissionelectron

microscope10 pm – 100 m

Atomic force microscope

0.1 nm – 10nm

Units of Measurement

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

• 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

Units of Measurement

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

Ocular lens(eyepiece)Remagnifies the image formed by the objective lens

Body tube Transmits the image from the objective lens to the ocular lensArm

Objective lensesPrimary 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

Coarse focusing knob

Base

Fine focusing knob

Principal parts and functions

Figure 3.1a The compound light microscope.

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.

Unrefractedlight

Immersion oil

Condenser

Light sourceIris diaphragm

Condenser lenses

Air

Without immersion oil most light is refracted and lost

Oil immersion objective lens

Glass slide

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 3.6b The principle of immunofluorescence.

• Uses electrons instead of light• The shorter wavelength of electrons gives

greater resolution

Electron Microscopy

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

• 10,000–100,000; resolution 2.5 nm

Transmission Electron Microscopy (TEM)

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

• 1,000–10,000; resolution 20 nm

Scanning Electron Microscopy (SEM)

Figure 3.22

• 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.

Preparing Smears for Staining

• Simple stain: use of a single basic dye• A mordant may be used to hold the stain or

coat the specimen to enlarge it

Simple Stains

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

Color of Gram-Positive Cells

Color ofGram-Negative Cells

Primary Stain:Crystal Violet Purple Purple

Mordant:Iodine Purple Purple

Decolorizing Agent:Alcohol-Acetone Purple Colorless

Counterstain:Safranin Purple Red

Gram Stain

Figure 3.12b Gram staining.

Cocci(gram-positive)

Rod(gram-negative)

Special Stains

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

• Primary stain: malachite green, usually with heat

• Decolorize cells: water• Counterstain: safranin

Endospore Staining

Figure 3.14b Special staining.

Endospore

Endospore staining