Tools of the Laboratory:The Methods for Studying Microorganisms
Chapter 2
What are the challenges if you want to study microbes?
• In their nature habitats microbes are found in complex associations with other microbes.
• Microbes are small so to study them you need to isolate them and grow them under artificial conditions.
• Microbes are invisible.• Microbes are everywhere and they often contaminate your isolated experimental microbes.
The Five I’s of Microbiology
Inoculation
Incubation
Isolation
Inspection
Identification
Major Techniques Performed by Microbiologists to Locate, Grow, Observe, and Characterize Microorganisms
Isolation:One result of inoculation and incubation isisolation of the microbe. Isolated microbesmay take the form of separate colonies (discretemounds of cells) on solid media, or turbidity(free-floating cells) in broths. Further isolation bysubculturing involves taking a bit of growth froman isolated colony and inoculating a separatemedium. This is one way to make a pure culturethat contains only a single species of microbe.
3Inspection:The colonies or broth cultures are observedmacroscopically for growth characteristics(color, texture, size) that could be useful inanalyzing the specimen contents. Slides aremade to assess microscopic details such ascell shape, size, and motility. Staining techniquesmay be used to gather specific information onmicroscopic morphology.
4Identification:A major purpose of the Five I’s is to determinethe type of microbe, usually to the level ofspecies. Information used in identification caninclude relevant data already taken during initialinspection and additional tests that furtherdescribe and differentiate the microbes.Specialized tests include biochemical tests todetermine metabolic activities specific to themicrobe, immunologic tests, and genetic analysis.
5
MMicroscopic morphology:shape, staining reactions
Inoculation:The sample is placed into a container of sterile medium containing appropriate nutrientsto sustain growth. Inoculation involves spreading the sample on the surface of a solid medium or introducing the sample into a flask or tube. Selection of media with specialized functions can improve later steps of isolation and identification. Some microbes may require a live organism (animal, egg) as the growth medium.
1Incubation:An incubator creates the proper growth temperature and otherconditions. This promotes multiplication of the microbes over aperiod of hours, days, and even weeks. Incubation produces aculture—the visible growth of the microbe in or on the medium.
2
1 2
Specimen Collection:Nearly any object or material can serve as a source ofmicrobes. Common ones are body fluids and tissues,foods, water, or soil. Specimens are removed by someform of sampling device: a swab, syringe, or a specialtransport system that holds, maintains, and preservesthe microbes in the sample.
A GUIDE TO THE FIVE I’s: How the Sample Is Processed and Profiled
Major Techniques Performed by Microbiologists to Locate,Grow, Observe, and Characterize Microorganisms
Birdembryo
Blood bottle IncubatorSubculture
Isolation Biochemicaltests
Immunologictests
DNAanalysis
Streak plate
Syringe
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Inoculation
Culture: the propagation of microorganisms with various media
Medium (pl. media): a nutrient used to grow microorganisms outside their natural habitat
Inoculation: the implantation of microorganisms into or onto culture media
Inoculation – Clinical Specimens Include
Blood
cerebrospinal fluid
Sputum
Urine
Feces
diseased tissue
Incubation
The Incubator: media containing inoculants are placed in temperature‐controlled chambers
Usual laboratory propagation temperatures fall between 20°C and 40°C
Atmospheric gases such as O2 and CO2 may be required for the growth of certain microbes
During incubation, microbes grow and multiply, producing visible growth in the media
Various Conditions of Cultures
Pure Culture Mixed Culture Contaminated Culture
(c)(b)(a)
Various conditions of cultures. (a) Three tubes containing pure cultures of Escherichia coli (white), Micrococcus luteus (yellow), and Serratia marcescens (red). A pure culture is a container of medium that grows only a single known species or type of microorganism. This type of culture is most frequently used for laboratory study, because it allows the systematic examination and control of one microorganism by itself.
(b) A mixed culture is a container that holds two or more identified, easily differentiated species of microorganisms, not unlike a garden plot containing both carrots and onions. Pictured here is a mixed culture of M. luteus (bright yellow colonies) and E. coli (faint white colonies).
(c) A contaminated culture was once pure or mixed (and thus a known entity) but has since had contaminants (unwanted microbes of uncertain identity) introduced into it, like weeds into a garden. Contaminants get into cultures when the lids of tubes or Petri dishes are left off for too long, allowing airborne microbes tosettle into the medium. They can also enter on an incompletely sterilized inoculating loop or on an instrument that you have inadvertently reused or touched to the table or your skin. This plate of S. marcescens was overexposed to room air, and it has developed a large, white colony. Because this intruder is not desirable andnot identified, the culture is now contaminated.
Media in Different Physical Forms
1 2 3 4 (c)(b)(a)
Liquid Semisolid Solid/Reversible to Liquid
Media in different physical forms. (a) Liquid media are water-based solutions that do not solidify at temperatures above freezing and that tend to flow freely when the container is tilted. Growth occurs throughout the container and can then present a dispersed, cloudy, or particulate appearance. Urea broth is used to show a biochemical reaction in which the enzyme urease digests urea and releases ammonium. This raises the pH of the solution and causes the dye to become increasingly pink. Left: uninoculated broth, pH 7; middle: weak positive, pH 7.5; right: strong positive, pH 8.0.
(b) Semisolid media have more body than liquid media but less body than solid media. They do not flow freely and have a soft, clotlikeconsistency at room temperature. Semisolid media are used to determine the motility of bacteria and to localize a reaction at a specific site. Here, sulfur indolemotility medium (SIM) is pictured. The (1) medium is stabbed with an inoculum and incubated. Location of growth indicates nonmotility (2) or motility (3). If H2S gas is released, a black precipitate forms (4).
(c) Media containing 1%–5% agar aresolid enough to remain in place when containers are tilted or inverted. They are reversibly solid and can be liquefied withheat, poured into a different container, and resolidified. Solid media provide a firm surface on which cells can form discretecolonies. Nutrient gelatin contains enough gelatin (12%) to take on a solid consistency. The top tube shows it as a solid. The bottom tube indicates what happens when it is warmed or when microbial enzymes digest the gelatin and liquefy it.
The MediaFood for Microbes in the Laboratory
(Physical states of media)
liquid
semisolid
solid (can be converted to liquid)
solid (cannot be liquefied)
The MediaFood for Microbes in the Laboratory (cont.)
Agar ‐ complex polysaccharide from the alga Gellidium
liquefies at 100°C and solidifies at 42°C and can be poured in liquid form that will not harm the microbe or the handler
flexible and moldable; can hold moisture and nutrients
not a digestible nutrient for microorganisms
Chemical Content of Media
Chemically Defined
Media where chemical composition of media’s composition are precisely defined
Contain pure organic and inorganic compounds that vary little from one source to another
Molecular content specified by an exact formula
Chemical Content of Media
Minimal media
contain nothing more than a few essential compounds such as salts and amino acids
some contain a variety of defined organic and inorganic chemicals
Chemical Content of Media
Complex media
contain at least one ingredient that is not chemically definable
extracts of animals, plants, or yeasts
blood, serum, meat extracts, or infusions
present a rich mixture of nutrients for microbes that have complex nutritional needs
Chemically Defined and Complex Media
Selective and Differential Media
Selective media
contains one or more agents that inhibit the growth of a certain microbes but not others
important in the primary isolation of a specific type of microorganism from samples containing dozens of species
Selective and Differential Media
Differential media
allow multiple types of microorganisms to grow but are designed to display differences among those microorganisms
differentiation shows as variations in colony
size or colormedia color changesformation of gas bubblesprecipitates
Selective and Differential Media
Media can be both selective and differential
Dyes are often used as pH indicators that change colors in response to the production of an acid or base
Comparison of Selective and Differential Media
Mixedsample
Mixedsample
Differential medium(All 3 species grow but mayshow different reactions.)
General-purposenondifferential medium
(All species have a similarappearance.)
Selective medium(One species grows.)
(b)(a)
General-purposenonselective medium
(All species grow.)
Miscellaneous Media
Reducing medium
contains a substance (thioglycolic acid or cystine) that absorbs oxygen or slows the penetration of oxygen
important for growing anaerobic bacteria
Carbohydrate fermentation media
contain sugars that can be fermentedand a pH indicator that shows this reaction
can contain a Durham tube to collect gas bubbles
Miscellaneous Media
Transport media
used to maintain and preserve specimens that have to be held for a period of time before clinical analysis
sustain delicate species that die rapidly if not held under stable conditions
Isolation
Based on the concept that if an individual cell is separated from other cells on a nutrient surface, it will form a colony
Colony: a macroscopic cluster of cells appearing on a solid medium arising from the multiplication of a single cell
Requires the following
- a medium with a firm surface
- a Petri dish
- inoculating tools
Methods for Isolating Bacteria
1 2 3
1 2
1 2
3
1 2 3
4 5
Steps in a Streak Plate
Note: This method only works if the spreading tool (usually aninoculating loop) is resterilized after each of steps 1–5.
Steps in Loop Dilution
(b)
Steps in a Spread Plate
(c)
(a)
“Hockey stick”
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Inspection and Identification
Microbes can be identified through
microscopic appearance
characterization of cellular metabolism
determination of products given offduring growth, presence of enzymes, and mechanisms for deriving energy
genetic and immunological characteristics
details of these techniques will be covered in chapter 15
Maintenance and Disposal of Cultures
Cultures and specimens constitute a potential hazard
Prompt disposal is required
Stock cultures represent a “living catalog”for study and experimentation
The American Type Culture Collection (ATCC) in Manassas, VA is the largest culture collection in the U.S.
Microbial Size
Macroscopic organisms can be measured in the range from meters (m) to centimeters (cm)
Microscopic organisms fall into the range from millimeters (mm) to micrometers (μm) to nanometers (nm)
viruses measure between 20 – 800 nm
smallest bacteria measure around 200 nm
protozoa and algae measure 3 – 4 mm
1 micron = 1 x 10 meters = 0.000001 meters-6
The Size of Things
Hydrogen atom
Amino acid(small molecule)
Diameter of DNA
Large proteinFlagellum
Polio virus
AIDS virus
Mycoplasma bacteria
Escherichia coli bacteria
Most bacteria fallbetween 1 and10 µm insize
Red blood cell
Colonial alga(Pediastrum)
Reproductivestructure
of bread mold
Louse
Macroscopic View
Microscopic View
100 µm
Range ofhuman eye
Rangeoflight microscope
10 µm
1 µm
200 nm
100 nm
Rangeofelectronmicroscope
10 nm
1 nm
Require specialmicroscopes
0.1 nm(1 Angstrom)
1 mm
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Principles of Light Microscopy
Magnification
objective lens: closest to the specimen, forms the initial image called the real image
ocular lens: forms the second image called the virtual image that will be received by the eye and converted to the retinal and visual image
Total magnification (see next slide)
Principles of Light Microscopy
Power of Objective Usual power of ocular Total magnification
4x scanning objective 10x 40x
10x low power objective 10x 100x
40x high dry objective 10x 400x
100x oil immersion objective 10x 1000x
Principles of Light Microscopy
Resolution = resolving power
the capacity of an optical system to distinguish or separate two adjacent points or objects from one another
the human eye can resolve two objects that are no closer than 0.2 mm apart
The Effect of Wavelength on Resolution
High resolutionLow resolution
(a) (b)
Principles of Light Microscopy
Oil Immersion Lens
uses oil to capture light that would otherwise be lost to scatter
reducing scatter increases resolution
oil immersion lens can resolve images that are at least 0.2 μmin diameter and at least 0.2 μmapart
Objective lens
SlideOilAir
Principles of Microscopy
Contrast
refractive index: a measurement of the degree of bending that light undergoes as it passes from one medium to another
the higher the difference in refractive indexes, the greater the contrast
the iris diaphragm can control the amount of light entering the condenser and increase contrast
special lenses and dyes are also used to increase contrast
Comparison of Types of MicroscopyTable 2.6 Comparison of Types of MicroscopyVisible light as source of illuminationMicroscopeBright Field The bright-field microscope is the most widely used type of light microscope. Although we ordinarily view objects like the words on this page with light reflected off the surface, a bright-field microscope forms its image when light is transmitted through the specimen. The specimen, being denser and more opaque than its surroundings, absorbs some of this light, and the rest of the light is transmitted directly up through the ocular. As a result, the specimen will produce an image that is darker than the surrounding brightly illuminated field. The bright-field microscope is a multipurpose instrument that can be used for both live, unstained material and preserved, stained material.
Dark Field A bright-field microscope can be adapted as a dark-field microscope by adding a special disc called a stop to the condenser. The stop blocks all light from entering the objective lens—except peripheral light that is reflected off the sides of the specimen itself. The resulting image is a particularly striking one: brightly illuminated specimens surrounded by a dark (black) field. The most effective use of dark-field microscopy is to visualize living cells that would be distorted by drying or heat or that cannot be stained with the usual methods. Dark-field microscopy can outline the organism’s shape and permit rapid recognition of swimming cells that might appear in dental and other infections, but it does not reveal fine internal details.
Phase-Contrast If similar objects made of clear glass, ice, cellophane, or plastic are immersed in the same container of water, an observer would have difficulty telling them apart because they have similar optical properties. Internal components of a live, unstained cell also lack contrast and can be difficult to distinguish. But cell structures do differ slightly in density, enough that they can alter the light that passes through them in subtle ways. The phase-contrast microscope has been constructed to take advantage of this characteristic. This microscope contains devices that transform the subtle changes in light waves passing through the specimen into differences in light intensity. For example, denser cell parts such as organelles alter the pathway of light more than less dense regions (the cytoplasm). Light patterns coming from these regions will vary in contrast. The amount of internal detail visible by this method is greater than by either bright-field or dark-field methods. The phase-contrast microscope is most useful for observing intracellular structures such as bacterial spores, granules, and organelles, as well as the locomotor structures of eukaryotic cells such as cilia.
Differential Interference Like the phase-contrast microscope, the differential interference contrast (DIC) microscope provides a detailed view of unstained, live specimens by manipulating the light. But this microscope has additional refinements, including two prisms that add contrasting colors to the image and two beams of light rather than a single one. DIC microscopes produce extremely well-defined images that are vividly colored and appear three-dimensional.
2,000x 0.2 µm(200 nm)
2,000x 0.2 µm
2,000x 0.2 µm
2,000x 0.2 µm
Amoeba proteus (160x)
Paramecium (400x)
Paramecium (400x)
Paramecium (400x)
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Comparison of Types of Microscopy
2,000x 0.2 µm
Algae showing cell walls made of calcium discs (10,000x)
Table 2.6 (continued)Ultraviolet rays as source of illuminationMicroscope
Confocal The scanning confocal microscope overcomes the problem of cells or structures being too thick, a problem resulting in other microscopes being unable to focus on all their levels. This microscope uses a laser beam of light to scan various depths in the specimen and deliver a sharp image focusing on just a single plane. It is thus able to capture a highly focused view at any level, ranging from the surface to the middle of the cell. It is most often used on fluorescently stained specimens but it can also be used to visualize live unstained cells and tissues
Scanning Electron Microscope (SEM) The scanning electon microscope provides some of the most dramatic and realistic images in existence. This instrument is designed to create an extremely detailed three-dimensional view of all kinds of objects—from plaque on teeth to tapeworm heads. To produce its images, the SEM bombards the surface of a whole metal-coated specimen with electrons while scanning back and forth over it. A shower of electrons deflected from the surface is picked up with great fidelity by a sophisticated detector, and the electron pattern is displayed as an image on a television screen.
You will often see these images in vivid colors. The color is always added afterwards; the actual microscopic image is black and white.
Electron beam forms image of specimenMicroscope
Maximum Practical Magnification Resolution
Maximum Practical Magnification Resolution
Myofibroblasts, cells involved in tissue repair (400x)
100,000,000x 10 nm
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Preparing Specimens for the Microscope
Specimens are usually prepared by mounting a sample on a suitable glass slide that sits on the stage between the condenser and the objective lens
The manner in which it is prepared depends on
the condition of the specimen, either living or preserved
the aims of the examiner: to observe overall structure, identify microorganisms, or see movement
the type of microscopy available: brightfield, dark‐field, phase‐contrast, orfluorescence
Fresh, Living Preparations
Placed on wet mounts or in hanging drop mounts to observe as near to the natural state as possible
Cells are suspended in water, broth, or saline to maintain viability and provide space for locomotion
Wet mount
consists of a drop or two of culture placed ona slide and overlaid with a cover slip
Hanging drop
a drop of culture is placed in a concave (depression) slide, Vaseline adhesive or sealant, and cover slip are used to suspend the sample
Short‐term mounts such as these provide a true assessment of size, shape, arrangement, color, and motility
Fixed, Stained Smears
More permanent mounts used for long‐term study
Smear technique developed by Robert Koch over 100 years ago
spread a thin film made from a liquid suspension of cells on a slide
air dry
heat fix: heat gently to kill the specimen and attach to the slide
Stains
Unstained cells in a fixed smear are difficult to see regardless of magnification and resolving power
Staining is any procedure that applies colored chemicals (dyes) to specimens
basic dyes have a positive charge
acidic dyes have a negative charge
Bacteria have numerous negatively charged substances and attract basic dyes
Acidic dyes are repelled by cells
Negative vs. Positive Staining
Positive stain: dye sticks to the specimen and gives it color
Negative stain: does not stick to the specimen but settles some distance from its outer boundary, forming a silhouette
- negatively charged cells repel the negatively charged dye and remain unstained
- smear is not heat fixed so there is reduced distortion and shrinkage of cells
- also used to accentuate a capsule
- nigrosin and India ink are used
Simple vs. Differential Staining
Simple stains:
only require a single dye and an incomplicated procedure
cause all the cells in the smear to appear more or less the same color, regardless of type
reveal shape, size, and arrangement
Differential stains:
use two differently colored dyes: the primary dye and the counterstain
distinguish cell types or parts
more complex and require additional chemical reagents to produce the desired reaction
Simple Stains
(b) Methylene blue stain of Corynebacterium(a) Crystal violet stain of Escherichia coli
Simple Stains
Types of Differential Stains
Gram stain ‐ developed in 1884 by Hans Christian Gram
consists of sequential applications ofcrystal violet (the primary stain), iodine (the mordant), an alcohol rinse (decolorizer), and safranin (the counterstain)
different results in the Gram stain are due to differences in the structure of the cellwall and how it reacts to the series ofreagents applied to the cells
remains the universal basis for bacterial classification and identification
a practical aid in diagnosing infection andguiding drug treatment
Types of Differential Stains
Acid‐fast stain
- differentiates acid‐fast bacteria (pink) from non‐acid‐fast bacteria (blue)
- originated as a method to detect Mycobacterium tuberculosis
- these bacteria cell walls have a particularly impervious cell wall that holds fast (tightly or tenaciously) to the dye (carbol fuschin) when washed with an acid alcohol decolorizer
- also used for other medically important bacteria, fungi, and protozoa
Types of Differential Stains
Endospore stain
similar to the acid fast stain in that adye is forced by heat into resistantbodies called spores or endospores
stain distinguishes between sporesand vegetative cells
significant in identifying gram‐positive, spore‐forming members ofthe genus Bacillus and Clostridium
Differential Stains
(c) Spore stain, showing endospores(red) and vegetative cells (blue)
(b) Acid-fast stain. Red cells areacid-fast. Blue cells are non-acid-fast.
(a) Gram stain. Purple cells aregram-positive. Pink cells aregram-negative.
Differential Stains
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Special Stains
Used to emphasize cell parts that are not revealed by conventional staining methods
Capsule staining
used to observe the microbial capsule
an unstructured protective layer surrounding thecells of some bacteria and fungi
negatively stained with India ink
Flagellar staining
used to reveal tiny, slender filaments used by bacteria for locomotion
flagella are enlarged by depositing a coating onthe outside of the filament and then staining it