2. Microbiological Culture Media

Question 1

The types of physical Culture Media

Answer 1

Solid media

Liquid Media

Semi Solid Media

Question 2

The 2 types of Solid Media

Answer 2

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

Question 3

example of thermoplastic agents

Answer 3

agar and gelatin

Question 4

example of non liquefiable solid media - natural products

Answer 4

grains, potato slices, cooked meat

Question 5

what is the most important thermoplastic gelling agent

Answer 5

agar because it is inert, stable once gelled and inexpensive

Question 6

why is agar the most important thermoplastic gelling agent

Answer 6

because it is inert, stable once gelled and inexpensive

Question 7

liquid media is also known as

Answer 7

broth media

Question 8

SEMI-SOLID MEDIA contains

Answer 8

a small % of agar (usually less than 0.5%)

Question 9

semi solid media is used for

Answer 9

bacterial motility; biochemical tests; growing microaerophiles

Question 10

Chemical Composition of media reflects the

Answer 10

the nutritional and physiological requirements of the microorganism being cultured

Question 11

Chemical components of media include

Answer 11

water
buffers (pH)
vitamins
amino acids
sugars (carbohydrate source/s)
fats/cholesterol
nitrogen source/s
salts/inorganic compounds & trace elements
growth factors (hormones)

Question 12

There are two major classes of media

Answer 12

Chemically Defined Media (synthetic)

Complex or Undefined Media (non-synthetic)

Question 13

CHEMICALLY DEFINED MEDIA

Answer 13

Constituents and their exact concentrations are known

Can be used for metabolic/physiological/genetic studies or production of metabolites

Question 14

COMPLEX MEDIA

Answer 14

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

Question 15

artificial and natural complex media examples

Answer 15

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

Question 16

Culture media can serve a number of different purposes:

such as

Answer 16

1. general purpose media
2. selective media
3. differential media
4. minimal media
5. enriched media
6. enrichment media
7. transport media
8. enumeration media
9. assays

Question 17

General Purpose Media

Answer 17

Multiple functions including
Storage
Enumeration
Isolation
General Culture

Example Nutrient Agar

Question 18

Selective Media

Answer 18

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

Question 19

differential media

Answer 19

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

Question 20

example of differential media

Answer 20

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

Question 21

MacConkey agar

Answer 21

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

Question 22

Minimal Media

Answer 22

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

Question 23

Enriched Media

Answer 23

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

Question 24

Enrichment Media

Answer 24

Broth medium containing substances that allow target group to multiply before plating onto selective or differential agar

Question 25

Transport Media

Answer 25

Maintains specific conditions for stabilizing cells during transport to lab

Cary-Blair semi-solid transport medium

Question 26

Enumeration Media

Answer 26

Used for assessing or monitoring the total and/or viable cell count

Plate count agar and R2A are two examples

Question 27

Assays

Answer 27

Used to measure specific biochemical activities

Example: Resistance to antibiotics

Question 28

Medium influences:

Answer 28

Rate of cell growth/proliferation
Metabolic pathways utilized, hence metabolites produced
Gene product quantity (regulation of gene expression)

Question 29

Medium optimization studies involve the

Answer 29

construction of growth curves to monitor cell proliferation rates

Question 30

Simultaneous culture sampling is performed to

Answer 30

gauge productivity using a range of analytic techniques

The process can be costly, long & tedious, but is necessary

Question 31

Preservation techniques for microbial cultures

Answer 31

1) periodic transfer
2) mineral oil slant
3) minimal medium/distilled water
4) drying
5) Lyophilization
6) Cryo-storage

Question 32

Once a cell line has been developed, culture preservation is CRUCIAL

This allows a stock of producer cells to be maintained:

Answer 32

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)

Question 33

A number of preservation techniques can be employed

They all require

Answer 33

GOOD ASEPTIC TECHNIQUE. Sterility is mandatory

Question 34

explain periodic transfer culture preservation

Answer 34

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.

Question 35

explain mineral oil slant culture preservation

Answer 35

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.

Question 36

explain Minimal Medium / Distilled Water culture preservation

Answer 36

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.

Question 37

explain drying culture preservation

Answer 37

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.

Question 38

explain Lyophilization culture preservation

Answer 38

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

Question 39

explain cryo-storage culture preservation

Answer 39

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.

Question 40

complex media include

Answer 40

Include plant and animal tissue extracts

Question 41

E.g. MacConkey agar used to isolate

Answer 41

Gram Negatives contains crystal violet which tends to be inhibitory towards most Gram Positive bacteria

Question 42

Thioglycolate broth is an example of what type of media

Answer 42

differential media