LESOON 12: CULTURE MEDIA Flashcards
Usually confirmed by isolating and
culturing microorganisms in artificial media.
Laboratory diagnosis of an infection
Bacteria and fungi are cultured in either
liquid (broth) or on solid (agar) artificial media.
Pioneered the use of
agar as a base for culture media as suggested by Fannie Eilshemius Hesse, the wife
of Walther Hesse, one of Koch’s a assistance
Robert koch
Developed the Petri dish (plate), a container for solid culture.
Richard Petri
developed the pour plate method and was the first to use solid culture media
for culture of bacteria.
Robert Koch
A nutrient material prepared for the growth of microorganisms in a
laboratory is called a
culture medium.
When microbes are introduced into a culture medium to initiate growth,
they are called an
Inoculum
The microbes that grow and multiply in or on a culture medium are referred
to as
Culture
: is the process of growing microorganisms in culture by taking bacteria
from the infection site (i.e. in vivo environment)
bacteria culture within the living
some means of specimen
collection and growing them in the artificial environment of the laboratory (i.e. the in
vitro environment)
bacteria culture growth outside laboratory
a laboratory culture containing a single species of organism.
Pure culture: a
Bacterial cultivation has three main purposes :
i. To grow and isolate all bacteria present in an infection.
ii. Infection and contaminants or colonizers:
To determine which of the bacteria that
grow are most likely causing infection and which are likely contaminants or
colonizers.
iii. Identification and characterization:
To obtain sufficient growth of clinically relevant
bacteria to allow identification and
Ingredients of Culture Media
Some of the components of culture media are as follows:
- water
- agar
- peptone
- Yeast Extract
- malt Extract
- Blood And Serum
often suitable for culture media, particularly if it has a low
mineral content, however glass-distilled or demineralized water
Tap water
Prepared from a variety of seaweeds and is now universally used for preparing solid media.
Agar does not add to the nutritive
properties of a medium and is not affected by the growth of bacteria.
Agar
the nutritive properties of a medium and is not affected by the growth of bacteria. The melting and solidifying points of agar solutions.
At the concentrations
normally used,
most bacteriological agars melt at about 95°C and solidify only when
cooled to about 42°C.
Another almost universal ingredient of common media
peptone
It is a complex mixture of partially digested proteins.
Peptone
The important constituents are
peptones, proteoses , amino acids, a variety of inorganic salts including phosphates, potassium and magnesium, and certain accessory growth factors such as nicotinic
acid and riboflavin.
Commercially available peptones or digest broth can be used.
Is also available commercially and is known as LabLemco.
Meat Extract
It contains a wide range of amino acids, growth factors and
inorganic salts.
Yeast Extract
Used mainly as a comprehensive source of growth factors and may be substituted for meat extract in culture media.
Yeast Factors
It consists mainly of maltose (about 50%), starch, dextrins and
glucose, and contains about 5 percent of proteins and protein breakdown products, and a wide range of mineral salts.
Malt extract
These are used for enriching culture media
blood and serum
Usually 5-10 percent blood is used and the most usual
concentration is 10 percent.
Serum is used in certain media
CLASSIIFICATION OF MEDIA :
A. PHASES OF GROWTH MEDIQ
B. BASED ON NUTIRIONAL FACTORS
C. DEFINED MEDIA
D. SPECIAL MEDIA
A. PHASES OF GROWTH MEDIA
- LIQUID (BROTH) MEDIA
- SOLID (AGAR) MEDIA
- SEMISOLID MEDIA
The earliest culture media were liquid. The original media used by Louis Pasteur were liquids such as urine or meat broth.
LIQUID MEDIA
In broth media nutrients are dissolved in water, and bacterial growth is indicated by a change
in broth’s appearance from clear to turbid, (i.e. cloudy).
Uses
(I) for obtaining
bacterial growth from blood or water when large volumes have to be tested
(ii) for preparing bulk cultures of antigens or vaccines.
Are made by adding a solidifying agent to
the nutrients and water.
SOLID (AGAR) MEDIA
Is the most common solidifying agent.
AGAROSE
The Petri dish containing the agar is referred to as
AGAR
provide isolated colonies that can be quantified and identified.
Some genera and species can be recognized on the basis of their colony
morphologies.
Clones of cells originating from a single bacterial cell.
COLONIES
Derived from a single colony or clone are considered “pure”.
BACTERIAL CULTURES
Prepared by adding 2% agar to nutrient broth is the simplest and most
common medium used routinely in diagnostic laboratories.
NUTRIENT AGAR
Other examples of solid
media include blood agar, chocolate agar, MacConkey agar, etc
For special purposes where agar is added to media in concentrations that are too low to solidify them.
SEMISOLID
At 0.2 to 0.5 percent it yields a
semisolid medium through which motile, but not non motile, bacteria may spread.
The simple or basal media include nutrient broth and peptone water, which form the basis.
Simple (basal) media:
Is an example of a simple liquid medium that consists of peptone, meat extract, sodium chloride, and water. Addition of 0.5% glucose to nutrient broth makes it glucose broth.
■ Nutrient broth
An example of a simple solid medium. The medium is
used routinely for isolation of many bacteria from clinical specimen
Nutrient agar
Media that contain some ingredients of unknown chemical composition.
A complex medium contains a variety of ingredients such as meat juices and digested proteins.
Complex media:
The complex media contain
(1) Water,
(2) Acarbon source such as glucose for bacterial growth,
(3) Various salts needed for
bacterial growth, and
(4) A source of amino acids and nitrogen (e.g., beef and yeast
extract).
Is a synthetic kind of medium which contains known quantities of all
ingredients used but does not contain any animal, yeast, or plant tissue.
C. Defined Media
These media consist of
(1) Trace elements and vitamins;
(2) A defined carbon source and
nitrogen source required by certain microbes.
Examples are glucose or glycerol and ammonium salts or nitrates respectively such as Dubos’ medium with Tween 80
D. 6 Special Media
- Enriched media
2 . Enrichment media - Selective media
- Differential or indicator media
- Transport media
- Sugar media
Invariably solid media that
facilitate growth of certain fastidious bacteria.
Enriched media
These media are prepared by adding
substances like blood, serum, and egg to the basal media in order to meet the
nutritional requirements of more exacting and more fastidious bacteria. B
Example of enriched media.
Blood agar,
chocolate agar
Loeffler’s serum slope
LJ medium
an enriched medium in which nutritionally rich whole blood supplements constitute the basic nutrients.
Blood agar
is enriched with heat-treated blood (80°C), which turns brown and gives the medium the color for which it is named.
Chocolate agar
Are liquid media that stimulate the
growth of certain bacteria or suppress the growth of others for isolation of desired
pathogenic bacteria.
Enrichment media:
In such situations, enrichment
media (such as selenite-F broth or tetrathionate broth) are used for the isolation of Salmonella typhi and Shigella spp.
Solid media that contain substances that inhibit
the growth of all but a few bacteria but at the same time facilitate isolation of certain
bacteria.
Selective media
Examples of selective media include:
(a) Thiosulfate citrate bile salt
sucrose agar (TCBS) selective for the isolation of Vibrio cholerae,
(b) Deoxycholate
citrate agar (DCA) selective for enteric bacilli, such as Salmonella spp. and Shigella
spp.,
(c) LJ medium selective for Mycobacterium tuberculosis,
(d) Hektoen enteric
(HE) agar selective for Gram-negative bacteria,
(e) Mannitol salt agar (MSA)
selective for Gram-positive bacteria,
(f) Xylose lysine desoxycholate (XLD) agar
selective for Gram-negative bacteria,
(g) Buffered charcoal yeast extract agar
selective for certain Gram-negative bacteria, such as Legionella pneumophila.
Examples:
(a) Eosin methylene blue (EMB), differential for lactose
and sucrose fermentation;
(b) MacConkey, differential for lactose fermentation;
(c) Mannitol salt agar (MSA), differential for mannitol fermentation; and
(d) X-gal plates, differential for lac operon mutants for detection of recombinant strains of bacteria for
study in molecular microbiology
Distinguish one microorganism from another growing on the same media by their growth characteristics.
Differential or indicator media
They typically contain only buffers and salt. They lack carbon, nitrogen, and organic growth factors, hence do not facilitate microbial multiplication.
Trasnport media
used to maintain the viability of
certain delicate organisms in clinical specimens during their transport to the
laboratory.
Transport media
Examples :
are Stuart’s transport medium for Neisseria gonorrhoeae.
Basically contains 1% “sugar”, which in
microbiology denotes any fermentable substance, such as glucose, sucrose, lactose, and mannitol that is routinely used
Sugar media
Preserving Bacterial Culture
Can be used for the short-term storage of bacterial
cultures.
Refrigeration
A process in which a pure culture of microbes is placed in a suspending liquid and quick-frozen at temperatures ranging from - 50º to -95°C.
The culture can usually be thawed and cultured even several years later.
Deep freezing
A suspension of microbes is quickly frozen at temperatures ranging from -54º to -95ºC, and the water is removed by a high vacuum (sublimation).
The organisms can be revived at any time by hydration with a suitable liquid nutrient
Lyophilization (Freeze dying)
A common method of preserving strains of bacteria.
Cold Storage
A number of for drying suspensions of bacteria for preservation purposes have been developed.
Drying methods
