Simple, complex, defined, and special media are different types of media used for culture of the bacteria.
TABLE 4-1 Composition and uses of some common liquid media
Medium Composition Uses
Peptone water Peptone, sodium chloride, water
Routine culture, base for sugar fermentation test, indole test Nutrient broth Peptone water, meat extract Routine culture Glucose broth Nutrient broth, glucose Blood culture,
culture of fastidious organisms, such as streptococci Brain heart
infusion broth
Sodium citrate, sodium chloride, sodium phosphate, dextrose peptone, brain and heart infusion broth (ox), sodium polyanethol sulfonate (SPS)
Whole blood, bone marrow, body fluid culture
Alkaline peptone water
Peptone water (pH 8.6) Enrichment medium for Vibrio
Selenite-F broth
Peptone water, sodium selenite Enrichment medium for feces for Salmonella and Shigella Tetrathionate
broth
Nutrient broth, sodium thiosulfate, calcium carbonate, iodine solution
Culture of feces for Salmonella
Robertson’s cooked meat (RCM) broth
Nutrient broth, predigested cooked meat of ox heart
Anaerobic bacterial culture
FIG. 4-1. Nutrient agar showing colonies of Staphylococcus spp.
Medium Composition Uses
Nutrient agar Nutrient broth, agar 2% Routine culture MacConkey
medium
Peptone, lactose, sodium taurocholate, agar, neutral red
Culture of Gram- negative bacteria, such as Escherichia coli Blood agar Nutrient agar, 5% sheep or
human blood
Routine culture, culture of fastidious organisms, such as Streptococcus spp.
Chocolate agar Heated blood agar Culture of Haemophilus influenzae and Neisseria Deoxycholate
citrate agar
Nutrient agar, sodium deoxycholate, sodium citrate, lactose, neutral red, etc.
Culture of Shigella spp.
and Salmonella spp.
Thiosulfate citrate bile salt sucrose agar
Thiosulfate, citrate, bile salt, sucrose,
bromothymol blue, thymol blue
Culture of Vibrio cholerae
Loeffler’s serum slope
Nutrient broth, glucose, horse serum
Culture of Corynebacterium diphtheriae Lowenstein–
Jensen medium
Coagulated hen’s egg, mineral salt solution, asparagine, malachite green
Culture of Mycobacterium tuberculosis
TABLE 4-2 Composition and uses of some common solid media
S ection III Chapter 43
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S ection I Chapter 4
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Simple media
The simple or basal media include nutrient broth and peptone water, which form the basis of other media.
■ Nutrient broth is an example of a simple liquid medium that consists of peptone, meat extract, sodium chloride, and water. Addition of 0.5% glucose to nutrient broth makes it glucose broth.
■ Nutrient agar is an example of a simple solid medium. The medium is used routinely for isolation of many bacteria from clinical specimens.
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Complex media
Most of the media other than basal media are usually known as complex media [e.g., chocolate agar, MacConkey agar, Robertson’s cooked meat (RCM) medium, Lowenstein–Jensen (LJ) medium, etc.]. Complex media have some complex ingredi- ents, which consist of a mixture of many chemicals in unknown proportions. This is an undefined medium, because the amino acid source contains a variety of compounds with the exact composition unknown. The complex media contain:
■ Water,
■ A carbon source such as glucose for bacterial growth,
■ Various salts needed for bacterial growth, and
■ A source of amino acids and nitrogen (e.g., beef and yeast extract).
◗
Defi ned media
A defined medium, also known as synthetic medium, contains known quantities of all ingredients. All the chemicals used are known, and it does not contain any animal, yeast, or plant tissue. These media consist of:
■ Trace elements and vitamins;
■ A defined carbon source and nitrogen source required by certain microbes. Glucose or glycerol is often used as carbon sources and ammonium salts or nitrates as inorganic nitro- gen sources.
Dubos’ medium with Tween 80 is an example of this medium.
◗
Special media
These include (a) enriched media, (b) enrichment media, (c) selective media, (d) indicator or differential media, (e) trans- port media, and ( f ) sugar media.
Enriched media: The enriched media are invariably solid media that facilitate growth of certain fastidious bacteria. These media are prepared by adding substances like blood, serum, and egg to the basal media in order to meet the nutritional requirements of more exacting and more fastidious bacteria. Blood agar (Color Photo 1), chocolate agar, Loeffler’s serum slope (LSS), and LJ medium are some examples of enriched media. Blood agar is an enriched medium in which nutritionally rich whole blood supplements constitute the basic nutrients. Chocolate agar is enriched with heat-treated blood (80°C), which turns brown and gives the medium the color for which it is named.
Enrichment media: Enrichment media are liquid media that stimulate the growth of certain bacteria or suppress the growth of others for isolation of desired pathogenic bacteria.
Commensal bacteria, such as Escherichia coli present in feces, tend to overgrow pathogenic ones in stool specimen. In such situations, enrichment media (such as selenite-F broth or tetrathionate broth) are used for the isola tion of Salmonella Typhi and Shigella spp. from feces.
Key Points
Enrichment media are useful for isolation of wanted bacteria from stool and other specimens containing more than one spe- cies of bacteria.
Selective media: These are solid media that contain substances that inhibit the growth of all but a few bacteria but at the same time facilitate isolation of certain bacteria. Some examples of selective media include:
■ Thiosulfate citrate bile salt sucrose agar (TCBS) (Color Photo 35) selective for the isolation of Vibrio cholerae.
■ Deoxycholate citrate agar (DCA) selective for enteric bacilli, such as Salmonella spp. and Shigella spp.
■ LJ medium selective for Mycobacterium tuberculosis.
■ Hektoen enteric (HE) agar selective for Gram-negative bacteria.
■ Mannitol salt agar (MSA) selective for Gram-positive bacteria.
■ Xylose lysine desoxycholate (XLD) agar selective for Gram-negative bacteria.
■ Buffered charcoal yeast extract agar selective for certain Gram-negative bacteria, such as Legionella pneumophila.
Some selective media used in routine microbiology laborato- ries are summarized in Table 4-3.
Differential or indicator media: Differential or indicator media distinguish one microorganism from another growing on the same media by their growth characteristics.
Key Points
Differential or indicator media depend on the biochemi- cal properties of a microorganism growing in the presence of specific nutrients or indicators, such as neutral red, phe- nol red, eosin, or methylene blue. The medium changes color when a bacterium grows in them. For example, S. typhi grows as black colonies on Wilson and Blair medium containing sulfite. Lactose fermenting bacteria, such as E. coli produce pink colonies (Color Photo 2), whereas nonlactose fermenting bacteria, such as Salmonella spp. form pale or colorless colonies on MacConkey agar. Fermentation of lactose in the medium makes it acidic and leads to the formation of pink colonies in the presence of neutral red.
Examples of differential media include:
■ Eosin methylene blue (EMB), differential for lactose and sucrose fermentation;
■ MacConkey, differential for lactose fermentation;
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S ection III Chapter 43 S ection I Chapter 4
■ Mannitol salt agar (MSA), differential for mannitol fermentation; and
■ X-gal plates, differential for lac operon mutants for detection of recombinant strains of bacteria for study in molecular biology.
Transport media: Transport media are used to maintain the via- bility of certain delicate organisms in clinical specimens during their transport to the laboratory. They typically contain only buf- fers and salt. They lack carbon, nitrogen, and organic growth fac- tors, hence do not facilitate microbial multiplication. Examples of transport media are Stuart’s transport medium for Neisseria gonorrhoeae.
Sugar media: Sugar media, basically contains 1% “sugar”, which in microbiology denotes any fermentable substance, such as glucose, sucrose, lactose, and mannitol that is routinely used for fermentation tests. The sugar media shows the follow- ing characteristics:
■ It consists of 1% sugar in peptone
■ The indicator used in sugar media is Andrade’s indicator that consists of 0.005% acid fuchsin in 1 N NaOH. The production of acid after fermentation of sugar is indicated by the change of color of the medium to pink due to the presence of indicator.
■ Durham’s tube is kept inverted inside the sugar tube to demonstrate the production of gas. Production of gas is indi- cated by the demonstration of gas bubble in Durham’s tube.
Nowadays, dehydrated media are available for wide use in diagnostic laboratories because of simplicity of procedure and being less cumbersome to prepare. These dehydrated media are prepared by simply reconstituting in distilled water and sterilizing it before use.
Medium Colony characteristics Organisms inhibited Mannitol salt
agar
Big yellow colonies of Staphylococcus aureus
Small pink colonies of Staphylococcus epidermidis
Streptococcus spp.
Thayer–
Martin medium
Gray colonies of Neisseria meningitidis and Neisseria gonorrhoeae
Gram-positive cocci
MacConkey agar medium
Lactose fermenters: red colonies, e.g., Escherichia coli
Lactose nonfermenters:
colorless colonies of Salmonella spp.
Gram-positive cocci
Thiosulfate citrate bile salt sucrose agar
Sucrose fermenter:
yellow colonies of Vibrio cholerae
Sucrose nonfermenters:
green colonies of Vibrio parahaemolyticus
Enteric bacilli
Charcoal yeast extract
Cut glass colonies of Legionella spp.
Gram-positive cocci
Lowenstein–
Jensen medium
Rough, tough, and buff colonies of Mycobacterium tuberculosis
Smooth and pigmented colonies of atypical Mycobacterium spp.
Cocci
Sabouraud’s dextrose agar
Most fungi Most bacteria
TABLE 4-3 Some selective media used in routine microbiology laboratories
Culture Methods
5
Introduction
Culture methods are very crucial in a microbiology laboratory.
Various culture methods are carried out to:
1. Isolate bacteria in pure culture and identify the same by performing various tests.
2. Demonstrate biochemical, antigenic, and other phenotypic and genomic properties of the isolated colonies.
3. Demonstrate susceptibility of the isolated bacteria to antibiotics, bacteriophages, bacteriocins, etc.
4. Prepare antigens for various uses.
5. Maintain stock culture.
Methods of Culture
Various methods are used for culturing of bacteria. These include (a) streak culture, (b) lawn culture, (c) pour-plate culture, (d ) stroke culture, (e) stab culture, and ( f ) liquid culture.