2. Microbiological Culture Media Flashcards
The types of physical Culture Media
Solid media
Liquid Media
Semi Solid Media
The 2 types of Solid Media
Liquefiable media - contain thermoplastic agents such as agar and gelatin
Non liquefiable media- eg- silica as gelling agent or use of natural products such as grains, potato slices, cooked meat etc
example of thermoplastic agents
agar and gelatin
example of non liquefiable solid media - natural products
grains, potato slices, cooked meat
what is the most important thermoplastic gelling agent
agar because it is inert, stable once gelled and inexpensive
why is agar the most important thermoplastic gelling agent
because it is inert, stable once gelled and inexpensive
liquid media is also known as
broth media
SEMI-SOLID MEDIA contains
a small % of agar (usually less than 0.5%)
semi solid media is used for
bacterial motility; biochemical tests; growing microaerophiles
Chemical Composition of media reflects the
the nutritional and physiological requirements of the microorganism being cultured
Chemical components of media include
water
buffers (pH)
vitamins
amino acids
sugars (carbohydrate source/s)
fats/cholesterol
nitrogen source/s
salts/inorganic compounds & trace elements
growth factors (hormones)
There are two major classes of media
Chemically Defined Media (synthetic)
Complex or Undefined Media (non-synthetic)
CHEMICALLY DEFINED MEDIA
Constituents and their exact concentrations are known
Can be used for metabolic/physiological/genetic studies or production of metabolites
COMPLEX MEDIA
The exact chemical composition of these media are not known
Include plant and animal tissue extracts
Used to support the growth of most heterotrophs and are nutrient rich
artificial and natural complex media examples
Artificial :
Can include natural extracts (malt or meat)
Peptones (enzyme digests of high protein substances such as soy bean, casein or meat)
Yeast extract (vitamins and growth factors)
Natural:
Obtained from natural sources and can include fruit juices
These natural products are used as the media without modifications
Culture media can serve a number of different purposes:
such as
- general purpose media
- selective media
- differential media
- minimal media
- enriched media
- enrichment media
- transport media
- enumeration media
- assays
General Purpose Media
Multiple functions including
Storage
Enumeration
Isolation
General Culture
Example Nutrient Agar
Selective Media
Used for the isolation of specific types/groups of microorganisms
Contain substances that can inhibit the growth of specific types/groups of bacterium while permitting the growth of others
E.g. MacConkey agar used to isolate Gram Negatives contains crystal violet which tends to be inhibitory towards most Gram Positive bacteria
differential media
Used to distinguish among morphologically & biochemically related organisms
Contain compounds that either produce a change in the appearance of the organism cultured or the surrounding medium
example of differential media
E.g. Thioglycolate broth
used to differentiate oxygen requirement levels of various microbes
A reaction with sodium glycolate in the medium reduces oxygen availability
Gradual decrease in oxygen concentration with increased distance from the surface of the media
The presence of oxygen in the media is indicated by a pink colour change
MacConkey agar
Apart from bile salts contains lactose and pH indicator (neutral red)
Organisms that have the ability to ferment lactose produce acid and therefore appear red at the surface
By contrast, non-lactose fermenters remain uncoloured since they do not produce acid
Minimal Media
Chemically defined medium containing minimal requirements for growth
It does not support the growth of some or all auxotrophic strains of a given organism
Only growth of prototrophic strains is permitted
Often used in metabolic studies and in taxonomic descriptions of organisms
Enriched Media
These media are supplemented with highly nutritious materials, e.g. blood, serum, yeast extract
Mainly used for the cultivation of fastidious organisms
Blood Agar which contains blood as an enrichment ingredient is used for the culture of members of the Streptococcus genus
Blood agar also allows classification of streptococci based on their haemolytic activities :
Gamma haemolysis
Alpha haemolysis
Beta haemolysis
Enrichment Media
Broth medium containing substances that allow target group to multiply before plating onto selective or differential agar
Transport Media
Maintains specific conditions for stabilizing cells during transport to lab
Cary-Blair semi-solid transport medium
Enumeration Media
Used for assessing or monitoring the total and/or viable cell count
Plate count agar and R2A are two examples
Assays
Used to measure specific biochemical activities
Example: Resistance to antibiotics
Medium influences:
Rate of cell growth/proliferation
Metabolic pathways utilized, hence metabolites produced
Gene product quantity (regulation of gene expression)
Medium optimization studies involve the
construction of growth curves to monitor cell proliferation rates
Simultaneous culture sampling is performed to
gauge productivity using a range of analytic techniques
The process can be costly, long & tedious, but is necessary
Preservation techniques for microbial cultures
1) periodic transfer
2) mineral oil slant
3) minimal medium/distilled water
4) drying
5) Lyophilization
6) Cryo-storage
Once a cell line has been developed, culture preservation is CRUCIAL
This allows a stock of producer cells to be maintained:
For use over a long period of time
As a back-up in the event of errors in process or losses due to contamination
As a reference for regulatory bodies/scientists performing checks (traceability & accountability)
A number of preservation techniques can be employed
They all require
GOOD ASEPTIC TECHNIQUE. Sterility is mandatory
explain periodic transfer culture preservation
Cells are maintained in culture by periodic transfer to fresh medium.
Frequency of transfer is usually defined.
Transfers/passages are performed during the exponential phase of growth.
explain mineral oil slant culture preservation
Stock cultures are grown on a slant and covered with sterile mineral oil.
Slants are refrigerated at 2-8°C.
Cultures may be preserved for up to 15-20 years.
explain Minimal Medium / Distilled Water culture preservation
Cultures are washed and stored in either minimal medium or distilled water.
They are kept refrigerated at 2-8°C.
Cultures may be kept in storage for up to between 3-5 months or more.
explain drying culture preservation
Cultures are dried on suitable sterile surfaces (soil, filter paper disks, gelatin drops).
These are then stored in a desiccator at refrigeration temperature.
Alternatively, they may be frozen to improve viability.
explain Lyophilization culture preservation
This process is also known as freeze-drying.
Water is removed from cultures by sublimation in the presence of a cryoprotective agent (glycerol).
Cultures are subsequently sealed in sterile ampules and refrigerated or frozen.
Viability >15 years but up to 30 years has been reported
explain cryo-storage culture preservation
Cultures are stored in media/cryopreservant (usually Glycerol) and frozen down in a controlled rate freezer.
They are then stored in liquid nitrogen at a temperature of -196°C.
Cultures may be kept viable for >15 years.
complex media include
Include plant and animal tissue extracts
E.g. MacConkey agar used to isolate
Gram Negatives contains crystal violet which tends to be inhibitory towards most Gram Positive bacteria
Thioglycolate broth is an example of what type of media
differential media
