Culture media

Culture Medium Culture medium: Nutrients prepared for microbial growth Sterile: No living microbes Inoculum: Introduction of microbes into medium Culture: Microbes growing in/on culture medium

Criteria of a medium It must contain the right nutrients for the specific microorganism we want to grow Sufficient moisture A properly adjusted pH A suitable level of oxygen The medium must initially be sterile Proper temperature for incubation of growing culture

Agar Complex polysaccharide derived from a marine alga Used as solidifying agent for culture media in Petri plates, slants, and deeps Generally not metabolized by microbes Liquefies at 100°C Solidifies at ~40°C

Slants When the agar containing test tubes are allowed to solidify with the tube held at an angle so that a large surface area for growth is available Deep When the agar solidifies in a vertical tube Petri plate Shallow dishes with a lid that nests over the bottom to prevent contamination; when filled they are called Petri (or culture) plates.

Culture Media Chemically defined media: Exact chemical composition is known Complex media: Extracts and digests of yeasts, meat, or plants Nutrient broth Nutrient agar

Anaerobic Culture Methods Reducing media Contain chemicals (thioglycolate or oxyrase) that combine oxygen and deplete oxygen in the culture medium Heated to drive off O2

Anaerobic Culture Methods(cont) When the culture must be grown in Petri plates to observe individual colonies, special anaerobic jars are used The culture plates are placed in the jar A packet of chemicals (sodium bicarbonate & sodium borohydride) in the jar is moistened with a few milliliters of water & the jar is sealed H2 & CO2 are produced by the reaction of the chemicals with the water A palladium catalyst in the jar combines the oxygen in the jar with the H2 produced by the chemical reaction & water is formed As a result oxygen quickly disappears Moreover the CO2 that is produced aids the growth of many anaerobic bacteria

Anaerobic Jar

An Anaerobic Chamber

Anaerobic Culture Methods(cont) A new technique to provide an anaerobic environment makes use of an enzyme, oxyrase( a respiratory enzyme derived from plasma membrane of certain bacteria) reduces oxygen to water

Special Culture Techniques Many clinical laboratories have special carbon dioxide incubators in which to grow aerobic bacteria that require concentration of carbon dioxide higher or lower than that found in the atmosphere High CO2 levels are obtained with simple candle jars Cultures are placed in a large sealed jar containing a lighted candle, which consumes oxygen The candle stops burning when the air in the jar has a lowered concentration of oxygen( but one still adequate for the growth of aerobic bacteria) An elevated concentration of CO2 is also present Microbes that require high CO2 conditions are called capnophiles Gas packs can generate CO2 also and are generally used in place of candle jars.

Candle Jars

Carbon dioxide generating packet

Selective Media Suppress unwanted microbes and encourage desired microbes Provide nutrients that enhance the growth of a particular type of bacterium & do not enhance ( and may even inhibit) other types of organisms that may be present For instance, a medium in which cellulose is the only carbon source will specifically select for the growth of cellulose utilizing organisms when it is inoculated with a soil sample containing many kinds of bacteria

Selective Media Medium Selective organism Inhibitory effect of the medium Bismuth sulfite agar Salmonella typhi Bismuth sulfite inhibits gram-positive bacteria & most gram-negative intestinal bacteria (other than Salmoneela typhi) Sabouraud’s dextrose agar Fungi The medium has a pH of 5.6. At this low pH fungi outgrow most bacteria

Differential Media Make it easy to distinguish colonies of different microbes Certain reagents or supplements, when incorporated into culture media, may allow differentiation of various kinds of bacteria For example, if a mixture of bacteria is inoculated onto a blood agar, some of the bacteria may hemolyze the red blood cells; others do not. Thus one can distinguish between hemolytic & nonhemolytic bacteria on the same medium

Differential Media Blood agar is a medium that microbiologists often use to identify bacterial species that destroy red blood cells. These species such as Streptococcus pyogenes (that causes strep throat) show a clear ring around their colonies where they have lysed the surrounding blood cells

Differential Media

Sometimes selective & differential characteristics are combined in a single medium To isolate Staphylococcus aureus, found in the nasal passages, Mannitol salt agar is used This organism has a tolerance for high concentrations of sodium chloride. It can also ferment mannitol to form acid Mannitol salt agar contains 7.5% sodium chloride which will discourage the growth of competing organisms & thus select for S aureus

Mannitol salt agar Differentiation: A pH indicator changes colour if the mannitol in the medium is fermented to acid. Bacteria that grow at the high salt concentration & ferment mannitol to acid can be readily identified by the colour change

MacConkey Agar Selective for gram negative bacteria Growth of gram positive bacteria is inhibited by crystal violet dye & bile salts in the media Diffferentiation: This medium also contains lactose. Gram-negative bacteria that produce acid from the metabolism of lactose can be differentiated from similar bacteria that can not. The ability to distinguish between lactose fermenters (red or pink colonies) & non fermenters ( colourless colonies) is useful in distinguishing between the pathogenic Salmonella & other related bacteria

EMB (Eosin Methylene Blue) Agar Selective for gram-negative bacteria Gram-positive bacteria is inhibited by methylene blue in the media E. coli & Enterobacter aerogenes ferment lactose E. coli produces colonies with a characteristic green metallic sheen Enterobacter aerogenes forms pink colonies often with a central dark purple dot Salmonella does not ferment lactose & produces colonies that are noncolored

EMB (Eosin Methylene Blue) Agar

Enriched Media Media such as tryptic soy broth and tryptic soy agar are called general purpose media or supportive media because they sustain the growth of many microorganisms. Blood and other special nutrients may be added to general purpose media to encourage the growth of fastidious microbes. These specially fortified media (e.g., blood agar) are called enriched media Fig.Chocolate agar, an enriched medium used to grow fastidious organisms such as Neisseria gonorrhoeae.

Enrichment Culture Encourages growth of desired microbe Similar to selective media but designed to increase very small numbers of desired type of organism to detectable levels Assume a soil sample contains a few phenol-degrading bacteria and thousands of other bacteria Inoculate phenol-containing culture medium with the soil, and incubate Transfer 1 ml to another flask of the phenol medium, and incubate Only phenol-metabolizing bacteria will be growing

Culture Media