Tools of the Laboratory: The Methods for Studying ...academic.uprm.edu/~lrios/3725/Chapter 2.pdf · Otros Macronutrientes: K, Mg, Ca, Na Potasio : requerido para la actividad de ciertas

Tools of the

Laboratory:

The Methods for

Studying

MicroorganismsMicroorganismsChapter 2

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

1. Macronutrientes : requeridos en grandes cantidades

C, H, O, N, P, SCarbono: •elemento más abundante en todas las macromoléculas

Nitrógeno:• necesario para síntesis de proteínas y ácidos nucleicos• necesario para síntesis de proteínas y ácidos nucleicos

Oxígeno e hidrógeno:•presentes en macromoléculas y compuestos orgánicos que sirven de fuente de energía

Fósforo: • necesario para síntesis de fosfolípidos y ácidos nucleicos

Azufre:•necesario para síntesis de ciertos amino ácidos (cisteína y metionina) y vitaminas

Otros Macronutrientes: K, Mg, Ca, Na

Potasio: requerido para la actividad de ciertas enzimas, en particular aquellas envueltas en síntesis de proteínas

Magnesio: estabiliza ribosomas, ácidos nucléicos, requerido para la actividad de varias enzimas

Calcio : estabiliza la pared celular, confiere resistencia al calor en endoesporas

Sodio: necesario para el crecimiento de microorganismos adaptados a Presiones osmóticas asociadas ambientes marinos o hipersalinos

2. Micronutrientes : compuestos inorgánicos(metales) requeridos en pequeñas cantidades (elementos trazas)

Hierro : requerido en proteínas asociadas al transporte de electrones

necesarios como cofactores de enzimasFe, Mn, Cr, Ni, Zn, Se, Cu, Co

Hierro : requerido en proteínas asociadas al transporte de electronesdurante el proceso de respiración celular (citocromos, proteínas dehierro -azufre ). Hierro esta presente en cantidades muy bajas en ambientes naturales

Sideroforos : Agente quelante producido por células, capaz de fijar o secuestrar iones metálicos en el ambiente para translocarlos al interior

3. Factores de crecimiento : compuestos Orgánicos requeridos en pequeñas cantidades

vitaminas, amino ácidos, purinas, pirimidinas,

deben ser suplidos a ciertos microorganismos que no pueden sintetizarlos ej. bacterias productoras de ácido láctico•Streptococcus•Streptococcus•Lactobacillus•Leuconostoc

vitaminas : factores de crecimiento más requeridos, se utilizan como cofactores de enzimas (componente necesario para el funcionamiento de una enzima)

Otros grupos basados en su requerimiento de oxigeno

Anaerobios facultativos = estos son organismos aeróbicos que puedenrespirar anaeróbicamente o fermentar.

Ejemplos: Escherichia coli, Enterobacter, Salmonella

Anaerobios aerotolerantes = estos son organismos que no respiran oxigenosino que solo fermentan pero el oxigeno no los afecta o limita.

Ejemplos: LactobacillusEjemplos: Lactobacillus

Microaerofilicos = estos requieren oxigeno exclusivamente pero en concentraciones bajas 2% - 10% mas de esto seria toxico.

Ejemplo: Helicobacter pylori

Mas oxigeno

Medio de thioglycolato

Menos oxigeno

Temperatura optima de crecimiento

Clasificación de microorganismos de acuerdo a su preferencia en temperatura

Cla

sific

acio

nus

ando

pHC

lasi

ficac

ion

Cultivo de microorganismos

medio de cultivo: solución de nutrientes para crecer microorganismos

cultivo puro: una sola clase de microorganismo

medio químicamente definido : contiene cantidades precisas de químicos altamente purificados, la composición exacta se conoce

medio complejo : contiene extractos de material animal o vegetal altamente nutritivos pero de composición no definida

•extracto de carne•sangre de oveja•extracto de levadura•peptonas (mezcla de proteínas parcialmente digeridas)

microorganismos que tienen menos requisitos nutricionales tienen una mayor capacidad biosintética (pueden producir lo que necesitansin depender de la disponiblilidad de nutrientes previamente existentes)

Chemically Defined and Complex Media

Técnicas asépticas:

cultivo puro: una sola clase de microorganismo

Streak Plate

Pour Plate

Puro

No Puro

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.

© Kathy Park Talaro

Medios selectivos, diferenciales o ambos!!

NA

MacConkey

Blood Agar

Streptococcus pyogenesBeta hemolitico

Streptococcus pneumoniaeAlpha hemolitico

Enterococcus faecalisNo helitico

Que tipo de medio es este?

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.)

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 thanliquid media but less body than solid media. They do not flow freely and have a soft, clotlike consistency at room temperature. Semisolid media are used to determine the motility of bacteria and to localize a reaction at a specific site. Here, sulfur indole motility 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 whencontainers are tilted or inverted. They arereversibly solid and can be liquefied withheat, poured into a different container, andresolidified. Solid media provide a firmsurface on which cells can form discretecolonies. Nutrient gelatin contains enoughgelatin (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.

(all): © Kathy Park Talaro

The Five I’s of Microbiology

•Inoculation

•Incubation

•Isolation

•Inspection

•Identification

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- important for growing anaerobic bacteria

•Carbohydrate fermentation media

- contain sugars that can be fermented and a pH

indicator that shows this reaction

- can contain a Durham tube to collect gas

bubbles

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

Seen Through Microscope (Microscopic)Seen by Naked

Eye (Macroscopic)

Parentcells


from the multiplication of a single cell

•Requires the following

- a medium with a firm surface

- a Petri dish

- inoculating tools

Microbes becomevisible as isolatedcolonies containingmillions of cells.

Growth increasesthe number of cells.

Separation ofcells by spreadingor dilution on agarmedium

Mixture of cellsin sample

Methods for Isolating Bacteria

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 step s 1–5.

Steps in Loop Dilution

(b)

(a)


1 2

1 2

3

1 2 3

(b)

Steps in a Spread Plate

(c)

“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 off during - determination of products given off during

growth, presence of enzymes, and

mechanisms for deriving energy

- genetic and immunological characteristics

- details of these techniques will be covered in

chapter 15

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)nanometers (nm)

- viruses measure between

20 – 800 nm

- smallest bacteria

measure around 200 nm

- protozoa and algae

measure 3 – 4 mm

1mm=1000µµµµm=10-3mm1um=1000nm=10-6mm1nm=1000pm=10-9mm1pm=1000fm=10-12mm

The Size of Things

Red blood cell

Colonial alga(Pediastrum)

Reproductivestructure

of bread mold

Louse

Macroscopic View

Microscopic View

100 µm

Range ofhuman eye

Rangeoflight microscope

1 mm


Hydrogen atom

Amino acid(small molecule)

Diameter of DNA

Large protein

Flagellum

Polio virus

AIDS virus

Mycoplasma bacteria

Escherichia coli bacteria

Most bacteria fallbetween 1 and10 µm insize

light microscope

10 µm

1 µm

200 nm

100 nm

Rangeofelectronmicroscope

10 nm

1 nm

Require specialmicroscopes

0.1 nm(1 Angstrom)

I. Light Microscope

resolución: 0.2 µm

Microscopio compuesto de luz

ResoluciónResolución : capacidad de distinguir 2 objetos adyacentes como unidades distintasy separadas

El Microscopio Como HerramientaEl Microscopio Como Herramienta

MagnificaciónMagnificación : capacidad de aumentar el tamaño

ContrasteContraste : diferencia en color entre el espécimeny el campo de visión

MagnificaciónMagnificación : capacidad de aumentar el tamaño de una imagen en relación altamaño real del objeto

Principles of Light Microscopy (cont’d)

•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)


High resolutionLow resolution

Coutesy of Nikon Instruments Inc.

Principles of Light Microscopy

(cont’d)

•Oil Immersion Lens

- uses oil to capture light

that would otherwise be

lost to scatter

- reducing scatter increases


- reducing scatter increases

resolution

- oil immersion lens can

resolve images that are at

least 0.2 μm in diameter

and at least 0.2 μm apart

Objective lens

SlideOilAir

Principles of Microscopy (cont’d)

•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 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

Tinciones: • se usan para aumentar de contraste

(algas)

pigmentos presentes en células permiten su detección con microscopio de luz

levaduras (hongo unicelular)

la mayoría de los microorganismosno son pigmentados

Tinción diferencial: permite detectar tipos distintos de cé lulas

Tinción Gram

1. aplicar tinte azul

2. Yodo

blue red

3. decolorizar (alcohol)

4. aplicar tinte rojo

5.ver cuál tinte se retiene

6. Gram+ retiene el tinte azul, Gram- retienetinte rojo

(Streptococcus) (Escherichia)

Desventajas de técnicas de tinción:

• requieren fijar la muestra con calor:(células mueren)

• calor + químicos puede distorsionarla forma original de las células

2. Microscopio de contraste de fase (microscopio de luz modificado)

microscopio compuesto de luz microscopio de contraste de fase

Se amplifica el efecto de desplazamiento de fase de aquellos rayos de luz que se refractan al pasar sobre partes densas del espécimen. Esto,permite mayor contrate entre el objeto de interés y su alrededor en el campo óptico

2. Microscopia de contraste de fase

ventajas sobre microscopio de luz compuesto y tinciones:permite observar células vivas, movimiento, forma natural

células de levaduraluz contraste de fase

3. Microscopio de campo oscuro (microscopio de luz modificado)

método por el cuál la muestra es observada sobre un fondo oscuro, al dirigir la luz por los lados de la muestra

3. Microscopio de campo oscuro

Darkfield is the method whereby the sample being viewed is actually in front of a dark background and light is being angled onto the sample from the sides

células de levaduraluz campo oscuro

En Resumen…….

Luz Campo oscuro Contraste de fase

II. Microscopio de fluorescencia resolución similar a la del microscopio de luz, es otra técnica para lograr contraste usando tintes fluorescentes

II. Microscopio de fluorescencia (se utiliza un pigmento que genera lu z al absorber luz de un largo de onda específico)

Ejemplo: autofluorescencia, clorofila de cianobacterias (no es necesario añadir tinte)

absorve luz verde (λ 546nm) emite luz roja (λ 700nm)

ventaja: permite visualizar células en un medio complejo,suelo, agua, muestras ambientales

cianobacterias

Microscopio de fluorescencia

Cuantificación y Viabilidad Usando Técnicas de Tinción Fluorescentes

•tiñe el DNA de color azul brillante •enumeración de microorganismos en muestras de tipo:•clínico•ambiental•alimentos

desventaja: no discrimina entre células vivas y muertas

1. DAPI (4',6-diamidino-2-phenylindole )

desventaja: no discrimina entre células vivas y muertas

2. Tinción de Viabilidad

Sistema “Live /Dead Bac Light TM ” (comercialmente disponible)

permite discriminar entre células vivas y muertas

tinte verde : bacterias vivas(membrana celular intacta)

tinte rojo : bacterias muertastinte rojo : bacterias muertas(membrana celular dañada)

desventaja: apropiado para cultivospuros, tintes se pueden pegar a otras cosas que no son células en muestras ambientales o complejas

GFP (Green Fluorescent Protein)estructura 3-D

3. Green Fluorescent Protein

envuelve la manipulación genética de un microorganismo al cual se le inserta un gen codificante para una proteína verde-fluorescente extraído de una medusa

UV

medusa Aequorea victoria bacterias

gen codificantepara la proteínaverde-fluorescente

aplicación: detección y rastreo de organismos introducidos en ambientes naturales

bacteria introducida en el tejidovascular de le caña de azúcar

III. Microscopía en tres dimensiones de alta resolución

1.microscopia electrónica de rastreo

se utiliza para imágenes de alta resolución de partes externas de la célulao superficies de objetos

•la muestra se cubre con una capa fina de metal, y se rastrea con un rayo de electrones en presencia de un vació•el patrón de movimiento de los electrones sobrela muestra produce una imagenla muestra produce una imagen

2. microscopio electrónico de transmisión

visualizar estructuras internas de una célula

requiere el corte de las muestras en secciones delgadassecciones delgadas

2. Microscopio electrónico de transmisión

region nucleoideregion nucleoide

microscopio de luz Microscopio de luzVersus

Microcopio Electrónico

region nucleoide

luz → electrónico rastreo → electrónico transmisión

poder de resolución aumenta

microscopio electrónico de rastreo

microscopio electrónico de transmisión (resoluciónmáxima 0.2nm)

Cuanto hemos mejorado?

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

preservedpreserved

- the aims of the examiner: to observe overall

structure, identify microorganisms, or see

movement

- the type of microscopy available: bright-field,

dark-field, phase-contrast, or fluorescence

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 on a slide - consists of a drop or two of culture placed on a 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 slidesuspension 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 - 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

uncomplicated 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 •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

Simple Stains


(b) Methylene blue stain of Corynebacterium(a) Crystal violet stain of Escherichia colia: © Kathy Park Talaro; b: © Harold J. Benson

(-)

(-)

(-)(-)

(-)(-)

(-)

(-)

(-)(-)

(-)

(-)

(+)

methylene blue

Tinción simple: un solo tinte , afinidad por carga c on componentes de la superficie de la célula

Ejemplo : azul de metileno

(-)

(-)(-)

(-)

methylene blue

Types of Differential Stains

•Gram stain

- developed in 1884 by Hans Christian Gram

- consists of sequential applications of crystal violet

(the primary stain), iodine (the mordant), an alcohol

rinse (decolorizer), and safranin (the counterstain)

- different results in the Gram stain are due to - different results in the Gram stain are due to

differences in the structure of the cell wall and how

it reacts to the series of reagents applied to the cells

- remains the universal basis for bacterial classification

and identification

- a practical aid in diagnosing infection and guiding

drug treatment

Types of Differential Stains (cont’d)

•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 (cont’d)

• Endospore stain

- similar to the acid fast stain in that a dye is

forced by heat into resistant bodies called

spores or endospores

- stain distinguishes between spores and - stain distinguishes between spores and

vegetative cells

- significant in identifying gram-positive, spore-

forming members of the genus Bacillus and

Clostridium

Differential Stains

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.

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 the cells

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 on the

outside of the filament and then staining it

Documents

Tools of the Laboratory: The Methods for Studying ...academic.uprm.edu/~lrios/3725/Chapter 2.pdf · Otros Macronutrientes: K, Mg, Ca, Na Potasio : requerido para la actividad de ciertas