Instituto Tecnológico de Zacatepec

Departamento de Ingeniería Química y Bioquímica

UNIDAD II METODOS Y TECNICAS DE CULTIVO

MICROBIANO.

PROFESOR: M.B ABEL FLORES MORENO

2.1. Cultivo de microorganismos
2.1.1 Definicion y tipos de medios de cultivo
Much of the study of microbiology depends on the ability to grow and maintain
microorganisms in the laboratory, and this is possible only if suitable culture
media are available. A culture medium is a solid or liquid preparation used to
grow, transport, and store microorganisms. To be effective, the medium must
contain all the nutrients the microorganism requires for growth.
Specialized media are essential in the isolation and identification of
microorganisms, the testing of antibiotic sensitivities, water and food analysis,
industrial microbiology, and other activities.
Although all microorganisms need sources of energy, carbon, nitrogen,
phosphorus, sulfur, and various minerals, the precise composition of a satisfactory
medium will depend on the species one is trying to cultivate because nutritional
requirements vary so greatly. Knowledge of a microorganism’s normal habitat
often is useful in selecting an appropriate culture medium because its nutrient
requirements reflect its natural surroundings.
Frequently a medium is used to select and grow specific microorganisms or to
help identify a particular species. In such cases the function of the medium also
will determine its composition.
Los medios de cultivo son sustratos o soluciones de
nutrientes que permite el desarrollo de
microorganismos. En las condiciones de laboratorio
para realizar un cultivo, se debe sembrar sobre el
medio de cultivo elegido las muestras en las que los
microorganismos van a crecer y multiplicarse para
formar colonias.
 Medios de cultivo.
Tipos de los medios de cultivo .
Los medios de cultivo pueden clasificarse
según diferentes criterios, pero los más
importantes son aquellos que se basan en:
a) Su consistencia
b) Su utilización
c) Su composición
d) Su origen

Tarea investigar cada una de las

clasificaciones anteriores.
2.1.2 Preparación de medios de cultivo

Los medios de cultivo se preparan:

1. En el laboratorio a partir de sus diferentes
componentes.
2. A partir de productos en polvo deshidratados
comerciales o:
3. Pueden adquirirse medios listos para su uso.

Se puede tomar como base el documento

ISO 7218:1996(E) Microbiology of food and animal feeding
stuff-General rules for microbiological examinations para:
-La preparación y esterilización de medios.
-Tiempos de almacenamiento recomendados.
2.2. Morfología microscópica

Transparent living microorganisms,

such as the syphilis spirochaete, can
be seen much more easily when
observed in a dark field.
2.2.1 Preparaciones en fresco
Para medios líquidos
 2.2.2 Tinciones
Bacterial Staining
Fixed, stained preparations are most frequently used for the observation of the
morphological characterization of bacteria. The advantages of this procedure are
that:

a) The cells are made more clearly visible after they are colored.
b) Differences between cells of different species and within the same species can
be demonstrated by use of appropriate staining solutions (differntial or selective
staining).

The essential steps in the preparation of a fixed, stained smear are:

1.-Preparation of the film (or) smear
2.-Fixation and
3.-Application of one (or) more staining solutions.
Microbiological stains:
A large number of colored organic compounds (dyes) are available for staining
microorganisms.
On the basis may be classified into groups such as:
a) Triphenylmethane dyes
b) Oxazine dyes
c) Thiazine dyes

On the based on the chemical behavior of the dye; ion in which the charge on
the dye ion is negative; a basic (or) neutral. An acid (or anionic) dye is one
which the charge on the dye ion is negative; a basic (or cationic) dye is one in
which the charge carried by the dye ion is positive.
Acid dyes generally stain basic cell components, and basic dyes generally stain
acidic cell components.
Staining mechanism:

The process of staining may involve ion-exchange

reactions between the stain and active sites at the surface of
(or) within the cell. Certain chemical grouping of cell
proteins (or) nucleic acids may be involved in salt
formation with positively charged ions such as Na+ (or)
K+.
Peripheral (or) outer area of the cell as carrying a negative
in combination with positively charged ions.
Simple staining:

The coloration of bacteria by applying a single solution of

stain to a fixed smear is termed as “Simple staining”. The
fixed smear is flooded with a dye solution for a specified
period of time after which this solution for a specified
period of time after which this solution is washed off with
water and the alide blotted dry.
Differential staining:

Staining procedure that make visible differences

between bacterial cells (or) parts of a bacterial cell
are termed “Differential staining techniques”.
Gram- Staining:
One of the most important and widely used differential techniques in
microbiology is ‘Gram staining’. This technique was first introduced by “Christian
Gram’ in 1884.
In this process the fixed bacterial smear is subjected to the following staining
reagents in the order listed:
Crystal violet,
Iodine solution,
Alcohol(decolorizing agent), and
Safrain (or) some other suitable counterstain.
This gram method fall into two groups:
Gram positive bacteria – which retain the crystal violet and hence appear drop
violet in color.
Gram Negative bacteria – Which lose the crystal violet are counterstained by the
safranin and hence appear red in color.
Procedure:
1. Prepare the smear on the slides with the bacterial cultures as done for the
simple positive staining method.
2. Stain it for one minute with crystal violet solution (gram’s stain) wash it
in tap water.
3. Apply the iodine solution (Morbant) for one minute. Wash intap water.
4. Decolorize with alcohol by adding dropwise on the tilted slide untill all
free blue color has been removed (20 to 30 sec), wash it in tap water.
5. Flood the slide with safranin (counter stain) for one minute. Wash it in tap
water and air dry.
6. Examine the stained smear under the oil immersion objective to
determine, which organism is Gram positive(violet color) and which is gram
negative(Pink color)
2.3 Aislamiento y características para la identificación de microorganismos

Isolation of Pure Culture

Introduction to isolation of pure culture

Microorganisms are generally found in nature (air, soil and water) as

mixed populations. Even the diseased pans of plants and animals
contain a great number of microorganisms, which differ markedly
from the microorganisms of other environments. To study the specific
role played by a specific microorganism in its environment, one must
isolate the same in pure culture. Pure culture involves not only
isolation of individual microorganisms from a mixed population, but
also (he maintenance of such individuals and their progenies in
artificial media, where no other microorganisms find way to grow.
 However, it is not easy to isolate (he individual microorganism;;
from natural habitats and grow them under imposed laboratory
conditions. For this, great deal of laboratory manipulation is
required. If inoculums from any natural habitat is taken and allowed
to grow in a culture medium, a large number of diverse colonies
may develop that, due to crowdedness, may run together and,
thereby, may lose individuality. Therefore, it is necessary to make
the colonies well-isolated from each other so that each appears
distinct, large and shows characteristic growth forms. Such colonies
may be picked up easily and grown separately for detailed study.
Several method for obtaining pure cultures are in use. Some
common methods are in everyday-use by a majority of n special
purposes.
COMMON METHODS
Pure culture of microorganisms that form discrete colonies
on solid medium., yeasts, most bacteria, many other micro
fungi, and unicellular micro algae, may be most commonly
obtained by plating methods such as streak plate method,
pour plate method and spread plate method, But, the
microbes that have not yet been successfully cultivated on
solid media and are cultivable only in liquid media are
generally isolated by serial dilution method.
 1. Streak Plate Method
a) This method is used most commonly to isolate pure cultures of bacteria. A small amount of
mixed culture is placed on the tip of an inoculation loop/needle and is streaked across the
surface of the agar medium.
b) The successive streaks "thin out” the inoculums sufficiently and the microorganisms are
separated from each other.
c) It is usually advisable to streak cut a second plate by the samenloop/needle without
reinoculation.
d) These plates are incubated to allow the growth of colonies.
e) The key principle of this method is that, by streaking, a dilution gradient is established
across the face of the Petri plate as bacterial cells are deposited on the agar surface. Because
of this dilution gradient, confluent growth does not take place on that part of the medium
where few bacterial cells are deposited.
f) Presumably, colony is the progeny of a single microbial cell thus representing a clone of
pure culture.
g) Such isolated colonies are picked up separately us re-streaked onto fresh media to ensure
purity.
2. Pour Plate Method

1.-This method involves plating of diluted samples mixed with melted agar medium.

2.-The main principle is to dilute the inoculum insuccessive tubes Containing

liquefied agar medium so as lo permit a thorough distribution of bacterial Here, the
mixed culture of bacteria is diluted directly in tubes contain maintained in the liquid
state at a temperature of 42-45 ֯C (agar soli difies below 42֯C).0
3.-The Bacteria and melted medium are mixed well.
4.-The contents of each tube are poured into separate Petri plates, allowed to solidify,
and
then incubate. When bacterial colonies develop, one finds that isolated colonies
develop
both within agar medium and on the medium (surface colonies).
5.-These isolate colonies are then picked up by inoculation loop and streaked onto
another Petri plate to insure purity.
 3. Spread Plate Method
1.- In this method, the mixed culture of microorganisms is not diluted in the
melted agar medium (unlike the pour plate method); it is rather diluted in a
series of tubes containing sterile liquid, usually, water or physiological saline.
2.-A drop of so diluted liquid from each tube is placed on the centre of an agar
plate and spread evenly over the surface by means of a sterilized bent-glass-rod.
3.-The medium is now incubated. When the colonies develop on the agar
medium plates, it is found that there are some plates in which well-isolated
colonies grow.
4.-This happens as a result of separation of individual microorganisms by
spreading over the drop of diluted liquid on the medium of the plate.
5.-The isolated colonies are picked up and transferred onto fresh medium to
ensure purity. In contrast to pour plate method, only surface colonies develop in
this method and the microorganisms are not required to withstand the
temperature of the melted agar medium.
 2.3.1 Aislamiento por la técnica de diluciones y estría cruzada.
4. Serial Dilution Method

1.- This method is commonly used to obtain pure cultures of those

microorganisms that have not yet been successfully cultivated on
solid media and grow only in liquid media.

2.- A microorganism that predominates in a mixed culture can be

isolated in pure form by a series of dilutions. The inoculums is
subjected to serial dilution in a sterile liquid medium, and a large
number of tubes of sterile liquid medium are inoculated with
aliquots of each successive dilution.
3.-The aim of this dilution is to inoculate a series of tubes with a
microbial suspension so dilute that there are some tubes showing growth
of only one individual microbe. For convenience, suppose we have a
culture containing 10 ml of liquid medium, containing 1,000
microorganisms i.e., 100 microorganisms/ml of the medium.

4.- If we take out 1 ml of this medium and mix it with 9 ml of fresh

sterile liquid m, we would then have 100 microorganisms in 10 ml or 10
microorganisms /ml. If we ml of this suspension to another 9 ml. of fresh
sterile liquid medium, each ml would maintain a single microorganism. If
this tube shows any microbial! Growth, there is a very high probability
that this growth has resulted from the introduction of a single
microorganism medium and represents the pure culture of that
microorganism.
2.2. Morfología microscópica
Morfología colonial de las bacterias en las cajas petri.

Se realiza la descripción de cada una de las colonias:

TAMAÑO: Diámetro en mm, los límites de tamaño de
las colonias varían desde fracciones de milímetros hasta
10 mm de diámetro. La mayoría de los microorganismos
forman colonias de tamaño limitado al tiempo de
incubación.
FORMA: Es la apreciación general de su figura la cual puede ser:
ELEVACIÓN: Que puede ser:
BORDE O MARGEN: Se refiere al contorno de las colonias y puede ser:
Entero, ondulado, Lobulado, crenado, filamentoso o rizado.
COLOR: Blanco, amarillo, negro, marrón, anaranjado, etc
SUPERFICIE: Lisa o rugosa.

ASPECTO: Húmedo, seco, algodonoso, pulverulento, aterciopelado, velloso,

granuloso.

CONSISTENCIA: Suave (butirosa), viscosa, membranosa o dura, esta prueba se

tiene que realizar con la ayuda de una asa.

LUZ REFLEJADA: Las colonias pueden ser brillantes o mate.

LUZ TRANSMITIDA: Las colonias pueden ser translúcidas o transparentes.

2.3.3 Pruebas bioquímicas, moleculares y serológicas
 Métodos basados en pruebas bioquímicas

Las pruebas bioquímicas han sido

ampliamente utilizadas para
diferenciar bacterias. Estas
pruebas se fundamentan en
demostrar si el microorganismo
es capaz de fermentar azúcares, la
presencia de enzimas, la
degradación de compuestos, la
producción de compuestos
coloreados, etc.
Existen flujogramas, como el que se
describe a continuación para la
identificación bacteriana, mediante
pruebas bioquímicas de
microorganismos. Por ejemplo, la
presencia de un coco bacilo gram
negativo, facultativo, fermentador de
glucosa, oxidasa negativo, nos indica la
presencia de una enterobacteria, para
determinar su género podemos seguir el
siguiente esquema.
Los géneros clínicamente mas importantes de la familia
Enterobacteriaceae son: Escherichia, Salmonella,
Shigella, Citrobacter y Enterobacter. El género
Escherichia, Enterobacter y Citrobacter fermentan la
lactosa con producción de acidos y gas, a diferencia de
los géneros Salmonella y Shigella que no fermentan la
lactosa.
El tiempo necesario para la identificación de bacterias puede reducirse
considerablemente con el uso de sistemas miniaturizados basados en
pruebas bioquímicas. Estas herramientas que permiten reducir el tiempo del
reporte, en primer lugar fueron elaborados para bacterias de importancia
médica, tales como las enterobacterias. Estos sistemas han sido diseñados
para realizar varias pruebas bioquímicas simultáneamente y permitir la
identificación en un tiempo más corto. Cada uno de los ensayos, consta de
tubos miniaturizados que contienen el medio de cultivo que se hidratan al
inocularlos con la suspensión bacteriana pura. Las pruebas se clasifican en
grupos; a cada uno de los resultados positivos de los ensayos se le asigna
un determinado valor numérico, obteniéndose un código que corresponderá
a un determinado género o especie en un texto de la base de datos.
APIS
 Métodos moleculares

Modernamente adquiere más importancia el uso de

métodos basados en biología molecular donde, a
través de procedimientos y reactivos, se pueden
detectar determinadas secuencias de ADN que son
propias de un determinado agente microbiano.
El método que se está utilizando ampliamente en los laboratorios
de diagnostico es el PCR (Polymerase Chain Reaction), que se
aplica generalmente para la identificación de microorganismos
que no pueden ser cultivados por los métodos convencionales.
A través de este método, puede aumentarse la cantidad de ADN
hasta niveles detectables mediante electroforesis o mediante
sondas de ADN
 Métodos serológicos

Se basa en la detección en suero sanguíneo del paciente los anticuerpos o

inmunoglobulinas especificas para un tipo especifico de microorganismo
patógeno lo cual permite identificarlo.