Microbiology: A Microbiology: A Systems Approach, 2Systems Approach, 2ndnd

ed.ed.Chapter 3: Tools of the Chapter 3: Tools of the

LaboratoryLaboratory

3.1 Methods of Culturing 3.1 Methods of Culturing Microorganisms: The Five I’sMicroorganisms: The Five I’s

Microbiologists use five basic techniques Microbiologists use five basic techniques to manipulate, grow, examine, and to manipulate, grow, examine, and characterize microorganisms in the characterize microorganisms in the laboratory: inoculation, incubation, laboratory: inoculation, incubation, isolation, inspection, and identificationisolation, inspection, and identification

Figure 3.1

Inoculation and IsolationInoculation and Isolation

InoculationInoculation: producing a culture: producing a culture Introduce a tiny sample (the inoculums) into a Introduce a tiny sample (the inoculums) into a

container of nutrient container of nutrient medium medium

IsolationIsolation: separating one species from another: separating one species from another Separating a single bacterial cell from other cells Separating a single bacterial cell from other cells

and providing it space on a nutrient surface will and providing it space on a nutrient surface will allow that cell to grow in to a mound of cells (a allow that cell to grow in to a mound of cells (a colonycolony). ).

If formed from a single cell, the colony contains cells If formed from a single cell, the colony contains cells from just that species.from just that species.

Figure 3.2

Streak Plate MethodStreak Plate Method Streak plate method- small droplet of culture Streak plate method- small droplet of culture

or sample spread over surface of the medium or sample spread over surface of the medium with an inoculating loopwith an inoculating loop Uses a pattern that thins out the sample and Uses a pattern that thins out the sample and

separates the cellsseparates the cells

Figure 3.3 a,b

Loop Dilation MethodLoop Dilation Method Loop dilation, or pour plate, method- sample Loop dilation, or pour plate, method- sample

inoculated serially in to a series of liquid agar tues to inoculated serially in to a series of liquid agar tues to dilute the number of cells in each successive tubesdilute the number of cells in each successive tubes Tubes are then poured in to sterile Petri dishes and Tubes are then poured in to sterile Petri dishes and

allowed to solidifyallowed to solidify

Figure 3.3 c,d

Spread Plate MethodSpread Plate Method

• Spread plate method- small volume of liquid, diluted Spread plate method- small volume of liquid, diluted sample pipette on to surface of the medium and spread sample pipette on to surface of the medium and spread around evenly by a sterile spreading toolaround evenly by a sterile spreading tool

Figure 3.3 e,f

Media: Providing Nutrients in the Media: Providing Nutrients in the LaboratoryLaboratory

At least 500 different typesAt least 500 different types Contained in test tubes, flasks, or Petri dishesContained in test tubes, flasks, or Petri dishes Inoculated by loops, needles, pipettes, and Inoculated by loops, needles, pipettes, and

swabsswabs Sterile technique necessarySterile technique necessary Classification of mediaClassification of media

Physical statePhysical state Chemical compositionChemical composition Functional typeFunctional type

Classification of Media by Physical Classification of Media by Physical StateState

Liquid media: water-based solutions, do not solidify at Liquid media: water-based solutions, do not solidify at temperatures above freezing, flow freely when temperatures above freezing, flow freely when container is tiltedcontainer is tilted Broths, milks, or infusionsBroths, milks, or infusions Growth seen as cloudiness or particulatesGrowth seen as cloudiness or particulates

Semisolid media: clotlike consistency at room Semisolid media: clotlike consistency at room temperaturetemperature Used to determine motility and to localize reactions at a Used to determine motility and to localize reactions at a

specific sitespecific site Solid media: a firm surface on which cells can form Solid media: a firm surface on which cells can form

discrete coloniesdiscrete colonies Liquefiable and nonliquefiableLiquefiable and nonliquefiable Useful for isolating and culturing bacteria and fungiUseful for isolating and culturing bacteria and fungi

Figure 3.4

Classification of Media by Chemical Classification of Media by Chemical ContentContent

Synthetic media- compositions are Synthetic media- compositions are precisely chemically definedprecisely chemically defined

Complex (nonsynthetic) media- if even just Complex (nonsynthetic) media- if even just one component is not chemically definableone component is not chemically definable

Classification of Media by Classification of Media by FunctionFunction

General purpose media- to grow as broad General purpose media- to grow as broad a spectrum of microbes as possiblea spectrum of microbes as possible Usually nonsyntheticUsually nonsynthetic Contain a mixture of nutrients to support a Contain a mixture of nutrients to support a

variety of microbesvariety of microbes Examples: nutrient agar and broth, brain-Examples: nutrient agar and broth, brain-

heart infusion, trypticase soy agar (TSA).heart infusion, trypticase soy agar (TSA).

Enriched MediaEnriched Media

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

Figure 3.6

Selective and Differential MediaSelective and Differential Media

Selective media- Selective media- contains one or more contains one or more agents that inhibit the growth of certain agents that inhibit the growth of certain microbes but not others.microbes but not others. Example: Mannitol Example: Mannitol salt agar (MSA), MacConkey agar, Hektoen salt agar (MSA), MacConkey agar, Hektoen enteric (HE) agar.enteric (HE) agar.

Differential media-Differential media- allow multiple types of allow multiple types of microorganisms to grow but display visible microorganisms to grow but display visible differences among those microorganisms. differences among those microorganisms. MacConkey agar can be used as a differential MacConkey agar can be used as a differential medium as well.medium as well.

Figure 3.7

Figure 3.8

Figure 3.9

Miscellaneous MediaMiscellaneous Media Reducing media- absorbs oxygen or slows its Reducing media- absorbs oxygen or slows its

penetration in the medium; used for growing penetration in the medium; used for growing anaerobes or for determining oxygen requirementsanaerobes or for determining oxygen requirements

Carbohydrate fermentation media- contain sugars Carbohydrate fermentation media- contain sugars that can be fermented and a pH indicator; useful that can be fermented and a pH indicator; useful for identification of microorganismsfor identification of microorganisms

Transport media- used to maintain and preserve Transport media- used to maintain and preserve specimens that need to be held for a period of timespecimens that need to be held for a period of time

Assay media- used to test the effectiveness of Assay media- used to test the effectiveness of antibiotics, disinfectants, antiseptics, etc. antibiotics, disinfectants, antiseptics, etc.

Enumeration media- used to count the numbers of Enumeration media- used to count the numbers of organisms in a sample.organisms in a sample.

Figure 3.10

IncubationIncubation IncubationIncubation: an inoculated sample is placed in an : an inoculated sample is placed in an

incubator to encourage growth.incubator to encourage growth. Usually in laboratories, between 20Usually in laboratories, between 20°° and 40°C. and 40°C. Can control atmospheric gases as well.Can control atmospheric gases as well. Can visually recognize growth as cloudiness in liquid Can visually recognize growth as cloudiness in liquid

media and colonies on solid media.media and colonies on solid media. Pure culturePure culture- growth of only a single known species - growth of only a single known species

(also called (also called axenicaxenic))• Usually created by Usually created by subculturesubculture

Mixed culture- Mixed culture- holds two or more identified speciesholds two or more identified species Contaminated culture-Contaminated culture- includes unwanted includes unwanted

microorganisms of uncertain identity, or microorganisms of uncertain identity, or contaminantscontaminants. .

Inspection and IdentificationInspection and Identification

• Inspection and identification: Using appearance Inspection and identification: Using appearance as well as metabolism (biochemical tests) and as well as metabolism (biochemical tests) and sometimes genetic analysis or immunologic sometimes genetic analysis or immunologic testing to identify the organisms in a culture.testing to identify the organisms in a culture.

Cultures can be maintained using stock Cultures can be maintained using stock culturescultures

Once cultures are no longer being used, Once cultures are no longer being used, they must be sterilized and destroyed they must be sterilized and destroyed properly.properly.

3.2 The Microscope: Window on 3.2 The Microscope: Window on an Invisible Realman Invisible Realm

Two key characteristics of microscopes: Two key characteristics of microscopes: magnification and resolving powermagnification and resolving power

MagnificationMagnification Results when visible light waves pass through a Results when visible light waves pass through a

curved lenscurved lens The light experiences The light experiences refractionrefraction An image is formed by the refracted light when an An image is formed by the refracted light when an

object is placed a certain distance from the lens object is placed a certain distance from the lens and is illuminated with lightand is illuminated with light

The image is enlarged to a particular degree- the The image is enlarged to a particular degree- the power of magnificationpower of magnification

Figure 3.13

Principles of Light MicroscopyPrinciples of Light Microscopy

Magnification- occurs Magnification- occurs in two phasesin two phases Objective lens- forms Objective lens- forms

the the real imagereal image Ocular lens- forms the Ocular lens- forms the

virtual imagevirtual image Total power of Total power of

magnification- the magnification- the product of the power product of the power of the objective and of the objective and the power of the ocularthe power of the ocular

ResolutionResolution

Resolution- the ability to distinguish two adjacent objects or Resolution- the ability to distinguish two adjacent objects or points from one anotherpoints from one another

Also known as Also known as resolving powerresolving power Resolving power (RP) = Resolving power (RP) = Wavelength of light in nm Wavelength of light in nm

2 x Numerical aperture of 2 x Numerical aperture of objective lensobjective lens

Resolution distance= Resolution distance= 0.61 x wavelength of light in nm0.61 x wavelength of light in nm

Numerical aperture of objective Numerical aperture of objective lens lens Shorter wavelengths provide a better resolutionShorter wavelengths provide a better resolution Numerical apertureNumerical aperture- describes the relative efficiency of a lens in - describes the relative efficiency of a lens in

bending light raysbending light rays Oil immersion lenses increase the numerical aperture Oil immersion lenses increase the numerical aperture

Figure 3.15

Figure 3.16

Magnification and ResolutionMagnification and Resolution

Increased magnification decreases the Increased magnification decreases the resolutionresolution

• Adjusting the amount of light entering the Adjusting the amount of light entering the condenser using an adjustable iris condenser using an adjustable iris diaphragm or using special dyes help diaphragm or using special dyes help increase resolution at higher increase resolution at higher magnificationsmagnifications

Figure 3.17

Variations on the Optical Variations on the Optical MicroscopeMicroscope

Visible light microscopes- optical Visible light microscopes- optical microscopes that use visible light. microscopes that use visible light. Described by their field.Described by their field. Four types: bright-field, dark-field, phase-Four types: bright-field, dark-field, phase-

contrast, and interferencecontrast, and interference Other light microscopes include Other light microscopes include

fluorescence microscopes and confocal fluorescence microscopes and confocal microscopesmicroscopes

Bright-Field MicroscopyBright-Field Microscopy

Most widely usedMost widely used Forms its image when light is transmitted Forms its image when light is transmitted

through the specimenthrough the specimen The specimen produces an image that is The specimen produces an image that is

darker than the surrounding illuminated darker than the surrounding illuminated fieldfield

Can be used with live, unstained and Can be used with live, unstained and preserved, stain specimenspreserved, stain specimens

Dark-Field MicroscopyDark-Field Microscopy

A bright-field microscope can be adapted to a dark-A bright-field microscope can be adapted to a dark-field microscope by adding a stop to the condenserfield microscope by adding a stop to the condenser

The stop blocks all light from entering the objective The stop blocks all light from entering the objective lens except for peripheral lightlens except for peripheral light

The specimen produces an image that is brightly The specimen produces an image that is brightly illuminated against a dark fieldilluminated against a dark field

Effective for visualizing living cells that would be Effective for visualizing living cells that would be distorted by drying or heat or that can’t be stained distorted by drying or heat or that can’t be stained with usual methodswith usual methods

Does not allow for visualization of fine internal Does not allow for visualization of fine internal details of cellsdetails of cells

Phase-Contrast MicroscopyPhase-Contrast Microscopy

Transforms subtle changes in light waves Transforms subtle changes in light waves passing through a specimen into passing through a specimen into differences in light intensitydifferences in light intensity

Allows differentiation of internal Allows differentiation of internal components of live, unstained cellscomponents of live, unstained cells

Useful for viewing intracellular structures Useful for viewing intracellular structures such as bacterial spores, granules, and such as bacterial spores, granules, and organellesorganelles

Figure 3.18

Interference MicroscopyInterference Microscopy

Interference MicroscopyInterference Microscopy Uses a differential-interference contrast (DIC) Uses a differential-interference contrast (DIC)

microscopemicroscope Allows for detailed view of live, unstained Allows for detailed view of live, unstained

specimensspecimens Includes two prisms that add contrasting Includes two prisms that add contrasting

colors to the imagecolors to the image The image is colorful and three-dimensionalThe image is colorful and three-dimensional

Figure 3.19

Fluorescence MicroscopyFluorescence Microscopy

Includes a UV radiation source and a filter Includes a UV radiation source and a filter that protects the viewer’s eyesthat protects the viewer’s eyes

Used with dyes that show Used with dyes that show fluorescencefluorescence under UV raysunder UV rays

Forms a colored image against a black fieldForms a colored image against a black field Used in diagnosing infections caused by Used in diagnosing infections caused by

specific bacteria, protozoans, and viruses specific bacteria, protozoans, and viruses using fluorescent antibodiesusing fluorescent antibodies

Figure 3.20

Confocal MicroscopyConfocal Microscopy

Allows for viewing cells at higher Allows for viewing cells at higher magnifications using a laser beam of light magnifications using a laser beam of light to scan various depths in the specimento scan various depths in the specimen

Most often used on fluorescently stained Most often used on fluorescently stained specimensspecimens

Figure 3.21

Electron MicroscopyElectron Microscopy Originally developed for studying nonbiological Originally developed for studying nonbiological

materialsmaterials Biologists began using it in the early 1930sBiologists began using it in the early 1930s Forms an image with a beam of electrons Forms an image with a beam of electrons

Electrons travel in wavelike patterns 1,000 times Electrons travel in wavelike patterns 1,000 times shorter than visible light wavesshorter than visible light waves

This increases the resolving power tremendouslyThis increases the resolving power tremendously Magnification can be extremely high (between Magnification can be extremely high (between

5,000X and 1,000,000X for biological specimens)5,000X and 1,000,000X for biological specimens) Allows scientists to view the finest structure of cellsAllows scientists to view the finest structure of cells Two forms: transmission electron microscope Two forms: transmission electron microscope

(TEM) and scanning electron microscope (SEM)(TEM) and scanning electron microscope (SEM)

TEMTEM

Often used to view structures of cells and Often used to view structures of cells and virusesviruses

Electrons are transmitted through the Electrons are transmitted through the specimenspecimen

The specimen must be very thin (20-100 The specimen must be very thin (20-100 nm thick) and stained to increase image nm thick) and stained to increase image contrastcontrast

Dark areas of a TEM image represent Dark areas of a TEM image represent thicker or denser partsthicker or denser parts

Figure 3.22

SEMSEM

Creates an extremely detailed three-dimensional Creates an extremely detailed three-dimensional view of all kinds of objectsview of all kinds of objects

Electrons bombard the surface of a whole metal-Electrons bombard the surface of a whole metal-coated specimencoated specimen

Electrons deflected from the surface are picked Electrons deflected from the surface are picked up by a sophisticated detectorup by a sophisticated detector

The electron pattern is displayed as an image on The electron pattern is displayed as an image on a television screena television screen

Contours of specimens resolved with SEM are Contours of specimens resolved with SEM are very revealing and surprisingvery revealing and surprising

Figure 3.23

Preparing Specimens for Optical Preparing Specimens for Optical MicroscopesMicroscopes

Generally prepared by mounting a sample Generally prepared by mounting a sample on a glass slideon a glass slide

How the slide is prepared depends onHow the slide is prepared depends on The condition of the specimen (living or The condition of the specimen (living or

preserved)preserved) The aims of the examiner (to observe overall The aims of the examiner (to observe overall

structure, identify microorganisms, or see structure, identify microorganisms, or see movement)movement)

The type of microscopy availableThe type of microscopy available

Living PreparationsLiving Preparations

Wet mounts or hanging drop mountsWet mounts or hanging drop mounts Wet mount:Wet mount:

Cells suspended in fluid, a drop or two of the Cells suspended in fluid, a drop or two of the culture is then placed on a slide and overlaid with a culture is then placed on a slide and overlaid with a cover glasscover glass

Cover glass can damage larger cells and might dry Cover glass can damage larger cells and might dry or contaminate the observer’s fingersor contaminate the observer’s fingers

Hanging drop mount:Hanging drop mount: Uses a depression slide, Vaseline, and coverslipUses a depression slide, Vaseline, and coverslip The sample is suspended from the coverslipThe sample is suspended from the coverslip

Figure 3.24

Fixed, Stained SmearsFixed, Stained Smears Smear technique developed by Robert KochSmear technique developed by Robert Koch

Spread a thin film made from a liquid suspension of Spread a thin film made from a liquid suspension of cells and air-drying itcells and air-drying it

Heat the dried smear by a process called heat fixation Heat the dried smear by a process called heat fixation Some cells are fixed using chemicals Some cells are fixed using chemicals

Staining creates contrast and allows features of Staining creates contrast and allows features of the cells to stand outthe cells to stand out Applies colored chemicals to specimensApplies colored chemicals to specimens Dyes become affixed to the cells through a chemical Dyes become affixed to the cells through a chemical

reactionreaction Dyes are classified as basic (cationic) dyes, or acidic Dyes are classified as basic (cationic) dyes, or acidic

(anionic) dyes.(anionic) dyes.

Positive and Negative StainingPositive and Negative Staining

Positive stainingPositive staining: the dye sticks to the : the dye sticks to the specimen to give it colorspecimen to give it color

Negative stainingNegative staining: The dye does not stick to : The dye does not stick to the specimen, instead settles around its the specimen, instead settles around its boundaries, creating a silhouette.boundaries, creating a silhouette. Nigrosin and India ink commonly usedNigrosin and India ink commonly used Heat fixation not required, so there is less Heat fixation not required, so there is less

shrinkage or distortion of cellsshrinkage or distortion of cells Also used to accentuate the capsule surrounding Also used to accentuate the capsule surrounding

certain bacteria and yeastscertain bacteria and yeasts

Simple StainsSimple Stains

Require only a single dyeRequire only a single dye Examples include malachite green, crystal Examples include malachite green, crystal

violet, basic fuchsin, and safraninviolet, basic fuchsin, and safranin All cells appear the same color but can reveal All cells appear the same color but can reveal

shape, size, and arrangementshape, size, and arrangement

Differential StainsDifferential Stains

Use two differently colored dyes, the Use two differently colored dyes, the primary dye and the counterstainprimary dye and the counterstain Distinguishes between cell types or partsDistinguishes between cell types or parts Examples include Gram, acid-fast, and Examples include Gram, acid-fast, and

endospore stainsendospore stains

Gram StainingGram Staining

The most universal diagnostic staining The most universal diagnostic staining technique for bacteriatechnique for bacteria

Differentiation of microbes as gram Differentiation of microbes as gram positive(purple) or gram negative (red)positive(purple) or gram negative (red)

Acid-Fast StainingAcid-Fast Staining

Important diagnostic stainImportant diagnostic stain Differentiates acid-fast bacteria (pink) from Differentiates acid-fast bacteria (pink) from

non-acid-fast bacteria (blue)non-acid-fast bacteria (blue) Important in medical microbiologyImportant in medical microbiology

Endospore StainEndospore Stain

Dye is forced by heat into resistant bodies Dye is forced by heat into resistant bodies called spores or endosporescalled spores or endospores

Distinguishes between the stores and the Distinguishes between the stores and the cells they come from (the cells they come from (the vegetativevegetative cells) cells)

Significant in medical microbiologySignificant in medical microbiology

Special StainsSpecial Stains

Used to emphasize certain cell parts that Used to emphasize certain cell parts that aren’t revealed by conventional staining aren’t revealed by conventional staining methodsmethods

Examples: capsule staining, flagellar Examples: capsule staining, flagellar stainingstaining

Figure 3.25