CULTURE MEDIA

Microorganisms need nutrients and certain

environmental conditions in order to grow and
reproduce. In the environment, these
microorganisms have adapted to the habitats
most suitable to their needs. In the laboratory,
however, these requirements must be met by
a culture medium
Culture medium- is basically an aqueous
solution to which all the necessary nutrients
have been added.
Culture media can be classified according to 3
primary levels:
1. Physical state
2. Chemical composition
3. Functional type
A. Physical State
1. Liquid Media- these are water-based solutions
that do not solidify at temperatures above
freezing point. These media are commonly
termed broths, milk or infusions.
2. Semisolid Medica- these exhibits a clotlike
consistency at ordinary room temperature.
These media contain an amount of solidifying
agent ( agar or gelatin) which thickens them
but does not produce a firm substance.
3. Solid media- these provide a firm surface on
which cells can form discrete colonies and are
advantageous for culturing and isolating
bacteria and fungi. Solid media come in two
forms:liquefiable or reversible solid media and
nonliquefiable or non-reversible solid media
B. Chemical Composition:
1. Synthetic- are composed of pure organic and
inorganic compounds which have molecular
contents specified by means of an exact formula
2. Non-synthetic- these complex media contains at
least one ingredient that is not chemicallu
defined, not a simple compound, pure
compound and not representable by an exact
chemical formula. Most of these substances are
extracts of animals , plants or yeasts.
C. Functional Types:
1. General-purpose media- these contain
mixture of nutrients that could support the
growth of pathogens and nonpathogens alike,
they are designed to grown as broad a
spectrum of microbes as possible
.
Ex. Nutrient Agar- contains beef extract and
peptone, as well as 1.5% agar by weight
Nutrient Broth- contains beef extract and
peptone dissolved in water
Trypticase Soy Agar- contains partially digested
milk protein (casein), soybean digest, NaCl,
and agar
2. Enrichment Media- these are designed to increase the
number of desired miroorganisms to a detectable level
without stimulating the rest of the bacterial population. It
contains complex organic substances such as blood, serum,
hemoglobin or special growth factors.
Ex. Blood agar- contains general nutrients with 5% blood
added.
- It is used both as an enriched medium and as a differential
medium.
- Exotoxins- cause lysis of the red blood cells (hemolysins)
- The degree of the hemolysis is an especially useful tool for
identification of many of the gram positive cocci
These organisms are grouped into 3 categories
based on hemolytic reactions:
1. Beta hemolysis completely lyses the red blood
cells and hemoglobin; these results in complete
clearing around colonies
2. Alpha hemolysis refers to the partial lysis of
RBC’s and hemoglobin and produces a greenish
discoloration of the blood agar around the
colonies.
3. No hemolysis, sometimes called gamma
hemolysis results in no change of the medium
Chocolate agar- is a nutrient medium which is used
in culturing fastitidious organisms such as
Haemophillus species and Neisseria.
3. Selective media- these contain one or more
agents that inhibit the growth of a certain
microbe or microbes and thereby encourage only
certain microbes to grow. Selective media are
very importnt in primary isolation of a specific
type of microorganism from samples containing
many different species like feces, saliva skin,
water and soil.
Examples:
MacConkey Agar (MAC)- is a widely- used culture
medium which is both selective and differential.
The medium is primarily used to differentiate
between Gram negative bacteria while inhibiting
a the growth of most Gram positive bacteria.
Addition to the nutrient agar base of bile salts
and crystal violet will inhibit the growth of most
Gram positive bacteria, making MAC selective.
Eosin Methylene Blue (EMB)- is selective and
differential medium used for isolation and
differentiation among members of the
Enterobacteriaciaceae. Eosin methylyne blue
agar selects for Gram negative bacteria. It
contains methylene blue and eosin dyes to
inhibit the growth of Gram positive bacteria.
Mannitol salt agar ( MSA)- was developed by
microbiologist seeking a method for isolating
Staphyloccoccus aureus, a pathogen frequently
transmitted by contaminated food. This medium
contains 7.5% salt, which is inhibitory to most
bacteria other than Staphyloccus.
Thayer Martin Agar- is a chocolate agar designed to
isolate Neisseria gonorrheoeae
Sabourad’s Dextrose Agar (SDA)- is used for fungi. It
contains gentamicin and has a low pH that will kill
most bacteria
Hektoen Enteric Agar- is a selective meium with bile salts
added to inhibit Gram positive organisms. The
comparativelu high concentration of bile salts inhibits not
only Gram positive, but also some Gram negative organisms,
but not Salmonella and Shigella species.
Lowenstein- Jensen Medium (LJ) –is used to isolate
Mycobacterium.
Mueller- Hinton Agar (MHA)- is used to isolate Neisseria
gonorrhea and N. meningitidis and also for anti-microbial
sensitivity testing.
Cetrimide Agar- is used to isolate Pseudomonas aeroginosa
Salmonella- shigella agar (SSA)- is used to isolate Salmonella
And Shigella species.
4. Differential Media- these allow growth of several types of
microorganisms and are designed to display visible
differences among those microorganisms. Differentiation
shows up as variations in colony size or in color, in media
color changes or in the formation of gas bubbles and
precipitates.
Ex. MAC- differentiates between lactose- fermenting coliforms
and lactosr nonfermenters, which iclude potential
pathogens. Lactose, a fermentable carbohydrate, a neutral
red, a pH indicator, are added to differentia the lactose
nonfermenters. When lactos is fermented, acid products
lower the pH below 6.8, whith the resulting colonial grwth
turning pinkish-red. If an organism is unable to ferment
lactose, the colonies will be colorless.
Eosin Methylene Blue- differentiating those which
ferment lactose ( the coliforms) from the
coliforms which do not ferment lactose. It
contains lactose Small amounts of acid
production result in a pink colored growth, while
large amounts of acid cause the acid to
precipitate on the colony, resulting in a
characteristics greenish, metallic sheen.
Organisms which do not ferment lactose will be
colorless, taking on the color of the medium. This
medium has been widely used in the past to
screen for coliforms in the water.
Triple Sugar Iron Agar- differentiates bacteria based on their ability to
ferment glucose, lactose and /or sucrose, and to reduce sulfur to
hydrogen sulfide, it is used primarily to distinguish the
morphologically similar bacteria of Enterobacteriaceae, all of which
ferment glucose to an cid end product.
Mannitol Salt Agar- is used to differentiate pathogenic Staphyloccocus
species form nonpathogenic members of the genus Micrococcus, If
the organism does not use mannitol, the medium will remain red
( no change). If the organism does ferment mannitol, it will create
metabolic by- products which are acidic – and the surrounding
medium will be yellow.
Salmonella-Shigella Agar( SSA)- is used to differentiate Salmonella and
Shigella species whether they are lacotse ( red colonies) and non-
lactose fermenters (colorless colonies) as well a hydrogen sulfide
producers ( black color).