Selective media Lab 6
Types of Bacteriological Media • Basic nutritive media: • E.g. nutrient agar • Enriched media: • E.g. blood agar • Selective media: • Selective for the growth of particular bacteria • Used in diagnostic bacteriology • They contain inhibitory substances (e.g. antibacterials) that inhibit the growth of unwanted bacteria
Eosin Methylene Blue (EMB) • Constituents: Nutrients, lactose, neutral red, eosin and methylene blue. • Appearance: A clear red medium • A selective stain for Gram-negative bacteria. • It is a blend of two stains, eosin and methylene blue in the ratio of 6:1. • A common application of this stain is in the preparation of EMB agar, a differential microbiological medium, which inhibits the growth of Gram-positive bacteria and provides a colour indicator distinguishing between organisms that ferment lactose (e.g., E. coli) and those that do not (e.g., Salmonella, Shigella). • Lactose fermentation produces acids, which lower the pH. This encourages dye absorption by the colonies, which are now coloured purple-black. • Lactose non-fermenters may increase the pH by deamination of proteins. This ensures that the dye is not absorbed. • On EMB if E. coli is grown it will give a distinctive metallic green sheen
MacConkey Agar • Constituents: Nutrients, Agar, Bile Salts, Lactose, Neutral Red • Appearance: A clear red medium • Purpose: For the detection and counting of coliforms and as an aid to the identification of other bacteria. It inhibits many non enteric bacteria because of the presence of bile salts. Bacteria growing on the medium may ferment lactose to produce acid which turns the colony red/pink. Non lactose-fermenters may digest the protein in the medium which turns the colony yellow. • Examples: • Bacteria inhibited include Streptococcus and Pasteurella multocida. • Bacteria which grow include: • Escherichia coli (pink), lactose fermenter (LF), • Salmonella spp. (yellow), non lactose fermenter (NLF) • Staphylococcus spp small deep pink colonies (LF)
Diagnostic Sensitivity Test Agar • Constituents: Nutrients, Agar, Buffers, Added adenine, guanine, uracil and xanthine, 7% Blood. • Appearance: An opaque red medium indistinguishable from blood agar unless labelled. • Purpose: • For the growth of a similar range of bacteria to blood agar. • To determine the sensitivity of bacterial cultures to antimicrobials in standard conditions. • The presence of the added bases prevents folate antagonism to trimethoprim and sulphonamides.
DNAase Agar (DNA) • Constituents: Nutrients, Agar, Deoxyribonucleic acid, Sodium chloride • Appearance: A clear medium resembling Nutrient Agar. • Purpose: For the detection of deoxyribonuclease production by bacteria such as Staphylococci. Point inoculations of Staphylococci digest the DNA in the medium surrounding them during incubation. The digestion is revealed by flooding the plate with hydrochloric acid which precipitates intact DNA leaving a clear zone around colonies which have digested the DNA. Pathogenic staphylococci are DNAase positive and most non-pathogens are DNAase negative. • Examples: Staphylococcus aureus is DNAase positive. • Staphylococcus epidermidis is DNAase negative.
'Chocolate' or Heated Blood Agar • Constituents: Nutrients, Agar, 7% Blood (either Sheep or Horse). • Appearance: An opaque chocolate brown medium • Purpose: For the growth of bacteria which require the presence of serum and the nutrients to be found in red cells, but which cannot liberate these nutrients from the red cells themselves. The nutrients available include haemin, altered haemoglobin and NAD. Bacteria which show satellitism on blood agar grow freely on this medium. Many other bacteria grow on 'chocolate' agar and produce larger colonies than on blood agar. • Examples: Bacteria which grow on 'chocolate' agar but not on blood agar include the Haemophilus group such as H. parasuis and H. somnus and other NAD-requiring bacteria such as Actinobacillus pleuropneumoniae and Taylorella equigenitalis. • They may be distinguished from other bacteria only by their colony size and colour. Effects on the medium are limited to slight greening in some cases and adhesion to the medium.
Deoxycholate Citrate Agar (DCA) • Constituents: Nutrients, Agar, Lactose, Sodium citrate, Sodium thiosulphate, Ferric citrate, Sodium desoxycholate, Neutral red • Appearance: Slightly cloudy or clear pink medium • Purpose: To select Salmonella (and Shigella) from pathological material directly or after enrichment. Gram positive and most non-enteric coliform organisms are inhibited by the sodium desoxycholate, thiosulphate and citrate. Thiosulphate and iron act as indicators for sulphide production which gives a black colour. Lactose and the indicators allow the identification of lactose fermenters. This medium is slightly less selective than Salmonella Shigella Agar. • Examples: Escherichia coli is often inhibited, but if it survives, it appears as a pink or red colony. • Faecal streptococci do not grow. • Salmonella will grow and form colourless colonies. • Proteus and pseudomonas will also grow as colourless colonies and may have to be distinguished from salmonellae.
Salmonella Shigella Agar (SS) • Constituents: Nutrients, Agar, Lactose, Bile salts, Sodium citrate, Sodium thiosulphate, Ferric citrate, Brilliant green, Neutral red • Appearance: A clear pink medium. • Purpose: To select Salmonella (and Shigella) from pathological material directly or after enrichment. Gram positive and most coliform organisms such as Escherichia coli are inhibited by the brilliant green, bile salts, thiosulphate and citrate. Thiosulphate and iron act as indicators for sulphide production which gives a black colour. Lactose and the indicators allow the identification of lactose fermenters. • Examples: • Escherichia coli is usually inhibited, but if it survives, it appears as a pink or red colony. • Faecal streptococci do not grow. • Salmonella will grow and form colourless colonies. • Proteus and pseudomonas will also grow as colourless colonies and may have to be distinguished from salmonellae.
Edwards medium • Contains crystal violet to inhibit the growth of other bacteria than Streptococci, and sheep blood to indicate the haemolysis type. • S. agalactiae is β haemolytic and S. dysgalactiae is α haemolytic. S. uberis is γ (non haemolytic) or may be α haemolytic. • The presence of aesculin allows S. uberis to be distinguished from S. dysgalactiae because it splits aesculin to give a black colour. S. uberis colonies appear black.
Milk Agar Selective for bacteria that produce caseinase Proteolytic bacteria will be surrounded by a clear zone