Artificial cultivation of bacteria, culture media

Patrick

Cultivation
The process of growing microorganisms in culture involves taking bacteria from the infection site growing them in artificial environment in the laboratory

Introduction
In nature, bacteria exist as mixed populations. In the laboratory these populations must be separated so that characteristics of individual species may be observed. A number of basic techniques are used in microbiology with this end in mind.

Introduction
First, microorganisms must be removed from natural environments and cultured this requires artificial media and surfaces on which bacteria may grow this also requires knowledge of nutritional requirements and environmental requirements bacteria of interest must be separated from all other bacteria

Introduction
This requires separation techniques that allow isolation of a pure culture once a pure culture is achieved, it should be maintained This requires that all media and lab supplies be sterile techniques are needed that facilitate working with pure cultures This requires aseptic technique and techniques of storage for pure cultures

Uses of media & culture

media provide nutrients for the growth of microbes Culture; gives in vitro diagnosis of infectious diseases. Isolating bacteria from sterile sites of the body provides an initial step in studying morphology and its identification. provides antigens for developing serological assays or vaccines.

Uses of media & culture

For genetic studies and manipulations in vitro. provides a reliable way estimating their numbers (viable count). solid media is a convenient way of separating bacteria in mixtures.

Classification of media
Based on three factors consistency, nutritional component, functional use,

Based on Consistency
Culture media are liquid, semi-solid or solid and biphasic.

Liquid media
These are available for use in test-tubes, bottles. They also called broths (e.g nutrient broth). Here, bacteria grow uniformly producing general turbidity. Certain aerobic bacteria and those containing fimbriae (Vibrio & Bacillus) are known to grow as a thin film called surface pellicle on the surface of undisturbed broth. Sometimes the initial turbidity may be followed by clearing due to autolysis (pneumococci)

Liquid media
Mostly used when a large number of bacteria are needed When the number in sample is suspected to be low. This is the practical approach in blood cultures. It can be used to obtain viable count (dilution methods). However, properties of bacteria are not visible presence of more than one type of bacteria can not be detected.

Solid media
Any liquid medium can be converted to solid media by adding a solidifying agents. Agar agar; is the most commonly used solidifying agent. Agar, is a galactan obtained from marine algae It melts at 95oC and solidifies at 42oC, It doesnt contribute any nutrients it is not hydrolyzed by most bacteria It is free from growth promoting or growth retarding substances. it is used at 1-3% to make a solid agar medium.

Semi-solid agar
Made by reducing the amount of agar to 0.2-0.5% useful in demonstrating bacterial motility Also for transporting some bacteria e.g. Stuarts and Amies media mannitol motility medium.

Biphasic media
Sometimes, a culture system comprises of both liquid and solid medium in the same bottle. This is known as biphasic medium. The inoculum is added to the liquid medium subcultures are made, by tilting the bottle This allows the liquid to flow over the solid medium This eliminates frequent opening of the culture bottle to subculture

Other solidifying agents

Besides agar, egg yolk and serum too can be used to solidify culture media. While serum and egg yolk are normally liquid, they can be rendered solid by coagulation using heat. Serum containing medium; Loefflers serum slope egg containing media; Lowenstein Jensen medium Dorset egg medium are solidified

Based on nutritional component

Media can be classified as simple, complex and synthetic (or defined). non-fastidious bacteria grow with minimal requirements Fastidious bacteria require extra nutrients. Simple media; peptone water, nutrient agar Complex media; blood agar, chocolate agar. Synthetic or defined media; Davis & Mingioli medium are specially prepared media for research purposes

Based on functional use

These include basal media, enriched media, selective/enrichment media, indicator/differential media, transport media and holding media.

Basal media
These are basically simple media they support growth of a broad range of organisms They usually have complex constituents Constituents of such media are generally defined e.g. peptone water, nutrient broth and nutrient agar are considered basal medium

Enriched media
Addition of extra nutrients in the form of blood, serum, egg yolk etc, to basal medium Enriched media are used to grow fastidious bacteria Examples; Blood agar, chocolate agar, Loefflers serum slope etc

Selective media
These are designed to inhibit unwanted organisms or contaminating bacteria this helps to recover pathogen from a mixture of bacteria. selective media are agar based addition of certain inhibitory agents to agar media makes it selective These should not affect the pathogen in choice Examples; antibiotics, dyes, chemicals, alteration of pH or a combination of these.

Enrichment media
These are liquid media that also serves to inhibit commensals in the clinical specimen. Examples; Selenite F broth, tetrathionate broth alkaline peptone water These are used to recover pathogens from fecal specimens.

Differential or indicator media

Certain media are designed to different bacteria on the basis of their colony colour. by incorporating dyes, metabolic substrates etc, bacteria that utilize them appear as differently coloured colonies Such media are called differential media or indicator media Examples: MacConkeys agar, CLED agar, TCBS agar, XLD agar etc

Transport media
Clinical specimens must be transported to the laboratory immediately after collection to prevent overgrowth of contaminating organisms or commensals. This can be achieved by using transport media. Such media prevent drying of specimen, They also maintain the pathogens they inhibit overgrowth of unwanted bacteria.

Transport media
Some of these media (Stuarts & Amies) are semi-solid in consistency Addition of charcoal serves to neutralize inhibitory factors Cary Blair medium is used to transport samples from cholera suspects Sachs buffered glycerol saline is used to transport samples of bacillary dysentery suspects

Anaerobic media
Anaerobic bacteria need special media ( no oxygen) and extra nutrients They need to be supplemented with nutrients like hemin and vitamin K Boiling the medium expels any dissolved oxygen Addition of 1% glucose, 0.1% thioglycollate, 0.1% ascorbic acid, 0.05% cysteine or red hot iron filings can render a medium reduced Robertson cooked meat that is commonly used to grow Clostridium spps It contains heart meat and nutrient broth.

Anaerobic media
Before use the medium must be boiled in water bath to expel any dissolved oxygen and then sealed Methylene blue or resazurin is an oxidationreduction potential indicator it is incorporated in the thioglycollate medium Under reduced condition, methylene blue is colourless

Use of physical separation procedures Streak plate tech For achieving single colony characteristics streak plate technique pour plate; spread plate;

Growth conditions
Bacteria may be isolated from a variety of environments For cultivation of bacteria in the lab, the conditions of the environments must be mimicked Refer to previous lecture on growth requirements

Bacterial requirements
Water Oxygen Carbon dioxide Temperature PH Light Nutrients Growth factors Inorganic salts Osmotic effect

Salt conditions
Halophiles; these bacteria require relatively high concentrations of salt for growth (10-20%); Halophiles must possess special membranes and enzyme Some organisms are salt tolerant. These do not require salt for growth but can grow in its presence An example is Staphylococcus aureus. which is found on skin, which often has a high salt concentration (10% NaCl).

Preparation and preservation

Heat stable ingredients Care must be taken to adjust the pH of the medium before autoclaving. Dehydrated media are commercially available These must be reconstituted as per manufacturers recommendation. Most culture media are sterilized by autoclaving.

Heat labile ingredients

like glucose, antibiotics, urea, serum, blood are not autoclaved. These are filtered and added separately after the medium is autoclaved. a representation from each lot is tested for performance and contamination before use. Once prepared, media may be held at 4-5oC in the refrigerator for 1-2 weeks. Certain liquid media in screw capped bottles or tubes or cotton plugged can be held at room temperature for weeks.

Thank you