Tools of the Laboratory: The Methods for Studying Microorganisms Chapter 2

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1. Macronutrientes: requeridos en grandes cantidades C, H, O, N, P, S Carbono: •elemento más abundante en todas las macromoléculas Nitrógeno: • 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) Fe, Mn, Cr, Ni, Zn, Se, Cu, Co necesarios como cofactores de enzimas

Hierro: requerido en proteínas asociadas al transporte de electrones durante el proceso de respiración celular (citocromos, proteínas de hierro-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 •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 pueden respirar anaeróbicamente o fermentar. Ejemplos: Escherichia coli, Enterobacter, Salmonella

Anaerobios aerotolerantes = estos son organismos que no respiran oxigeno sino que solo fermentan pero el oxigeno no los afecta o limita. Ejemplos: Lactobacillus

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

Medio de thioglycolato

Mas oxigeno

Menos oxigeno

Temperatura optima de crecimiento

Clasificación de microorganismos de acuerdo a su preferencia en temperatura

Clasificacion usando pH

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

Pure Culture

(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.

Mixed Culture

(b)

Contaminated Culture

(c)

(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).

© Kathy Park Talaro

(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 to settle 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 and not identified, the culture is now contaminated.

Medios selectivos, diferenciales o ambos!!

NA

MacConkey

Blood Agar Streptococcus pyogenes Beta hemolitico

Streptococcus pneumoniae Alpha hemolitico

Enterococcus faecalis No helitico

Que tipo de medio es este?

Comparison of Selective and Differential Media Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Mixed sample

Mixed sample

General-purpose nonselective medium (All species grow.) Selective medium (One species grows.) (a)

General-purpose nondifferential medium (All species have a similar appearance.) (b)

Differential medium (All 3 species grow but may show different reactions.)

Media in Different Physical Forms Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Liquid

(a)

Semisolid

(b)

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.

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Solid/Reversible to Liquid

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(b) Semisolid media have more body than liquid 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).

(all): © Kathy Park Talaro

(c)

(c) Media containing 1%–5% agar are solid enough to remain in place when containers are tilted or inverted. They are reversibly solid and can be liquefied with heat, poured into a different container, and resolidified. Solid media provide a firm surface on which cells can form discrete colonies. 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 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

•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

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

Seen Through Microscope (Microscopic)

•Colony: a macroscopic cluster of cells appearing on a solid medium arising from the multiplication of a single cell

Parent cells

Mixture of cells in sample

•Requires the following - a medium with a firm surface

Microbes become visible as isolated colonies containing millions of cells.

Separation of cells by spreading or dilution on agar medium Growth increases the number of cells.

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a Petri dish

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inoculating tools

Seen by Naked Eye (Macroscopic)

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

Steps in a Streak Plate (a)

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4 5 Note: This method only works if the spreading tool (usually an inoculating loop) is resterilized after each of steps 1–5. Steps in Loop Dilution (b)

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Steps in a Spread Plate (c)

“Hockey stick”

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2 © Kathy Park Talaro and Harold Benson

Inspection and Identification •Microbes can be identified through - microscopic appearance -

characterization of cellular metabolism

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determination of products given off during growth, presence of enzymes, and mechanisms for deriving energy

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genetic and immunological characteristics

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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) - viruses measure between 20 – 800 nm -

smallest bacteria measure around 200 nm

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protozoa and algae measure 3 – 4 mm

1mm=1000µ µm=10-3mm 1um=1000nm=10-6mm 1nm=1000pm=10-9mm 1pm=1000fm=10-12mm

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

Macroscopic View 1 mm

Louse

Range of human eye Reproductive structure of bread mold Microscopic View

100 µm

Range of light microscope 10 µm

Colonial alga (Pediastrum)

Red blood cell Most bacteria fall between 1 and 10 µm insize

1 µm

Escherichia coli bacteria

200 nm

Mycoplasma bacteria

100 nm Range 10 nm of electron microscope

1 nm Require special microscopes 0.1 nm (1 Angstrom)

AIDS virus Polio virus Flagellum Large protein Diameter of DNA

Amino acid (small molecule) Hydrogen atom

I. Light Microscope Microscopio compuesto de luz resolución: 0.2 µm

El Microscopio Como Herramienta Resolución: capacidad de distinguir 2 objetos Resolución adyacentes como unidades distintas y separadas Magnificación: capacidad de aumentar el tamaño Magnificación de una imagen en relación al tamaño real del objeto Contraste: diferencia en color entre el espécimen Contraste y el campo de visión

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

(a)

(b)

Low resolution

High resolution

Coutesy of Nikon Instruments Inc.

Principles of Light Microscopy (cont’d) Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

•Oil Immersion Lens - uses oil to capture light that would otherwise be lost to scatter -

reducing scatter increases resolution

Objective lens

Air

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oil immersion lens can resolve images that are at least 0.2 μm in diameter and at least 0.2 μm apart

Oil Slide

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

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the iris diaphragm can control the amount of light entering the condenser and increase contrast

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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 microorganismos no son pigmentados

Tinción diferencial: permite detectar tipos distintos de células blue

red

Tinción Gram 1. aplicar tinte azul 2. Yodo 3. decolorizar (alcohol) 4. aplicar tinte rojo 5.ver cuál tinte se retiene 6. Gram+ retiene el tinte azul, Gram- retiene tinte rojo

(Streptococcus)

(Escherichia) Desventajas de técnicas de tinción:

• requieren fijar la muestra con calor: (células mueren) • calor + químicos puede distorsionar la 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

luz

contraste de fase células de levadura

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

luz

células de levadura

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 luz 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)

cianobacterias

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

Microscopio de fluorescencia

Cuantificación y Viabilidad Usando Técnicas de Tinción Fluorescentes 1. DAPI (4',6-diamidino-2-phenylindole ) •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

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 muertas (membrana celular dañada)

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

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 GFP (Green Fluorescent Protein) estructura 3-D

UV

medusa Aequorea victoria

gen codificante para la proteína verde-fluorescente

aplicación: detección y rastreo de organismos introducidos en ambientes naturales bacteria introducida en el tejido vascular de le caña de azúcar

bacterias

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 célula o 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 sobre la muestra produce una imagen

2. microscopio electrónico de transmisión

visualizar estructuras internas de una célula

requiere el corte de las muestras en secciones delgadas

2. Microscopio electrónico de transmisión

region nucleoide

microscopio de luz

Microscopio de luz Versus Microcopio Electrónico luz → electrónico rastreo → electrónico transmisión

poder de resolución aumenta region nucleoide

microscopio electrónico de rastreo

microscopio electrónico de transmisión (resolución máxima 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 preserved -

the aims of the examiner: to observe overall structure, identify microorganisms, or see movement

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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 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

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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

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also used to accentuate a capsule

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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 Differential stains - use two differently colored dyes: the primary dye and the counterstain -

distinguish cell types or parts

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more complex and require additional chemical reagents to produce the desired reaction

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

Simple Stains

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

Tinción simple: un solo tinte , afinidad por carga con componentes de la superficie de la célula Ejemplo : azul de metileno (+)

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(-) (-) (-)

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(-) methylene blue

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(-) (-)

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(-) (-) (-)

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)

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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

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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 vegetative cells

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significant in identifying gram-positive, sporeforming members of the genus Bacillus and Clostridium

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

Differential Stains

(a) Gram stain. Purple cells are gram-positive. Pink cells are gram-negative.

(b) Acid-fast stain. Red cells are acid-fast. Blue cells are non-acidfast.

(c) Spore stain, showing endospores (red) and vegetative cells (blue)

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