Laboratory Tools in MicrobiologyLaboratory Tools in Microbiology

Sofronio Agustin

Professor

Sofronio Agustin

Professor

LECTURES IN

MICROBIOLOGY

LECTURES IN

MICROBIOLOGY

LESSON 3LESSON 3

Topics CoveredTopics Covered

Microscopy

Staining Techniques

Methods of Culturing Microbes

Units of MeasurementUnits 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

Simple MicroscopeSimple Microscope

A simple microscope has only one lens

Compound MicroscopeCompound 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 : MagnificationOptics : 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.

Optics: ResolutionOptics: 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: ResolutionOptics: Resolution

Resolution can be increased

by using immersion oil and

shorter wavelengths of light.

Optics: RefractionOptics: 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 MicroscopyBrightfield Microscopy

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

Darkfield MicroscopyDarkfield 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 MicroscopyPhase-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

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 MicroscopyFluorescence 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 MicroscopyConfocal Microscopy

Uses fluorochromes and laser light.

The laser illuminates each

plane in a specimen to produce

a 3-D image.

Electron MicroscopyElectron 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 MicroscopyTransmission Electron Microscopy

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

Scanning Electron MicroscopyScanning Electron Microscopy

False-color Scanning Electron Micrograph of Paramecium sp.

Summary of Microscope TypesSummary of Microscope Types

Optical and Electron Microscopy:A Comparison

Optical and Electron Microscopy:A Comparison

TEM and SEM ComparedTEM and SEM Compared

Scanning Electron Microscope Transmission Electron Microscope

Scanning Probe MicroscopyScanning Probe Microscopy

Scanning tunneling microscopy uses a metal probe to scan a specimen.

Resolution 1/100 of

an atom.

Scanning Probe MicroscopyScanning Probe Microscopy

Atomic force

microscopy uses a

metal and diamond

probe inserted into

the specimen.

Produces 3-D

images.

Preparation of Specimens for Light MicroscopyPreparation 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.

StainsStains

Positive stains

Dye binds to the specimen

Negative stains

Dye does not bind to the specimen, but rather

around the specimen.

Positive and Negative StainsPositive 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 StainsPositive Stains

Simple- One dye

Differential- Two-different colored

dyes

Ex. Gram stain Special

- Emphasize certain cell partsEx. Capsule stain

Bacterial Stain TypesBacterial Stain Types

Simple StainSimple 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 StainDifferential 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 StainDifferential Stain: Gram Stain

Color of

Gram + cells

Color of

Gram – cells

Primary stain:

Crystal violet

Purple Purple

Mordant:

Iodine

Purple Purple

Decolorizing agent:

Alcohol-acetone

Purple Colorless

Counterstain:

Safranin

Purple Red

Differential Stain: Gram StainDifferential Stain: Gram Stain

Differential Stain: Acid Fast StainDifferential 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 MicrobesCulture of Microbes

Five basic techniques

Media

Microbial growth

Five Basic TechniquesFive Basic Techniques

1. Inoculate

2. Incubate

3. Isolation

4. Inspection

5. Identification

Summary of Laboratory TechniquesSummary of Laboratory Techniques

Isolation TechniqueIsolation Technique

A single visible colony

represents a pure

culture or single type of

bacterium isolated from

a mixed culture.

Isolation MethodsIsolation Methods

Culture MediaCulture Media

Classified according to three properties Physical state

Chemical composition

Functional types

Culture Media: Physical StateCulture Media: Physical State

Liquid media Semi-solid media Solid media

Liquid MediaLiquid Media

Liquid media are water-

based solutions that are

generally termed broths,

milks and infusions.

Semi-solid MediaSemi-solid Media

Semi-solid media contain a

low percentage (<1%) of agar,

which can be used for motility

testing.

Solid MediaSolid Media

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

Culture Media: Chemical CompositionCulture Media: Chemical Composition

Synthetic or chemically-defined media Nonsynthetic or complex media

Synthetic MediaSynthetic Media

Synthetic media contain

pure organic and

inorganic compounds

that are chemically

defined (i.e. known

molecular formula).

Complex MediaComplex Media

Complex or enriched

media contain

ingredients that are not

chemically defined or

pure (i.e. animal

extracts).

Culture Media: Functional TypesCulture Media: Functional Types

Enriched media

Selective media

Differential media

Enriched MediaEnriched Media

Enriched media are

used to grow

fastidious bacteria.

Differential and Selective MediaDifferential 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 MediaDifferential Media

Selective MediaSelective Media

Mannitol Salt Agar

MacConkey Agar

Miscellaneous MediaMiscellaneous Media

Examples of

miscellaneous media

are reducing,

fermentation and

transportation media.

Microbial GrowthMicrobial Growth

• Incubation Varied temperatures, atmospheric states

• Inspection Mixed culture Pure culture

• Identification Microscopic appearance

• Maintenance and disposal Stock cultures sterilization