Aim of this section:1-Why studying culture media? 2-Importance of culture media. 3-Classification of culture media. 4-Composition of culture media. 5-Detailed study of different types of culture media.
Why culture media? General scheme for identification of microorganisms:1-Staining (previous year) 2-Culture media 3-Biochemical reactions (biotyping) 4-Serology(antigen antibody reaction) 5-Animal inoculation 6-Phage typing 7-Molecular identification(DNA probes ,PCR,hybridization,……)
These are mixtures of nutrients on
Microbiological Culture Media:
which microorganisms can grow outside their natural habitat. The composition of these media is calculated to supply the organism with sources of energy and elements allowing its optimum growth.
Purpose of culture media
1-To study the properties of the microorganism . 2- To isolate the wanted M.O. from clinical (pathological) specimen e.g. blood, sputum,…... For diagnosis of infectious diseases. 3-To make antibiotic sensitivity tests to help in directing the lines of treatment.
Classification of culture media
Media can be classified according to their properties: 1-according to physical state into solid ,semi-solid .broth 2-according to chemical composition into
a) natural (emprical)media don’t have any addition of specific nutrients e.g. milk ,vegetable juices, diluted blood. it is Called empirical because the exact chemical composition of the constituents is not exactly known. b)semisynthetic media it is one in which the chemical composition of the medium is partially known, in other words the medium has natural component (unknown chemical composition) and certain specific nutrients (known chemical composition) e.g. potatodextrose agar
c)synthetic media (chemically defined media) one in which all chemical ingredients (of known composition) are mixed in definite proportions.e.g....
a living media consists of living cells or tissues which are used for the culture of strictly parasitic organisms like viruses or rickettsiae which can not be cultured on a non living medium
3-acoording to functional type general purpose in
selective ,enriched, enrichment,………….
Culture Media( physically)
adding small amounts of agar to fluid
And mainly used as transport media or in motility tests.
Solid Culture Media
Petri dish Stab
Essential requirements in culture media
Any culture medium must contains: • -A source of energy -Sources of carbon, nitrogen, sulfur, phosphorus -Minerals, e.g., Ca2+, Mg2+, Na+ -Vitamins and growth factors - Water
Many of the ingredients used in culture media are of natural origin, the most important are:
Beef extract Peptones Yeast extract Gelatin Agar
Agar: Chemically, it is sulfuric acid ester of unbranched
polysaccharide obtained from the cell membrane of some species of red algae or seaweed. o It is soluble in hot water at 98 C and resolidifies into a gel at 38o – 42oC. Not digestible by most microorganisms. Therefore it is ideal to use as a solidifying agent for culture media. Added to culture media in a concentration of 1.2% to 2% to render it solid and in a lower conc. to increase its viscosity.
Water soluble protein (milk,meat,soya)spray dried Product obtained from hydrolysis(acid,enzyme e.g. papin,pepsin,trypsin).
Meat extract e.g. concentrated aqueous infusion
of fresh beef.
Mineral salts e.g.sulphate, phosphate, iron,…. Carbohydrate provide the m.o. with carbon which
is the source of energy.
Water deionized or distilled water free from
chemicals that can suppress the growth of m.o.
Types of Culture Media(general purpose)
Basic or Ordinary Enriched Enrichment Selective Differential or Indicator Selective and Differential Transport media Media for Anaerobes Media for fungi
Basic Media: These media contain the essential
requirements for the growth of most microorganisms. Example: 1. Nutrient Broth (beef extract+peptone+Nacl) 2. Nutrient Agar(similar to nutrient broth but
supplemented with 1-2% agar )
3. Nutrient Gelatin
4. Peptone Water
added at 55c poured in plates identify hemolysis –some bacteria secrete enzymes that lyses red blood cells (hemolysins) such that clearing zones around the colonies appear.a)Grrenish discoloration of the medium surrounding the bacterial colonies (alpha heamolysis)or partial heamolysis.the green color is due to the breakdown of RBCs with reduction of hemoglobin to met hemoglobin.
Additional growth factors are added to the media to enhance the growth of one or more microorganisms(fastidious M.O.). These factors are usually body fluids . Example: Blood Agar(nutrient agar+10%blood )the sterile blood is
1. B)clearzone surrounding the bacterial colonies (beta
hemolysis)with complete destruction of RBCs and use of hemoglobin (complete hemolysis) 2. c)no hemolysis no reaction zone on the medium (gamma hemolysis).
1. 2-Chocolate Agar(heated blood agar above 100c to make rupture of R.B.CS. and liberation of nutrients) suitable for the growth of organisms of the Nisseria and Haemophilus groups. 2. 3-Looflers serum media specially for the growth of
Loeffler’s serum medium
Fluid culture media that increase the relative
concentration(number) of certain microorganisms(pathogens) by containing enrichments or sub.supress the growth of unwanted M.O. Example:
Tetrathionate broth for isolation of Salmonella in feces &urine. Thioglycollate broth for anaerobic M.O.
These media contain a substance that inhibits the growth of some microorganisms while allowing the growth of others (They select for certain microbes). Examples of substances added to the media to enhance selectivity: 1-dyes e.g. a) malachite green dye in LOWENESTEIN
JENSEN media inhibits the growth of bacteria other than T.B.
b) sodium azyde dye added to the medium to inhibit the growth of gram-negative organisms and enhance the growth of staph. and strep.
a) potassium tellurite in blood tellurite agar inhibits the growth of all bacteria except C.diphteria.
b)bile salt in Desoxcholate citrate agar more selective for isolation of
shigella and salmonella.
c)sodium chloride in high conc. In mannitol salt agar for isolation
and differentiation of staph.
3-antibiotics e.g. a)cetremide in cetremide agar for isolation of
pseudomonas aeruginosa. b)vancomycin,colistin,nystatin in modified Thayer-martin medium for isolation of N.GONORRHEA. Examples for selective media: Mannitol salt agar ,s-s agar,MacConkey agar,cetermide agar
Lactose fermentors on MacConkey Agar
Plane Mac conkey agar
Non Lactose fermentors on MacConkey Agar
Inoculated cetremide agar with pseudomonas with pigment production
1. These media contain a substance (indicator) that
is changed visibly as result of the metabolic activities of particular organisms. examples 1-litmus milk media 2-urea broth for isolation of PROTEUS which is urease +ve. 3-triple sugar iron agar(TSI) 4-xylose lysine deoxycholate agar(XLD) For isolation of salmonella and shigella 5- Eosine Methylene blue (EMB), Differentiation
between lactose fermenting and non fermenting enteric bacteria
Selective and Differential Media:
1. 2. 3. 4. 5. 6. 7.
These are both selective & differential. Example: Mannitol Salt Agar Eosin Methylene Blue Agar MacConkey’s Agar Medium Deoxycholate Citrate Agar T.C.B.S Medium Triple Sugar Iron Agar Bismuth Sulfite Agar
XLD inoculated with salmonella and shigella
Red pink black centered colonies salmonella due to H2S production
Red pink colonies shigella
↓FeS (Black ppt) Salmonella produce on DCA medium yellow colonies with dark center Thiosulphate Salmonella s2Fe2-
Mannitol salt agar for differentiation between satph species
Lactose non fermented enterobaceriacea
Lactose fomenter enetrobacetriacea
Bismuth Sulfite Agar plate
Inoculated bismuth sulfite agar with salmonella showing black colonies due to H2S
Plain DCA medium
DCA inoculated with salmonella
Media for anaerobes:
These media have little if any dissolved oxygen on which
anaerobes can thus grow. It contains “Reducing Agents” that are autooxidizable with great affinity to oxygen. It contains a low concentration of agar which increases the viscosity of the medium and decreases the rate of diffusion of oxygen. Major types of bacteria grow in the medium according to their respiratory requirements. Examples: 1- Fluid Thioglycollate Medium which contains thioglycolate (reducing agent) and methylene blue or resazurin to show the medium is reduced.
Resazurin Resazurin Layer
Fluid Thioglycollate Medium
2-Robertsons cooked meat consists of cooked minced
meat to which the broth is added it contains reducing substances e.g. hematin and glutathione which maintain anaerobic conditions in the depth of this fluid medium. suitable for clostridium species.
Mostly semi-solid media contain ingredients to improve
the survival of wanted both aerobic and anaerobic organisms and transports them where they are soon be cultured and prevent the growth of commensals. Examples
Amies transport media for gonococci in swab
collected specimens. Cary-Blair medium for enteric pathogens.
Media for fungi