Tools of the Laboratory: for Studying Microorganisms · The Methods for Studying Microorganisms...

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

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

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


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


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


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


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”


© Kathy Park Talaro and Harold Benson

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


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)


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


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)


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)


(first): © Carolina Biological Supply, Co/Visuals Unlimited; (second–fourth): © Michael Abbey/Visuals Unlimited

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


(Top): Courtesy of Dr. Jeremy Allen/University of Salford, Biosciences Research Institute; (bottom): © Science Photo Library RF/Getty Images

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


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


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


a,b: © Jack Bostrack/Visuals Unlimited; c: © Manfred Kage/Peter Arnold/Photolibrary

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

Tools of the Laboratory: for Studying Microorganisms · The Methods for Studying Microorganisms...

Documents

Transcript of Tools of the Laboratory: for Studying Microorganisms · The Methods for Studying Microorganisms...