b - Uprm

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
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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
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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
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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.
1
2
Solid/Reversible to Liquid
3
4
(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
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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.
-
a Petri dish
-
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)
1
2
3
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)
1
2
3
1
2
3
Steps in a Spread Plate
(c)
“Hockey stick”
1
2
© 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
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)
- viruses measure between
20 – 800 nm
-
smallest bacteria
measure around 200 nm
-
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
-
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
-
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
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
-
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
-
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
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
-
more complex and require additional chemical
reagents to produce the desired reaction
Simple Stains
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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
(+)
(-)
(-) (-)
(-)
(-)
(-)
(-)
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
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
vegetative cells
-
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