Cultivation Of Bacteria

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 Base for other media

Nutrient broth, peptone water

Enriched media

Support the growth of nutritionally demanding bacteria

Blood agar, chocolate agar Loeffler's serum slope

Selective media

Suppress growth of unwanted bacteria and encourage desired ones

MacConkey agar Bismuth sulphite agar

Enrichment media

Liquid media and increase number of desired microbes to detectable levels Both functions in same medium Sustain microbes during transportation

Tetrathionate broth, Selenite F Alkaline peptone water

Indicator media

Distinguish colonies of one type from another

MacConkey agar Stuart's medium Cary Blair medium

Storage media

Preserve bacteria

Nutrient agar, Dorset egg media

Selective + Indicator Transport media

MacConkey agar

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.