Lesson 12: CULTURE MEDIA Flashcards
Laboratory diagnosis of an infection is usually confirmed by isolating and culturing microorganisms in
Artificial media
developed the Petri dish (plate), a container for solid culture media.
Richard Petri
pioneered the use of
agar as a base for culture media
Robert Koch
the wife of Walther Hesse, one of Koch’s assistants who suggested the use of agar base for culture media.
Fannie Eilshemius Hesse,
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 a
Culture
is the process of growing microorganisms on culture media and obtained as pure cultures for study.
Cultivation
taking bacteria from the infection site
in vivo environment
by some means of specimen
collection and growing them in the artificial environment of the laboratory
in vitro environment
By appropriate procedures they have to
isolated cultivation
Purpose of bacterial cultivation
i. To grow and isolate all bacteria present in an infection.
ii. Infection and contaminants or colonizers
iii. Identification and characterization
Some of the components of culture media are as follows:
- Water
- Agar
- Peptone
- Yeast extract
- Malt extract
- Blood and serum
prepared from a variety of seaweeds and is now universally used for preparing solid media. Does not add to the nutritive properties of a medium and is not affected by the growth of bacteria. The melting and solidifying points of the solutions are not the same. At the concentrations normally used, most bacteriological melt at about 95°C and solidify only when
cooled to about 42°C.
Agar (or agar-agar)
Another almost universal ingredient of common media. It is a complex mixture of partially digested proteins. Commercially available digest broth can be used. Meat extract is also available commercially and is known as LabLemco.
Peptone
Tap is often suitable for culture media, particularly if it has a low mineral content, however glass-distilled or demineralized is best use
Water
It contains a wide range of amino acids, growth factors and inorganic salts. Used mainly as a comprehensive source of growth factors and may be substituted for meat extract in culture media.
Yeast extract
The important constituents peptone 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.
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 and growth factors.
Malt extract
These are used for enriching culture media. Either human or animal blood can be used. Usually 5-10 percent blood is used and the most usual
concentration is 10 percent. Used in certain media.
Blood and serum
Classification of media
A. Phase of growth media
B. Based on Nutritional factor
C. Defined media
D. Special media
The earliest culture media. The original media used by Louis Pasteur were such as urine or meat. In this media, nutrients are dissolved in water, and bacterial growth is indicated by a change in appearance from clear to turbid, (i.e. cloudy).
Liquid (broth) media
Uses of Liquid (broth) media
(I) for obtaining bacterial growth from blood or water when large volumes have to be tested
(ii) for preparing bulk cultures of antigens
made by adding a solidifying agent to the nutrients and water. Though somewhat less sensitive than liquid media, provide isolated colonies that can be quantified and identified. Some genera and species can be recognized on the basis of their colony morphologies. Other examples include blood agar, chocolate agar, MacConkey agar, etc
Solid (asgar) media
the most common solidifying agent.
Agarose
the simplest and most common medium used routinely in diagnostic laboratories.
Nutrient agar prepared by adding 2% agar to nutrient broth
For special purposes where agar is added to media in concentrations that are too low to solidify them. At 0.2 to 0.5 percent it yields, through which motile, but not non motile, bacteria may spread.
Semisolid media
a include nutrient broth and peptone water, which form the basis of the other media.
Simple (basal) media
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 specimens.
Nutrient Agar
The complex media contains
- Water
- A carbon source such as glucose for bacterial growth
- Various salts needed for
bacterial growth - A source of amino acids and nitrogen (e.g., beef and yeast
extract)
a synthetic kind of medium which contains known quantities of all ingredients used but does not contain any animal, yeast, or plant tissue. 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.
Defined Media
a 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
Invariably solid media that facilitate growth of certain fastidious bacteria. 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.
Enriched media
Some examples of enriched media
Blood agar, chocolate agar, Loeffler’s serum slope (LSS), and LJ medium
an enriched medium in which nutritionally rich whole
blood supplements constitute the basic nutrients.
Blood agar
enriched with
heat-treated blood (80°C), which turns brown and gives the medium the color for which it is named
Chocolate agar
Liquid media that stimulate the growth of certain bacteria or suppress the growth of others for isolation of desired
pathogenic bacteria. Commensal bacteria, such as Escherichia coli present in feces, tend to overgrow pathogenic ones in stool specimen. In such situations, enrichment media (such as selenite-F broth or tetrathionate broth) are used for the isolation of
Salmonella typhi and Shigella spp. from feces
Enrichment media
These are 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
selective for the isolation of Vibrio cholerae
Thiosulfate citrate bile salt
sucrose agar (TCBS)
selective for enteric bacilli, such as Salmonella spp. and Shigella spp.
Deoxycholate citrate agar (DCA)
selective for Mycobacterium tuberculosis
LJ medium
selective for Gram-negative bacteria
Hektoen enteric (HE) agar
selective for Gram-positive bacteria
Mannitol salt agar (MSA
Xylose lysine desoxycholate (XLD) agar selective for
Gram-positive bacteria
selective for certain Gram-negative bacteria, such as Legionella pneumophila
Buffered charcoal yeast extract agar
distinguish one microorganism from another growing on the same media by their growth characteristics.
Differential or indicator media
differential for lactose
and sucrose fermentation
Eosin methylene blue (EMB)
differential for lactose fermentation
MacConkey
differential for mannitol fermentation
Mannitol salt agar (MSA)
differential for lac operon mutants for detection of recombinant strains of bacteria for study in molecular biology
X-gal plates
basically contains 1% “sugar”, which in microbiology denotes any fermentable substance, such as glucose, sucrose, lactose, and mannitol that is routinely used for fermentation tests.
Sugar media
used to maintain the viability of certain delicate organisms in clinical specimens during their transport to the laboratory. They typically contain only buffers and salt. They lack carbon, nitrogen, and organic growth factors, hence do not facilitate microbial multiplication. Examples are Stuart’s transport medium for Neisseria gonorrhoeae.
Transport media
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 medium.
Lyophilization (freeze drying)
drying suspensions of bacteria for preservation purposes have been developed
Drying methods
a common method of preserving strains of bacteria
Cold storage
