II.

MÉTODOS PARA LA
OBSERVACIÓN BACTERIANA
Métodos para la observación de bacterias
Estructura general de los procariotes
Morfología bacteriana
EXUDADO FARINGEO
 MÉTODOS PARA LA
OBSERVACIÓN DE BACTERIAS

Microscopía.

Preparaciones.

Tinciones.
 MICROSCOPÍA

El microscopio óptico utiliza lentes de vidrio para

desviar y enfocar los rayos de luz, produciendo
imágenes aumentadas de objetos pequeños.

La resolución máxima de un microscopio óptico es de

aproximadamente 0.2 um.

Se han desarrollado muchos tipos de microscopios

ópticos, incluyendo microscopio de campo claro, de
campo oscuro, de contraste de fases y de fluorescencia.
 UNIDAD DE MEDIDA

Unidad Abreviatura Valor

1 centímetro cm 10-2 metros

1 milímetro mm 10-3 metros

1 micrómetro um 10-6 metros

1 nanómetro nm 10-9 metros

1 Angstrom A 10-10 metros

1 picómetro pm 10-12 metros

 MICROSCOPÍA

El microscopio de campo claro requiere la aplicación

de colorantes sobre los microorganismos para
facilitar su visualización.

Los colorantes se utilizan para visualizar estructuras

procariotas específicas como flagelos o cápsulas.

Los aumentos útiles de un microscopio óptico están

limitados por su poder de resolución.
 MICROSCOPÍA

El poder de resolución está limitado por la longitud

de onda del haz iluminador.

Los microscopios electrónicos utilizan haces de

electrones en vez de luz para lograr una resolución
(hasta 0.5 nm) y una ampliación muy elevadas.
DESVIACIÓN DE LA LUZ

Cuando un rayo de luz pasa de un medio a otro se

produce refracción, es decir, el rayo se desvía en la
interfase.

El índice de refracción es una medida de la intensidad

con la que una sustancia disminuye la velocidad de la luz.

La dirección y la magnitud de la desviación se

determinan por los índices de refracción de los dos
medios que forman la interfase.
DESVIACIÓN
DE LA LUZ
 FUNCIONAMIENTO DE
UNA LENTE

Los rayos de luz producidos por una fuente distante

se enfocan en un punto focal (F).

La distancia entre el centro de una lente y el punto

focal se denomina distancia focal (f).
FUNCIONAMIENTO DE UNA LENTE
 FUNCIONAMIENTO DE
UNA LENTE

Los ojos humanos no pueden enfocar objetos que

estén a una distancia menor de 25 cm.

La potencia de la lente está relacionada con la

distancia focal; una lente con una distancia focal
corta aumentará un objeto más que una lente más
débil con una mayor distancia focal.
MICROSCOPIO DE CAMPO
CLARO

Recibe este nombre porque forma una imagen oscura

sobre un fondo más claro.

El aumento total se calcula multiplicando los

aumentos del objetivo y del ocular.
MICROSCOPIO DE CAMPO CLARO
 OBJETIVOS DE UN
MICROSCOPIO

Objetivo 4x (Rojo) ——— Lupa — 40x totales

Objetivo 10x (Amarillo) ——— Seco débil — 100x

Objetivo 40x (Azul) ——— Seco fuerte — 400x

Objetivo 100x (Blanco) ——— Inmersión — 1,000x

 TRAYECTORIA DE LUZ CHAPTER 3 Observing Microorganisms Through a Microscope 55

CHAPTER 3 Observing Microorganisms Through a Microscope 55

Ocular lens
(eyepiece)
Remagnifies the lens
Ocular
image formed by
(eyepiece) Ocular lens
the objective lens
Remagnifies the Line of vision
image formed by Ocular lens
the objective lens Line of vision
Path of light
Body tube Transmits
Path of light
Prism
the image from the
Body tube Transmits
objective lens to the Prism
the image from the
ocular lens
objective lens to the
ocular lens
Arm
Arm
Body tube
Objective lenses Body tube
Objective
Primary lenses thatlenses
Primary
magnify the lenses that
specimen
magnify the specimen
Objective
Objective
Stage Holds theHolds the
Stage lenses
lenses
microscope slide
microscope slide
in positionin position

Condenser Specimen
Specimen
Condenser FocusesFocuses
lightspecimen
light through through specimen Condenser
Condenser
lenses
lenses
Diaphragm Controls the amount
Diaphragm Controls
of light thethe
entering amount
condenser
of light entering the condenser Illuminator
Illuminator Light source Illuminator
Illuminator Light source
Coarse focusing knob Base with
Coarse focusing knob source of
Base with
Base
illumination
source of
Base
Fine focusing knob illumination
Fine focusing knob parts and functions
(a) Principal (b) The path of light (bottom to top)

(a) Principal parts and functions (b) The path of light (bottom to top)
Figure 3.1 The compound light microscope.
 OBJETIVO DE INMERSIÓN
CHAPTER 3 Observing Microorganisms Through a Microscope 59

Unrefracted Oil immersion

is simi- light objective lens
ences in
eams of
Without immersion oil
m, add- most light is refracted
ution of and lost
contrast
s nearly Immersion oil
Air

Glass slide

the abil-
raviolet)
Condenser
ganisms lenses
n to be
Sin aceite de inmersión Con aceite de inmersión
a group Condenser
ganisms
ence mi-
they ap- Iris diaphragm
nd. Light source
micro-
, which Figure 3.3 Refraction in the compound microscope using an oil
ngly ab- immersion objective lens. Because the refractive indexes of the glass
m that microscope slide and immersion oil are the same, the light rays do not
 of aspecimen
specimen effectively.
determines which microscopes can be used to view the
specimen effectively.
1m Most micrographs shown in this textbook (like the ones below) have
Unaided eye 1m Most micrographs
size shown intothis
bars and symbols textbook
help (like the
you identify theones below)
actual size have
of the

ranges
≥ 200
Unaided μm
eye sizespecimen
bars and and
symbols to help you identify the actual size
the type of microscope used for that image. of the

microscopy ranges
≥ 200 μm specimen and the type of microscope used for that image.
A red icon indicates that a micrograph has been artificially colorized.
A red icon indicates that a micrograph has been artificially colorized.
0.1 m
0.1 m

1 cm
1 cm
microscopy Light microscope
Light
200microscope
nm–10 mm
200 nm–10 mm
LM
LM

Tick
Tick
Actual size
Actual size
1 mm
1 mm
Scanning
Scanning
electron microscope SEM
electron microscope SEM
10 nm–1
10 nm–1 mmmm

μ mμ m
100100

Red cells
Red blood blood cells
LM LM
5 μm 5 μm
mμm
10 μ10
range of organisms in this book
range of organisms in this book

Transmission
Transmission
electron
electronmicroscope
microscope TEM
TEM
1010pm
pm–100
–100μm
μm
1 μm
1 μm
E. coliE.
bacteria
coli bacteria
SEM SEM
2 μm 2 μm
100100
nm nm

T-even bacteriophages
10 nm T-even bacteriophages
10 nm (viruses)
(viruses)
Atomic force
Atomic force AFM
microscope
TEM
50 nm
TEM
microscope AFM 50 nm
0.1 nm–10 nm
0.1 nm–10 nm
micro
1 nm tip micro
1 nm tip
If a bacterium is one
If a bacterium
micrometer in lengthisandone
your index finger is 6.5 cm and
micrometer in length
0.1 nm DNA double helix long, your index of
how many finger
the is 6.5 cm
0.1 nm long,
DNA double helix bacteria canhow
you many
place of the
AFM
10 nm bacteria
end-to-end on can
your you place
finger?
AFM
10 nm end-to-end
Answer: 32,500. on your finger?
Answer: 32,500.
10 pm
10 pm
58
 MICROSCOPIO DE CAMPO
Ocular lens

OSCURO 60 PART ONE Fundamentals of Microbiology

Objective lens

Specimen

Eye Eye
Condenser lens
Fundamentals of Microbiology
Ocular lens

Eye Eye Eye Light

Only light reflected
Objective lens by the specimen is
captured by the
objective lens
Ocular lens Ocular lens
Specimen
Unreflected light
Diffraction plate
Condenser lens
Undiffracted light
(unaltered by specimen)
Opaque disk
Only light reflected LM
20 μ m
Objective lens by the specimen is Objective lens
(a) Brightfield. (Top) The path of light in (b) Darkfiel
captured
Light by the Light
brightfield microscopy, the type of uses a spec
Refracted or diffracted
objective lens illumination produced by regular compound disk that elim
lightBrightfield
light microscopes. (Bottom) (altered by the beam. T
illumination shows internal structures
specimen) and the specimen co
Specimen Unreflected light outline of the transparent pellicle (external light reflecte
covering). reaches the
Specimen the black ba
microscopy,
some intern
Condenser lens Condenser lens the pellicle i

Opaque disk LM
Annular diaphragm
LM
20 μ m 20 μ m
Figure 3.4 Brightfield, darkfield, and phase-contrast micro
(a) Brightfield. (Top) The path of light in (b) Darkfield.
contrasting (Top)
light The darkfield
pathways of eachmicroscope (c) Phase-c
of these types of microscopy
brightfield microscopy, the type of uses a special condenser with an opaque microscopyt
protozoan Paramecium using these three different microscopy
illumination produced by regular compound disk that eliminates all light in the center of light passin
Light Light Lightonly light that reaches the
light microscopes. (Bottom) Brightfield
illumination shows internal structures and the
Q
the beam.
WhatThe shaped)
are the advantages of brightfield, darkfield,
specimen comes in at an angle; thus, only
and dia
ph
(unaltered b
outline of the transparent pellicle (external light reflected by the specimen (blue rays) path from li
covering). reaches the objective lens. (Bottom) Against diffracted a
can be rotated
the black and seen
background viewedwithin any orientation. This
darkfield Thesetechn
two s
microscopy, edges of the cell are bright,
been used to obtain three-dimensional images of entire eye. Reflec
some internal structures seem to sparkle, and indicated in
cellular components
the pellicle (Figure 3.7). In addition, confocal
is almost visible. Phase-contmi
can be used to evaluate cellular physiology by monito differentiatio
 Ocular lens Ocular lens

MICROSCOPIO DE Diffraction plate

Undiffracted light
(unaltered by specimen)

CONTRASTE DE FASES
Only light reflected
Objective lens by the specimen is Objective lens
captured by the
Refracted or diffracted
objective lens
light (altered by
Specimen specimen)
Unreflected light
Specimen

Condenser lens Condenser lens

Eye
Opaque disk Annular diaphragm

Ocular lens
ght Light Light
Diffraction plate
Undiffracted light
(unaltered by specimen)
nly light reflected
the specimen is Objective lens
ptured by the
Refracted or diffracted
jective lens
light (altered by
specimen)
nreflected light
Specimen
LM LM LM
20 μ m 20 μ m 20 μ m
Condenser lens
he path of light in (b) Darkfield. (Top) The darkfield microscope (c) Phase-contrast. (Top) In phase-contrast
he type of uses a special condenser with an opaque microscopy, the specimen is illuminated by
paque disk
y regular compound
Annular diaphragm
disk that eliminates all light in the center of light passing through an annular (ring-
om) Brightfield the beam. The only light that reaches the shaped) diaphragm. Direct light rays
nal structures and the specimen comes in at an angle; thus, only (unaltered by the specimen) travel a different
nt pellicle (external light reflected by the specimen (blue rays) path from light rays that are reflected or
Light
reaches the objective lens. (Bottom) Against diffracted as they pass through the specimen.
the black background seen with darkfield These two sets of rays are combined at the
microscopy, edges of the cell are bright, eye. Reflected or diffracted light rays are
some internal structures seem to sparkle, and indicated in blue; direct rays are red. (Bottom)
the pellicle is almost visible. Phase-contrast microscopy shows greater
 TIPOS DE MICROSCOPIOS CHAPTER 3 Observing Microorganisms Through a Microscope 65

TABLE 3.2 A Summary of Various Types of Microscopes

Microscope Type Distinguishing Features Typical Image Principal Uses

Light
Brightfield Uses visible light as a source To observe various stained
of illumination; cannot resolve specimens and to count
structures smaller than about microbes; does not resolve
0.2 μm; specimen appears very small specimens, such
against a bright background. as viruses.
Inexpensive and easy to use.

Paramecium LM
25 μ m
Darkfield Uses a special condenser with To examine living
an opaque disk that blocks microorganisms that are
light from entering the objective invisible in brightfield
lens directly; light reflected by microscopy, do not stain
specimen enters the objective lens, easily, or are distorted by
and the specimen appears light staining; frequently used to
against a black background. detect Treponema pallidum
in the diagnosis of syphilis.

Paramecium LM
25 μ m

Phase-contrast Uses a special condenser To facilitate detailed examination

containing an annular (ring-shaped) of the internal structures of
diaphragm. The diaphragm allows living specimens.
direct light to pass through the
condenser, focusing light on the
specimen and a diffraction plate
 Paramecium LM
25 μ m
Darkfield Uses a special condenser with To examine living

TIPOS DE MICROSCOPIOS
an opaque disk that blocks
light from entering the objective
lens directly; light reflected by
specimen enters the objective lens,
microorganisms that are
invisible in brightfield
microscopy, do not stain
easily, or are distorted by
and the specimen appears light staining; frequently used to
against a black background. detect Treponema pallidum
in the diagnosis of syphilis.

Paramecium LM
25 μ m

Phase-contrast Uses a special condenser To facilitate detailed examination

containing an annular (ring-shaped) of the internal structures of
diaphragm. The diaphragm allows living specimens.
direct light to pass through the
condenser, focusing light on the
specimen and a diffraction plate
in the objective lens. Direct and
reflected or diffracted light rays are
brought together to produce the
image. No staining required.
Paramecium LM
25 μ m

Differential Like phase-contrast, uses To provide three-dimensional

interference differences in refractive indexes images.
contrast (DIC) to produce images. Uses two
beams of light separated by
prisms; the specimen appears
colored as a result of the prism
effect. No staining required.

Paramecium LM
23 μ m

Fluorescence Uses an ultraviolet or near-ultraviolet For fluorescent-antibody

source of illumination that causes techniques
fluorescent compounds (green-colored) (immunofluorescence)
in a specimen to emit light. to rapidly detect and
identify microbes in tissues
or clinical specimens.
 image. No staining required.
Paramecium LM
25 μ m

Differential Like phase-contrast, uses To provide three-dimensional

TIPOS DE MICROSCOPIOS
interference
contrast (DIC)
differences in refractive indexes
to produce images. Uses two
beams of light separated by
prisms; the specimen appears
images.

colored as a result of the prism

effect. No staining required.

Paramecium LM
23 μ m

Fluorescence Uses an ultraviolet or near-ultraviolet For fluorescent-antibody

source of illumination that causes techniques
fluorescent compounds (green-colored) (immunofluorescence)
in a specimen to emit light. to rapidly detect and
identify microbes in tissues
66 PART ONE Fundamentals of Microbiology or clinical specimens.

TABLE 3.2 A Summary of Various Types of Microscopes (continued)

Treponema pallidum LM
Microscope Type Distinguishing Features Typical Image 2 μm Principal Uses

Confocal Uses a single photon to illuminate To obtain two- and (continued)

one plane of a specimen at a time. three-dimensional images
of cells for biomedical
applications.

Paramecium CF
25 μ m

Two-Photon Uses two photons to illuminate To image living cells, up to depth

a specimen. of 1 mm, reduce phototoxicity,
and observe cell activity in real
time.
Microscope Type Distinguishing Features Typical Image Principal Uses

Confocal Uses a single photon to illuminate To obtain two- and

one plane of a specimen at a time. three-dimensional images

TIPOS DE MICROSCOPIOS
of cells for biomedical
applications.

Paramecium CF
25 μ m

Two-Photon Uses two photons to illuminate To image living cells, up to depth

a specimen. of 1 mm, reduce phototoxicity,
and observe cell activity in real
time.

Paramecium TPM
22 μ m

Scanning Acoustic Uses a sound wave of specific To examine living cells attached
frequency that travels through the to another surface, such as
specimen with a portion being cancer cells, artery plaque, and
reflected when it hits an interface biofilms.
within the material.

Biofilm SAM
180 μ m

Electron
Transmission Uses a beam of electrons instead To examine viruses or the
of light; electrons pass through the internal ultrastructure in thin
specimen; because of the shorter sections of cells (usually
wavelength of electrons, structures magnified10,000–100,000×).
 TPM
22 μ m

Scanning Acoustic Uses a sound wave of specific To examine living cells attached

TIPOS DE MICROSCOPIOS
frequency that travels through the to another surface, such as
specimen with a portion being cancer cells, artery plaque, and
reflected when it hits an interface biofilms.
within the material.

Biofilm SAM
180 μ m

Electron
Transmission Uses a beam of electrons instead To examine viruses or the
of light; electrons pass through the internal ultrastructure in thin
specimen; because of the shorter sections of cells (usually
wavelength of electrons, structures magnified10,000–100,000×).
smaller than 0.2 μm can be
resolved. The image produced is
two-dimensional.

Paramecium TEM
25 μ m

Scanning Uses a beam of electrons instead To study the surface features

of light; electrons are reflected from of cells and viruses (usually
the specimen; because of the shorter magnified 1000–10,000×).
wavelength of electrons, structures
smaller than 0.2 μm can be resolved.
The image produced appears
three-dimensional.

Paramecium SEM
25 μ m
 TIPOS DE MICROSCOPIOS CHAPTER 3 Observing Microorganisms Through a Microscope 67

TABLE 3.2 (continued)

Microscope Type Distinguishing Features Typical Image Principal Uses

Scanned-Probe
Scanning Uses a thin metal probe that scans Provides very detailed views of
tunneling a specimen and produces an image molecules inside cells.
revealing the bumps and depressions
of the atoms on the surface of the
specimen. Resolving power is much
greater than that of an electron
microscope. No special preparation
required.

RecA protein STM

from E. coli 45 nm

Atomic force Uses a metal-and-diamond probe Provides three-dimensional

gently forced down along the images of biological specimens
surface of the specimen. Produces a at high resolution in nearly
three-dimensional image. No special atomic detail and can measure
preparation required. physical properties of biological
specimens and molecular
processes.

Perfringolysin O AFM
toxin from Clostri- 9 nm
dium perfringens

Because most microorganisms appear almost colorless when color of so-called basic dyes is in the positive ion; in acidic
viewed through a standard light microscope, we often must pre- dyes, it is in the negative ion. Bacteria are slightly negatively
pare them for observation. One way to do this is to stain (color) charged at pH 7. Thus, the colored positive ion in a basic dye
PREPARACIONES DE
MUESTRAS
 FIJACIÓN

La fijación es el proceso por el que se preservan y se

fijan en una posición las estructuras internas y
externas de las células y los microorganismos.

Inactiva enzimas que podrían alterar la morfología

celular y endurece las estructuras celulares.

Durante la fijación los microorganismos se inactivan

y se unen firmemente al portaobjetos.
 FIJACIÓN

La fijación por calor preserva la morfología global

pero no las estructuras intracelulares.

La fijación química se utiliza para proteger

subestructuras celulares finas y la morfología de
microorganismos más grandes.
 TINCIONES

Tinción simple.

Tinción diferencial.

Tinción especial.
TINCIÓN
SIMPLE
TINCIÓN SIMPLE
 TINCIÓN
DIFERENCIAL
 TINCION DE GRAM
68 PART ONE Fundamentals of Microbiology

KEY

Crystal violet
Iodine
Alcohol
Safranin

Gram-positive

Gram-negative

1 Application of 2 Application of 3 Alcohol wash 4 Application of

crystal violet iodine (mordant) (decolorization) safranin (counterstain)
(a) (purple dye)

Rod
(gram-negative)

Cocci
(gram-positive)
TINCION DE GRAM

1 Application of 2 Application of 3 Alcohol wash 4 A

crystal violet iodine (mordant) (decolorization) s
(a) (purple dye)

Rod
(gram-negative)

Cocci
(gram-positive)

Figure 3.12 Gram stain

gram-stained bacteria. The
rods (pink) are gram-negat

(b)
Q How can the Gram r
LM
1.5 μ m treatment?

examined. Occasionally, a chemical is added to the solution to procedures because it

intensify the stain; such an additive is called a mordant. One gram-positive and gram
function of a mordant is to increase the affinity of a stain for a bi- In this procedure (F
 TINCION ACIDA

70 PART ONE Fundamentals of Microbiology

Chapter 4. In medical microbio

M. bovis a capsule is a means of determ
degree to which a pathogen can
Capsule staining is more d
procedures because capsular
may be dislodged or removed d
strate the presence of capsules
teria in a solution containing a
particles (usually India ink or
background and then stain th
as safranin (Figure 3.14a). Beca
capsules do not accept most bi
LM
8 μm thus appear as halos surroundi

Figure 3.13 Acid-fast bacteria. The Mycobacterium bovis bacteria that Endospore (Spore) Staining
have infected this tissue have been stained pink or red with an acid-fast
An endospore is a special re
stain. Non–acid-fast cells (Staphylococcus) are stained with the methylene
blue counterstain. within a cell that protects a bac
conditions. Although endospo
 ZIEHL-NEELSEN
Baciloscopía Ácido-Alcohol Resistente (BAAR)
TINCIONES
ESPECIALES
 have infected this tissue have been stained pink or red with an acid-fast
An endospore is a special
stain. Non–acid-fast cells (Staphylococcus) are stained with the methylene
blue counterstain. within a cell that protects a ba

TINCION
Q NEGATIVA
Why is Mycobacterium tuberculosis easily identified by the
acid-fast stain ?
conditions. Although endosp
terial cells, they can be form
dospores cannot be stained
staining and Gram staining,
Negative Staining for Capsules wall of the endospore.
Many microorganisms contain a gelatinous covering called a capsule, The most commonly us
which we will discuss in our examination of the prokaryotic cell in Fulton endospore stain (F

Capsules

Endospore

(a) Negative staining LM (b) Endospore staining

10 μ m
k or red with an acid-fast
An endospore is a special resistant, dormant structure formed
tained with the methylene
within a cell that protects a bacterium from adverse environmental

SCHAEFFER-FULTON
conditions. Although endospores are relatively uncommon in bac-
identified by the terial cells, they can be formed by a few genera of bacteria. En-
dospores cannot be stained by ordinary methods, such as simple
staining and Gram staining, because the dyes do not penetrate the
wall of the endospore.
covering called a capsule, The most commonly used endospore stain is the Schaeffer-
of the prokaryotic cell in Fulton endospore stain (Figure 3.14b). Malachite green, the

Capsules

Endospore

LM (b) Endospore staining LM

10 μ m 12 μ m
 TINCION DE FLAGELOS
Endospore

LM (b) Endospore staining LM

10 μ m 12 μ m

ning provides a
bacteria, Klebsiella
the stained cells.
od-shaped cells of the
ton endospore stain. Flagellum
nds of these cells of
e body of the cell, the
yers of the stain have
th a mordant.
(c) Flagella staining LM
nd flagella to bacteria? 4 μm