1.18/MHS06/011 1. Blood Agar Plates (BAP) This is a differential medium. It is a rich, complex medium that contains 5% sheep red blood cells. BAP tests the ability of an organism to produce hemolysins, enzymes that damage/lyse red blood cells (erythrocytes). The degree of hemolysis by these hemolysins is helpful in differentiating members of the genera Staphylococcus, Streptococcus and Enterococcus. Beta-hemolysis is complete hemolysis. It is characterized by a clear (transparent) zone surrounding the colonies. Staphylococcus aureus, Streptococcus pyogenes and Streptococcus agalactiae are b-hemolytic (the picture on the left below shows the beta-hemolysis of S. pyogenes). Partial hemolysis is termed alpha-hemolysis. Colonies typically are surrounded by a green, opaque zone. Streptococcus pneumoniae and Streptococcus mitis are a-hemolytic (the picture on the right below shows the a-hemolysis of S. mitis). If no hemolysis occurs, this is termed gamma-hemolysis. There are no notable zones around the colonies. Staphylococcus epidermidis is gamma-hemolytic. 2. Sulfur Indole Motility Media (SIM) This is a differential medium. It tests the ability of an organism to do several things: reduce sulfur, produce indole and swim through the agar (be motile). SIM is commonly used to differentiate members of Enterobacteriaceae. Sulfur can be reduced to H2S (hydrogen sulfide) either by catabolism of the amino acid cysteine by the enzyme cysteine desulfurase or by reduction of thiosulfate in anaerobic respiration. If hydrogen sulfide is produced, a black color forms in the medium. Proteus mirabilis is positive for H2S production. The organism pictured on the far left is positive for hydrogen sulfide production. Bacteria that have the enzyme tryptophanase, can convert the amino acid, tryptophane to indole. Indole reacts with added Kovac’s reagent to form rosindole dye which is red in color (indole +). Escherichia coli is indole positive. The organism pictured second from left is E. coli and is indole positive. SIM tubes are inoculated with a single stab to the bottom of the tube. If an organism is motile than the growth will radiate from the stab mark and make the entire tube appear turbid. Pseudomonas aeruginosa and the strain of Proteus mirabilis that we work with are motile 3. Oxidase Test This test is used to identify microorganisms containing the enzyme cytochrome oxidase (important in the electron transport chain). It is commonly used to distinguish between oxidase negative Enterobacteriaceae and oxidase positive Pseudomadaceae. Cytochrome oxidase transfers electrons from the electron transport chain to oxygen (the final electron acceptor) and reduces it to water. In the oxidase test, artificial electron donors and acceptors are provided. When the electron donor is oxidized by cytochrome oxidase it turns a dark purple. This is considered a positive result. 4. Coagulase test Coagulase is an enzyme that clots blood plasma. This test is performed on Gram-positive, catalase positive species to identify the coagulase positive Staphylococcus aureus. Coagulase is a virulence factor of S. aureus. The formation of clot around an infection caused by this bacteria likely protects it from phagocytosis. This test differentiates Staphylococcus aureus from other coagulase negative Staphylococcus species 5. Starch hydrolysis test This test is used to identify bacteria that can hydrolyze starch (amylose and amylopectin) using the enzymes a-amylase and oligo-1,6-glucosidase. Often used to differentiate species from the genera Clostridium and Bacillus. Because of the large size of amylose and amylopectin molecules, these organisms can not pass through the bacterial cell wall. In order to use these starches as a carbon source, bacteria must secrete a-amylase and oligo-1,6-glucosidase into the extracellular space. These enzymes break the starch molecules into smaller glucose subunits which can then enter directly into the glycolytic pathway. In order to interpret the results of the starch hydrolysis test, iodine must be added to the agar. The iodine reacts with the starch to form a dark brown color. Thus, hydrolysis of the starch will create a clear zone around the bacterial growth. 6. Methyl Red / Voges-Proskauer (MR/VP) This test is used to determine which fermentation pathway is used to utilize glucose. In the mixed acid fermentation pathway, glucose is fermented and produces several organic acids (lactic, acetic, succinic, and formic acids). The stable production of enough acid to overcome the phosphate buffer will result in a pH of below 4.4. If the pH indicator (methyl red) is added to an aliquot of the culture broth and the pH is below 4.4, a red color will appear (first picture, tube on the left). If the MR turns yellow, the pH is above 6.0 and the mixed acid fermentation pathway has not been utilized (first picture, tube on the right). The 2,3 butanediol fermentation pathway will ferment glucose and produce a 2,3 butanediol end product instead of organic acids. In order to test this pathway, an aliquot of the MR/VP culture is removed and a-naphthol and KOH are added. They are shaken together vigorously and set aside for about one hour until the results can be read. The Voges-Proskauer test detects the presence of acetoin, a precursor of 2,3 butanediol. If the culture is positive for acetoin, it will turn “brownish-red to pink. Note: A culture will usually only be positive for one pathway: either MR+ or VP+. Escherichia coli is MR+ and VP-. In contrast, Enterobacter aerogenes and Klebsiella pneumoniae are MR- and VP+. Pseudomonas aeruginosa is a glucose nonfermenter and is thus MR- and VP- 7. Urease test This test is used to identify bacteria capable of hydrolyzing urea using the enzyme urease. It is commonly used to distinguish the genus Proteus from other enteric bacteria. The hydrolysis of urea forms the weak base, ammonia, as one of its products. This weak base raises the pH of the media above 8.4 and the pH indicator, phenol red, turns from yellow to pink. 8. Motility agar is a differential medium used to determine whether an organism is equipped with flagella and thus capable of swimming away from a stab mark. The results of motility agar are often difficult to interpret. Generally, if the entire tube is turbid, this indicates that the bacteria have moved away from the stab mark (are motile) 9. MacConkey agar This medium is both selective and differential. The selective ingredients are the bile salts and the dye, crystal violet which inhibit the growth of Gram-positive bacteria. The differential ingredient is lactose. Fermentation of this sugar results in an acidic pH and causes the pH indicator, neutral red, to turn a bright pinky-red color. Thus organisms capable of lactose fermentation such as Escherichia coli, form bright pinky-red colonies. MacConkey agar is commonly used to differentiate between the Enterobacteriaceae. 10. Spirit Blue agar This agar is used to identify organisms that are capable of producing the enzyme lipase. This enzyme is secreted and hydrolyzes triglycerides to glycerol and three long chain fatty acids. These compounds are small enough to pass through the bacterial cell wall. Glycerol can be converted into a glycolysis intermediate. The fatty acids can be catabolized and their fragments can eventually enter the Kreb’s cycle. Spirit blue agar contains an emulsion of olive oil and spirit blue dye. Bacteria that produce lipase will hydrolyze the olive oil and produce a halo around the bacterial growth. The Gram-positive rod, Bacillus subtilis is lipase positive (pictured on the right) The plate pictured on the left is lipase negative. 2. Giemsa Staining 1. Flood the smear with methyl alcohol and leave for 3–5 min for fixation. 2. Add prepared Giemsa stain and leave for 45 min. 3. Wash slide thoroughly with running tap water. 4. Blot dry with absorbent paper. 5. Observe under oil immersion. 6. Look for intracellular budding yeasts; fungi stain with purplish-blue.