In Vitro Cultivation of Bacteria

BACTERIOLOGICAL MEDIA is the substrate on which bacterial culture is done and it provides nutrition
and pH to the organism
Liquid and Solid Bacteriological Media
Both are extensively used in bacteriological laboratories. To obtain a pure growth one has to use solid
medium because of the following disadvantages with the liquid media:
1. Growth does not show character on the basis of which a tentative diagnosis can be established.
2. If it is a mixed culture of more than one organisms, their separation cannot be performed.
However, once a pure growth has been obtained on solid media, liquid media are used to perform further
tests because of the homogenous growth in these media.

Agar Agar
Agar agar (commonly called as agar) is used as the most effective solidifying agent.
Properties of Agar are:
• Derived from certain seaweeds
• Melts at 95°C and solidifies at 42°C
• Remains firm at temperatures of incubation
• Heat labile material can be added to it even at temperature of 45°C thus retaining their potency
• Bacteriologically inert
• Resistant to action of all medically important bacteria
• Easily available
• Economical

Types of Culture Media

Some bacteria can grow with minimum and ordinary available growth conditions whereas many others are
very specific in their demands. These can be in liquid or solid state; with or without oxygen. Some media
have simple composition whereas others may be either synthetic or complex in composition. Media can be
divided into two broad groups:
1. Defined Synthetic Media
These are prepared from chemicals and their exact composition is known. These are used for research
purposes and are of two types: Simple synthetic media contain a carbon and energy source, an
organic source of nitrogen and various inorganic salts in buffered aqueous solution. The complex
synthetic media possess, in addition to components of simple media, amino acids, purines,
pyramidines and many other growth factors depending upon the nature and growth requirements of
the organism.
2. Routine Laboratory Media
These media attempt to provide nutrition and pH to the organisms which are akin to that of tissues
and body fluids. Many of these nutrients are supplied by aqueous extracts of meat and peptone. There
are various types of these media.

Name Purpose Examples

Basal media Simple liquid media Nutrient broth, peptone

Base for other media water

Enriched media Support the growth of Blood agar, chocolate

nutritionally demanding agar Loeffler's serum
bacteria slope

Selective media Suppress growth of MacConkey agar

unwanted bacteria and Bismuth sulphite agar
encourage desired ones
Enrichment media Liquid media and Tetrathionate broth,
increase Selenite F
number of desired Alkaline peptone water
microbes
to detectable levels
Selective + Indicator Both functions in same MacConkey agar
Transport media medium Sustain
microbes during
transportation

Indicator media Distinguish colonies of MacConkey agar

one type from another Stuart's medium
Cary Blair medium

Storage media Preserve bacteria Nutrient agar, Dorset

egg media

Sugar Media
The term 'sugar' denotes fermentable substance. The usual sugar consists of 1 % of the chosen type of
sugar (lactose, sucrose, mannose, dextrose inulin, arabinose, etc) in peptone water alongwith an
appropriate indicator. A small tube (Durham's tube) is kept inverted in the sugar tube to detect gas
production.
Anaerobic Media
Medium such as Robertson's cooked meat medium is used for growing anaerobic organisms.

Media Recommended for Routine Use In Laboratory

Common experience suggests the use of following media for almost all the clinical samples in the initial
phase of isolation:
a. Nutrient broth
b. Nutrient agar (or still better blood agar)
c. MacConkey agar
d. Sensitivity test agar (this can be nutrient agar for most of the commonly used antibiotics except for
sulpha or sulpha containing drugs).

Preparation of Media
The components of any medium are combined together in recommended proportion without sterile
precautions. Sterilization process is then followed. All media are distributed as liquids. Blood for use in
bacteriology laboratory must be collected under sterile precautions from natural source and should be
preserved in sterile container. It should be rendered noncoagulable by defibrination or by the addition of
anti-coagulants such as citrates or oxalates. Serum can be sterilized by filtration.
The prepared medium is distributed in test tubes (prior to sterilization) and in petridishes (usually called
as plates).
Storage of Media
If the sterilized medium is in screw capped bottles, it can be stored at room temperature for weeks.
Medium in petri dishes deteriorates rather quickly at room temperature. Blood, serum and media can be
preserved for longer time if these are kept at low temperatures. Plates of media can be kept in a
refrigerator for 7 to 10 days. Screw capped bottles having media can be stored for months in refrigerator.
Blood and serum can be stored for still longer periods at -10°C to -40°C. Media should never be frozen
since it is detrimental to quality of nutrition.

CULTIVATION OF BACTERIA
The indications for culture of bacteria include:
· To obtain pure culture
· To characterize and confirm the isolate
· To carry out antibiotic sensitivity tests
· To carry out other epidemiological marker studies
· To estimate viable counts
· To store the bacterial isolates for future use.

Instrument for Seeding Media

This is selected according to the nature of the medium and inoculum. Platinum or nichrome wires of
different gauges are used. Nichrome is oxidising and hence in some cases nichrome wire is used.

Aerobic Incubation of Cultures

Incubation is usually done at 37°C. Some bacteria require special temperature such as Campylobacters
grow better at 43°C and leptospires at 30°C. For prolonged incubations as are required for the growth of
Mycobacterium tuberculosis screw capped bottles should be used instead of petri dishes or tubes to
prevent the drying of medium.
Incubation in an atmosphere with added carbon dioxide
Extra carbon dioxide is needed for optimal growth of organisms such as Brucella abortus, Pneumococcus
and Gonococcus, etc. The concentration of additional carbon dioxide nedded is 5-10 per cent. The simplest
method for having this environment is to put the plates in a container and generate C02 inside by lighting
a candle in it just before putting on the lid. Pure C02 can also be introduced in a container.

Methods of Isolating pure cultures

When there is a mixture of bacteria, following methods can be utilised to isolate bacteria in pure cultures.
1) Use of enrichment, selective and indicator media as described above can help in isolation of pure
cultures.
2) Pretreatment of clinical sample with substances which destroy unwanted bacteria e.g. culture of
tubercle bacilli. The sputum is pretreated with alkali / acids.
3) Plating on solid media to obtain isolated colonies, for selecting the desired ones.
4) Obligate aerobes and anaerobes can be separated by incubating in appropriate conditions.
5) The vegetative forms and spore forms can be separated by heating at 80°C which will kill the
vegetative forms.
6) Motile and non-motile bacteria can be separated by inoculating the Craigie's tube which permits
travel of motile bacteria to appear in the outside of the tube.
7) Bacteria of different sizes can be separated by use of selective filters with different pore sizes.
8) Pathogenic organisms can be separated from non pathogenic by animal inoculation tests.

ANAEROBIC CULTURES
For obligatory anaerobic bacteria, oxygen acts as a lethal poison and hence for the growth of these an
environment which is free of oxygen has to be created. The MacIntosh and Fildes technique of 1916 is still
widely practised with some modifications. In this the inoculated medium to be incubated anaerobically is
placed in the air tight jar and with the help of vacuum pump it is evacuated to -660 mm Hg. Hydrogen
(90%) and carbon dioxide (10%) are added to this through built in valves. The oxygen is removed by its
combination with hydrogen in the presence of palladium catalyst which is present in the jar. More
hydrogen or gas mixture is then introduced to equilibrate the pressure and the jar is incubated at 37°C.
Glove box or anaerobic chamber is used in modem laboratories with high quantum of work.