Sie sind auf Seite 1von 9

Culture media contains nutrients and physical growth parameters necessary for microbial

growth. All microorganisms cannot grow in a single culture medium and in fact many can’t
grow in any known culture medium.
Organisms that cannot grow in artificial culture medium are known as obligate
parasites. Mycobacterium leprae, rickettsias, Chlamydias, and Treponema pallidum are
obligate parasites. Bacterial culture media can be distinguished on the basis of composition,
consistency and purpose.

Classification of culture media used in Microbiology


laboratory on the basis of consistency
1. Solid medium
Solid medium contains agar at a concentration of 1.5-2.0% or some other, mostly inert
solidifying agent. Solid medium has physical structure and allows bacteria to grow in
physically informative or useful ways (e.g. as colonies or in streaks). Solid medium is
useful for isolating bacteria or for determining the colony characteristics of the
isolate.
2. Semisolid media
They are prepared with agar at concentrations of 0.5% or less. They have soft custard
like consistency and are useful for the cultivation of microaerophilic bacteria or
for determination of bacterial motility.
3. Liquid (Broth) medium
These media contains specific amounts of nutrients but don’t have trace of gelling
agents such as gelatin or agar. Broth medium serves various purposes such as
propagation of large number of organisms, fermentation studies, and various other
tests. e.g. sugar fermentation tests, MR-VR broth.

Classification of culture media based on the basis of composition

1. Synthetic or chemically defined medium


A chemically defined medium is one prepared from purified ingredients and therefore
whose exact composition is known.
2. Non synthetic or chemically undefined medium

Non-synthetic medium contains at least one component that is neither purified nor completely
characterized nor even completely consistent from batch to batch. Often these are partially
digested proteins from various organism sources. Nutrient broth, for example, is derived from
cultures of yeasts.

Synthetic medium may be simple or complex depending up on the supplement incorporated


in it. A simple non-synthetic medium is capable of meeting the nutrient requirements of
organisms requiring relatively few growth factors where as complex non-synthetic medium
support the growth of more fastidious microorganisms.

Classification of Bacterial Culture Media based on the basis of purpose/


functional use/ application

Many special purpose media are needed to facilitate recognition, enumeration, and isolation
of certain types of bacteria. To meet these needs, numerous media are available.
1. General purpose media/ Basic media
Basal media are basically simple media that supports most non-fastidious bacteria. Peptone
water,nutrient broth and nutrient agar are considered as basal medium. These media are
generally used for the primary isolation of microorganisms.

Nutrient Agar

2. Enriched medium (Added growth factors):

Blood Agar

Addition of extra nutrients in the form of blood, serum, egg yolk etc, to basal medium makes
them enriched media. Enriched media are used to grow nutritionally exacting (fastidious)
bacteria. Blood agar, chocolate agar, Loeffler’s serum slope etc are few of the enriched media.
Blood agar is prepared by adding 5-10% (by volume) blood to a blood agar base. Chocolate
agar is also known as heated blood agar or lysed blood agar.

3. Selective and enrichment media are designed to inhibit unwanted commensal or contaminating
bacteria and help to recover pathogen from a mixture of bacteria. While selective media are agar
based, enrichment media are liquid in consistency. Both these media serve the same purpose.
Any agar media can be made selective by addition of certain inhibitory agents that don’t affect
the pathogen of interest. Various approaches to make a medium selective include addition of
antibiotics, dyes, chemicals, alteration of pH or a combination of these.

a. Selective medium
Principle: Differential growth suppression
Selective medium is designed to suppress the growth of some microorganisms while allowing the
growth of others. Selective medium are agar based (solid) medium so that individual colonies
may be isolated.

Examples of selective media include:

1. Thayer Martin Agar used to recover Neisseria gonorrhoeae contains antibiotics;


vancomycin, colistin and nystatin.
2. Mannitol Salt Agar and Salt Milk Agar used to recover S.aureus contains 10% NaCl.
3. Potassium tellurite medium used to recover C.diphtheriae contains 0.04% potassium

tellurite.
4. MacConkey’s Agar used for Enterobacteriaceae members contains bile salt that inhibits
most gram positive bacteria.
5. Pseudosel Agar (Cetrimide Agar) used to recover P. aeruginosa contains cetrimide
(antiseptic agent).
6. Crystal Violet Blood Agar used to recover S. pyogenes contains 0.0002% crystal violet.
7. Lowenstein Jensen Medium used to recover M.tuberculosis is made selective by
incorporating malachite green.
8. Wilson and Blair’s Agar for recovering S. typhi is rendered selective by the addition of dye
brilliant green.
9. Selective media such as TCBS Agar used for isolating V. cholerae from fecal specimens
have elevated pH (8.5-8.6), which inhibits most other bacteria.

b. Enrichment culture medium


Enrichment medium is used to increase the relative concentration of certain microorganisms in
the culture prior to plating on solid selective medium. Unlike selective media, enrichment culture
is typically used as broth medium. Enrichment media are liquid media that also serves to inhibit
commensals in the clinical specimen. Selenite F broth, tetrathionate broth and alkaline peptone
water (APW) are used to recover pathogens from fecal specimens.

4. Differential/ indicator medium: differential appearance:


Certain media are designed in such a way that different bacteria can be recognized on the basis
of their colony colour. Various approaches include incorporation of dyes, metabolic substrates
etc, so that those bacteria that utilize them appear as differently coloured colonies. Such media
are called differential media or indicator media. Differential media allow the growth of more than
one microorganism of interest but with morphologically distinguishable colonies.
Examples of differential media include:

1. Mannitol salts agar (mannitol fermentation = yellow)


2. Blood agar (various kinds of hemolysis i.e. α, β and γ hemolysis)
3. Mac Conkey agar (lactose fermenters, pink colonies whereas non- lactose fermenter
produces pale or colorless colonies.
4. TCBS (Vibrio cholerae produces yellow colonies due to fermentation of sucrose)

5. Transport media:
Clinical specimens must be transported to the laboratory immediately after collection to prevent
overgrowth of contaminating organisms or commensals. This can be achieved by using transport
media. Such media prevent drying (desiccation) of specimen, maintain the pathogen to
commensal ratio and inhibit overgrowth of unwanted bacteria. Some of these media (Stuart’s &
Amie’s) are semi-solid in consistency. Addition of charcoal serves to neutralize inhibitory factors.

 Cary Blair transport medium and Venkatraman Ramakrishnan (VR) medium are used to
transport feces from suspected cholera patients.
 Sach’s buffered glycerol saline is used to transport feces from patients suspected to be
suffering from bacillary dysentery.
 Pike’s medium is used to transport streptococci from throat specimens.

6. Anaerobic media:
Anaerobic bacteria need special media for growth because they need low oxygen content,
reduced oxidation –reduction potential and extra nutrients.
Media for anaerobes may have to be supplemented with nutrients like hemin and vitamin K. Such
media may also have to be reduced by physical or chemical means. Boiling the medium serves
to expel any dissolved oxygen. Addition of 1% glucose, 0.1% thioglycollate, 0.1% ascorbic acid,
0.05% cysteine or red hot iron filings can render a medium reduced. Before use the medium
must be boiled in water bath to expel any dissolved oxygen and then sealed with sterile liquid
paraffin.

Robertson Cooked Meat (RCM) medium that is commonly used to grow Clostridium spps contains
a 2.5 cm column of bullock heart meat and 15 ml of nutrient broth. Thioglycollate broth contains
sodium thioglycollate, glucose, cystine, yeast extract and casein hydrolysate.

Methylene blue or resazurin is an oxidation-reduction potential indicator that is incorporated in


the medium. Under reduced condition, methylene blue is colorless.
7. Assay media
These media are used for the assay of vitamins, amino acids and antibiotics. E.g. antibiotic
assay media are used for determining antibiotic potency by the microbiological assay technique.
Other types of medium includes;

 Media for enumeration of Bacteria,


 Media for characterization of Bacteria,
 Maintenance media etc
https://microbeonline.com/types-of-bacteriological-culture-medium/

Common Fungal Culture Media and their uses


This post was most recently updated on May 2nd, 2019

Two general types of culture media are essential to ensure the primary recovery of all clinically significant
fungi from clinical specimens. One medium should be non-selective (such as Brain Heart Infusion
Agar; i.e., one that will permit the growth of virtually all clinically relevant fungi) and other medium
should be selective, specially tailored to isolate specific pathogenic fungi of interest.

For optimal recovery of fungal pathogen, a battery of media should be used, and the followings
are recommended:

1. Media with or without cyclohexamide (Cycloheximide is added to inhibit the growth of


rapidly growing contaminating molds.)
2. Media with or without an antibacterial agent (Chloramphenicol, Gentamicin and
Ciprofloxacin are commonly used antibacterial for this purpose).

Antibacterial agents are used to kill the contaminating bacterial species. If the sample is taken
from sterile site, it is not necessary to use media containing antibacterial agents.

1. Brain-heart infusion (BHI) agar: It is a non-selective fungal culture medium that permits the
growth of virtually all clinically relevant fungi. It is used for the primary recovery of
saprophytic and dimorphic fungi
2. Czapek’s agar: It is used for the subculture of Aspergillus species for their differential
diagnosis.
3. Inhibitory mold agar (IMA): Primary recovery of dimorphic pathogenic fungi. Saprophytic
fungi and dermatophytes will not be recovered.
4. Mycosel/Mycobiotic agar:
1. It is generally Sabouraud’s dextrose agar with cycloheximide and chloramphenicol
added.
2. It is used for the primary recovery of dermatophytes.
3. Niger Seed Agar: It is used for the identification of Cryptococcus neoformans.
5. Potato Dextrose Agar (PDA): It is a relatively rich medium for growing a wide range of
fungi.
6. Sabouraud’s Heart Infusion (SABHI) agar: Primary recovery of saprophytic and dimorphic
fungi, particularly fastidious strains.

Penicillium notatum on Sabouraud agar


Image source: ASM

7. Sabouraud’s dextrose agar (SDA):


1. Sabouraud’s agar is sufficient for the recovery of dermatophytes from cutaneous
samples and yeasts from genital cultures.
2. Not recommended as a primary isolation medium because it is insufficiently rich to
recover certain fastidious pathogenic species, particularly most of the dimorphic
fungi.
3. Sabouraud’s dextrose agar (2%) is most useful as a medium for the subculture of
fungi recovered on enriched medium to enhance typical sporulation and provide the
more characteristic colony morphology.
8. Potato flake agar: Primary recovery of saprophytic and dimorphic fungi, particularly
fastidious and slow growing strains.
Further reading and sources: For more information please read the
books:
1. Koneman’s color atlas and textbook of diagnostic microbiology and
2. Bailey and Scott’s Diagnostic Microbiology.

Cultivation of Viruses
1. 1. Cultivation of Viruses MICROBIOLOGY Islam Ghassan Sarakbi : 39
2. 2. A virus is a biological agent that reproduces inside the cells of living hosts. When infected by a
virus, a host cell is forced to produce many thousands of identical copies of the original virus, at
an extraordinary rate. Unlike most living things, viruses do not have cells that divide; new viruses
are assembled in the infected host cell.
3. 3. Viruses can't be grown in culture media or on agar plates alone, they must have living cells to
support their replication. The easiest viruses to grow are bacteriophages (because the easiest cells
to grow in the lab are bacteria).
4. 4. On the other hand, Viruses can be cultivated within suitable hosts, such as a living cell. To
study bacteriophages, for example, bacteria are grown in a suitable growth medium, then
bacteriophages are added. The bacteriophages multiply within the bacteria and increase their
numbers substantially.
5. 5. Purpose Of Virus Cultivation  The primary purposes of viral cultivation are: 1.To isolate and
identify viruses in clinical specimens. 2. To prepare viruses for vaccines. 3. To do detailed
research on viral structure, multiplication cycles, genetics and effects on host cells.
6. 6. Virus Cultivation Systems  Tissue culture system.  Embryonated eggs system.  Whole
animal systems.
7. 7. Tissue culture system For Cultivation Of Viruses Animal and plant viruses are cultivated in cell
cultures. A cell culture is prepared by encouraging cell growth outside the animal or plant source.
The cells are kept alive in a suspension of growth factors within a Petri dish. A thin layer of cells,
or monolayer, is then inoculated with viruses, and replication takes place.
8. 8. Tissue culture system For Cultivation Of Viruses (Fertilized eggs and living animals can also be
used to cultivate viruses.) For research study, viruses can be cultivated in large volumes by
inoculations to tissue culture systems. After a time, the cells are degenerated, and viruses are
harvested.
9. 9. Growing Bacteriophages in the Laboratory: Bacteriophages can be grown either in suspension
of bacteria in liquid media or in bacterial cultures on solid media. Solid media makes possible the
plaque method for detecting and counting viruses. A sample of bacteriophage is mixed with host
bacteria and melted agar. The agar containing the bacteriophages and host bacteria is then poured
into a Petri plate containing a hardened layer of agar growth medium.
10. 10. Growing Bacteriophages In The Laboratory (cont.) : The viral particles are concentrated by
precipitation methods and purified by repeated centrifugations. Highly purified viruses can be
obtained by crystallization and concentration under established conditions.
11. 11. Flasks containing tissue culture growth medium which provides nourishment to growing cells.
12. 12. The Cell Cultures • In modern usage, tissue culture generally refers to the growth of cells from
a tissue from a multicellular organism in vitro. • These cells may be cells isolated from a donor
organism, primary cells, or an immortalised cell line. • The cells are bathed in a culture medium,
which contains essential nutrients and energy sources necessary for the cells' survival.
13. 13. The Cell Cultures  The term tissue culture is often used interchangeably with Cell Culture. 
Cell cultures have replaced embryonated eggs as the preferred type of growth medium for many
viruses.  The literal meaning of tissue culture refers to the culturing of tissue pieces.
14. 14. The Cell Cultures Tissue culture is an important tool for the study of the biology of cells from
multicellular organisms. It provides an in vitro model of the tissue in a well defined environment
which can be easily manipulated and analysed.
15. 15. The Cell Cultures  Contain cell lines, called diploid cell lines, developed from human
embryos can be maintained for about 100 generations and are widely used for culturing viruses
that require a human host.  When viruses are routinely grown in the laboratory, continuous cell
lines are used. These are transformed (cancerous) cells that can be maintained through an
indefinite number of generations, and they are sometimes called mortal cell lines.
16. 16. The Cell Cultures  This cell deterioration is called cytopathic effect (CPE), can be detected
and counted in much the same way as plaques caused by bacteriophages on a lawn of bacteria. 
Primary cell lines, derived from tissue slices, tend to die out after only a few generations.
17. 17. Cell cultures
18. 18. CULTURE CONDITIONS • Culture conditions vary widely for each cell type. • The artificial
media invariably consist of a substrate or medium that supplies the essential nutrients (amino
acids, carbohydrates, vitamins,minerals), growth factors, hormones, and gases (O2, CO2). • It also
regulates the physicochemicalenvironment (pH, osmotic pressure, temperature). • Most cells are
anchoragedependent and must be cultured while attached to a solid or semi-solid
substrate(adherent or monolayer culture), • Others can be grown floating in the culture
(suspension culture).
19. 19. Suspension Adherent
20. 20. Differences between Adherent and Suspension Cultures ADHERENT CULTURES
SUSPENSION CULTURES Most cells can be cultured this way Cells which are adapted to
suspension cultures or non-adhesive cultures Passaging required at certain intervals Passaging is
much easier, can dilute culture to stimulate growth Allows easy visualisation of cells Harder to
view cells Cells dissociated enzymatically or mechanically Not require enzymatic or mechanical
dissociation
21. 21. DISADVANTAGES OF CELL CULTURES • Long period (up to 4 weeks) required for
result. • Often very poor sensitivity, sensitivity depends on a large extent on the condition of the
specimen. • Susceptible to bacterial contamination. • Susceptible to toxic substances which may be
present in the specimen. • Many viruses will not grow in cell culture e.g. Hepatitis B, Diarrhoeal
viruses, parvovirus, papillomavirus.
22. 22. Steps of cultivating animal viruses using tissue culture technique:  Cultivating animal viruses
using tissue culture technique involves following three main steps: 1. Monolayer preparation. 2.
Clonal cell line preparation. 3. Infection with virus.
23. 23. 1,2) Monolayer & Clonal Cell Line Preparation: CELL CULTURE • Cell culture refers to the
removal of cells from an animal or plant and their subsequent growth in a favourable artificial
environment. • The cells may be removed from the tissue directly and disaggregated by enzymatic
or mechanical means before cultivation, or they may be derived from a cell line or cell strain that
has already been already established. • Can be classified under the following cell lines. i) Primary
culture. ii) Diploid cell lines. iii) Continuous cell lines.
24. 24. i) PRIMARY CULTURE: Primary culture refers to the stage of the culture after the cells are
isolated from the tissue and proliferated under the appropriate conditions until they occupy all of
the available substrate (i.e., reach confluence) (e.g. Primary monkey kidney and mice fibroblasts).
25. 25. ii) Diploid Cel Lines: • After the first subculture, the primary culture becomes known as a
Diploid cell line or subclone. (e.g. human fetal lung).
26. 26. iii) Continuous Cell Lines: When a finite cell line undergoes transformation and acquires the
ability to divide indefinitely, it becomes a continuous cell line. Become immortal through a
process called transformation. Can occur spontaneously or can be chemically or virally induced.
27. 27. 3) Infection with virus • The clonal cell lines suspended in suitable media are infected with
any desired virus which replicates inside the multiplying cells. If the virus is virulent, they cause
lysis of cells and virus particles are released in the surrounding medium. • These newly produced
virus particles (virions) infect the adjacent cells. As a result localized areas of cellular destruction
and lysis (called plaques) often are formed.
28. 28. Growing Bacteriophages In Living Animals: Animal inoculation may be used as a diagnostic
procedure for identifying and isolating a virus from a clinical specimen. After the animal is
inoculated with the specimen, the animal is observed for signs of disease, or is killed so that
infected tissues can be examined for the virus.
29. 29. Growing Bacteriophages In Embryonated Eggs: Growing viruses in an embryonated egg can
be a fairly convenient and inexpensive form of host for many animal viruses. A hole is drilled in
the shell of embryonated egg, and a viral suspension or suspected virus-containing tissue is
injected into the fluid of the egg.
30. 30. Growing Bacteriophages In Embryonated Eggs (cont.): There are several membranes in an
egg, and the virus is injected near the one most appropriate for its growth. Viral growth is signaled
by the death of the embryo, by embryo cell damage, or by the formation of typical pocks or
lesions on the egg membranes. This method was once the most widely used method of viral
isolation and growth, and it is still used to grow viruses for some vaccines.
31. 31. Detection Of Viral Growth In Embryonated Eggs: The signs of viral growth include: i) Death
of the embryo, ii) Defects in embryonic development, and iii) Localized areas of damage in the
membranes, resulting in discrete, opaque spots called pocks (a variant of pox). iv)The embryonic
fluid and tissue can be prepared for examination with an electron microscope. v) Some can also be
detected by their ability to agglutinate red blood cells or by their reaction with an antibody of
known specificity that will affix to its corresponding virus, if it is present.
32. 32. HARVESTED EMBRYOS
33. 33. Viral Measurements Viruses are generally too small to be seen under the light microscope, and
an electron microscope is usually necessary to make them visible. Once viruses have replicated
and been harvested the concentration of viral particles (virions) in the viral stock solution must be
determined.
34. 34. Viral Measurements The most important method and the most commonly used to determine
the concentration of a stock solution of bacteriophages is the Plaque Assay (plaque method).
35. 35. The Plaque Essay (plaque method) : The plaque assay is performed by cultivating viruses on a
“lawn” of host cells and noting the presence of clear areas where viruses have replicated and
destroyed the cells (the virus lyses the bacteria). Each plaque is assumed to come from a single
viral particle.
36. 36. The concentration of the stock solution of the virus is given in plaque forming units.
37. 37. Viral Measurements Another way of determining virus infectious units is by cultivating
viruses in living animals and determining which dilution of virus is lethal to the animals. The end-
point dilution can be calculated by this method.
Recommended

Das könnte Ihnen auch gefallen