Culture media lecture (2) Mrs. Dalia kamal eldien Msc in microbiology

For a culture medium to be successful in growing the pathogen sought it must provide all essential nutrients, ions, and moisture, maintain the correct pH and osmotic pressure, and neutralize any toxic materials produced. It is also essential to incubate the inoculated medium in the correct atmosphere, at the optimum temperature and for an adequate period. The main types of culture media based on nutritional component are: ● Basic ● Enriched ● Selective ● Indicator ● Transport ● Identification

Basic media These are simple media such as nutrient agar and nutrient broth that will support the growth of microorganisms, do not have special nutritional requirements. They are often used in the preparation of enriched media, to maintain stock cultures of control strains of bacteria, and for sub culturing pathogens from differential or selective media prior to performing biochemical and serological identification tests. Common examples include Peptone water nutrient broth nutrient agar

Peptone water

Enriched media Media contain the nutrients required to support the growth of a wide variety of organisms, including some of the more fastidious one. Basic media may be enriched with whole or lyzed blood, serum, peptones, yeast extract, vitamins and other growth factors. Such a medium is often used for specimens collected from sites which are normally sterile to ensure the rapid multiplication of a pathogen Common examples Blood agar chocolate agar Loeffler’s serum

Blood agar& chocolate blood agar

Enrichment media This term is usually applied to fluid selective media which contain substances that inhibit the growth of unwanted organisms Common examples Selenite F broth tetrathionate broth alkaline peptone water

Selenite F broth

Selective media These are solid media which contain substances(e.g. bile salts or other chemicals, dyes, antibiotics) which inhibit the growth of one organism to allow the growth of another to be more clearly demonstrated. A selective medium is used when culturing a specimen from a site having a normal microbial flora to prevent unwanted contaminants overgrowing a pathogen. Common example Crystal violet blood agar Neomycin blood agar

Differential media Certain media are designed in such a way that different bacteria can be recognized on the basis of their colony color. Various approaches include incorporation of dyes, metabolic substrates etc, so that those bacteria that utilize them appear as differently colored colonies. Examples: MacConkey’s agar CLED agar TCBS agar XLD agar Blood agar

CLED& MAC

What are those?

Transport media Clinical specimens must be transported to the laboratory immediately, this can be achieved by using transport media. Such media prevent drying of specimen, contain ingredients to prevent the overgrowth of commensals maintain the viability of all organisms in the specimen without altering their concentration. Examples Stuart’s Amie’s Cary-Blair medium Thioglycolate broth for strict anaerobes.

Anaerobic media Anaerobic bacteria need special media for growth because they need low oxygen content, Examples Robertson cooked meat that is commonly used to grow Clostridium spps

cooked meat media

Identification media These include media to which substrates or chemicals are added to help identify bacteria isolated on primary cultures. Examples peptone water sugars urea broth Kligler iron agar. Organisms are mainly identified by a change in the color of the medium and or the production of gas. Organisms used to inoculate identification media must be first isolated in pure culture.

KIA

Sugar fermentation test

Preparation of culture media Use of dehydrated culture media In the preparation of complex culture media, it is advisable for district laboratories to use ready-made standardized dehydrated media to ensure good performance and reproducibility. In most instances it will also be less expensive than buying the individual chemical constituents. Some chemicals may also be difficult to obtain, not available in small amounts, and may have a short shelf-life. Dehydrated media is hygroscopic, i.e. it absorbs water When exposed to moisture

Commercial culture media

The media are prepared by simply dissolving the powder in water, sterilizing the solution, and then dispensing it into culture vessels. Steps of preparation: Weighing the medium according to the formula in the bottle rapidly, and tightly capping the bottle as soon as possible after removing the approximate amount. Once the ingredients are weighed, do not delay in making up the medium. Follow exactly the manufacturer’s instructions. Use distilled water from a glass still.

Steps of media preparation

Add the powdered or granular ingredients to the water and stir to dissolve. Do not shake a medium but mix by stirring or by rotating the container. When heating is required to dissolve the medium, stir while heating and control the heat to prevent boiling and foaming which can be dangerous and damage the medium, e.g. DCA or TCBS agar. Overheating a medium can alter its nutritional and gelling properties, and also its pH. Autoclave a medium only when the ingredients are completely dissolved. Dispense medium in sterile bottles or tubes in amounts convenient for use.

Pouring of sterile media in petridishs

autoclave

Checking the pH of a culture medium The pH of most culture media is near neutral. An exception is alkaline peptone water. The simplest way of testing the pH of a culture medium is to use narrow range pH papers or a pH meter A fluid medium can be tested by dipping a narrow range pH paper into a sample of the medium An agar medium can be tested by pouring a sample of the molten medium into a small beaker or petri dish and when it has solidified, laying a narrow range pH paper on its surface.

Sterilizing culture media The following methods are used to sterilize culture media: – Autoclaving – Steaming at 100 C – Filtration autoclaving Most culture media are sterililized by autoclaving. Steaming at 100 C This is used to sterilize media containing ingredients that would be broken down or inactivated at temperatures over 100 C, e.g. Cary-Blair transport medium.

Filtration It is used mainly to sterilize additives that are heat-sensitive and cannot be autoclaved, or less stable substances that need to be added to a sterile medium immediately before it is used. Examples include serum and solutions containing urea and certain carbohydrates Several different types of filters can be used including those made from sintered glass or inert cellulose esters. Cellulose filters are referred to as membrane filters. They are preferred to other types of filters because they filter more rapidly, do not affect the filtrate in any way, and absorb very little of the substance being filtered. Certain highly selective media such as Wilson and Blair’s medium and TCBS agar need not be sterilized.

Reference District Laboratory Practice in Tropical Countries Part 2- Monica Cheesbrough Culture Conditions and Types of Growth Media for Mammalian Cells- Zhanqiu Yang and Hai-Rong Xiong