MODULE G- BACTERIA

• Specimens


  • Urine
  • Blood
  • Pus
  • Spinal fluid
  • Sputum
  • Other material, as indicated by the localization of the disease process
• Gram staining

  • Gram-negative bacilli; they resemble each other morphologically
  • The presence of large capsules, which can be observed as colorless to light pink halo around the bacilli, is suggestive of Klebsiella species
• Wayson staining


  • A basic fuchsin-methylene blue, ethyl alcohol-phenol microscopic staining procedure
  • Useful alternative for Gram staining
  • Yersiniae show bipolar purple staining with a central vacuole giving a characteristic "safety pin" appearance
• Selenite broth


  • Used as an enrichment medium for the isolation of Salmonella from feces, urine, water, food and other materials
  • Sodium selenite inhibits many species of gram-positive and gram-negative bacteria including the coliforms and many strains of Shigella
  • Subcultures should be made within 8-12 hours at 35 oC, because coliforms or other intestinal flora may overgrow the pathogens within a few hours
  • During preparation, heat to boiling. DO NOT OVERHEAT. DO NOT AUTOCLAVE. Overheating may produce a visible precipitate, making it unsatisfactory for use
• GN (Hajna) broth


  • Used as an enrichment medium for the recovery of Salmonella and Shigella from clinical and nonclinical specimens
  • Mannitol, in a higher concentration than glucose, enhances the growth of Salmonella and Shigella
  • Sodium citrate and sodium desoxycholate inhibit gram-positive and some gram-negative bacteria
  • Subcultures should be made within 4-6 hours at 35 oC, because of relatively low concentration of desoxycholate, it is less inhibitory to E. coli and other coliforms
• Tetrathionate broth, with iodine-iodide solution


  • Used as a selective enrichment for Salmonella species
  • Bile inhibits gram-positive bacteria, and tetrathionate which is formed in the medium by the addition of iodine-iodide solution inhibits the normal intestinal flora of fecal specimens
  • Subcultures should be made within 18-24 hours at 35 oC
  • During preparation, heat the broth base to boiling, add the iodine solution. DO NOT REHEAT AFTER ADDITION OF IODINE. DO NOT AUTOCLAVE
• Blood agar medium (BAM)


  Colonies are usually large, white or gray, smooth, shiny, circular, raised colonies which may or may not be hemolytic
• Selective media


  • Used for selective isolation of gram-negative enteric bacilli by incorporation of agent/s inhibitory to gram-positive bacteria; may be slightly selective, moderately selective, or highly selective
  • Are also generally differential plating media to distinguish between coliforms (lactose-fermenting) from noncoliforms (non-lactose-fermenting) enteric bacilli
• MacConkey Agar (MAC)


  • Crystal violet dye and bile salts inhibit gram-positive bacteria and allow gram-negative bacteria to grow
  • Bacteria that ferment lactose, the sole carbohydrate in the medium, produce acids, which, in the presence of neutral red as the pH indicator, results in the formation of pink to red colonies. Non-lactose fermenters form colorless colonies
• Eosin-methylene blue Agar (EMB)


  • Eosin Y and methylene blue dyes inhibit gram-positive bacteria and allow gram-negative bacteria to grow
  • The same dyes play a role in differentiating between lactose fermenters and non-lactose fermenters due to the presence or absence of dye uptake in the bacterial colonies. Lactose-fermenting bacteria produce pink to purple colonies due to the taking up of an eosin-methylene blue dye complex by the bacterial cells when the pH drops. Escherichia coli colonies show a characteristic green metallic sheen (GMS) due to the rapid fermentation of lactose. Non-lactose-fermenting bacteria produce colonies that are colorless, or transparent
• Salmonella Shigella Agar (SSA)


  • Bile salt salts, brilliant green and citrates inhibit all gram-positive bacteria and many gram-negative bacteria, including the coliforms
  • Bacteria that ferment lactose, the sole carbohydrate in the medium, produce acids which, in the presence of neutral red as the pH, results in the formation of pink to red colonies. Non-lactose fermenters form colorless colonies
  • Sodium thiosulfate is a source of sulfur. Any bacteria that produce hydrogen sulfide (H2S) are detected by the black precipitate formed with ferric citrate
  • During preparation, heat to boiling to completely dissolve the agar. DO NOT OVERHEAT. DO NOT AUTOCLAVE
• Xylose Lysine Desoxycholate Agar (XLD)

  • Bile salts (sodium desoxycholate) inhibit gram-positive bacteria
  • Xylose, lactose, and sucrose are the fermentable carbohydrates available for acid production, and phenol red is the pH indicator. Xylose is fermented by practically all enterics except for the shigellae. This property enables differentiation of Shigella
  • Lysine decarboxylation by Salmonella, which initially produce yellow colonies due to xylose fermentation, results in delayed red colonies due to alkaline amines produced
  • Sodium thiosulfate and ferric ammonium citrate are the H2S indicator system. H2S production results in the formation of colonies with black centers
  • During preparation, heat to boiling to completely dissolve the agar. DO NOT OVERHEAT. DO NOT AUTOCLAVE
• Hektoen Enteric Agar (HEA)


  • Bile salts inhibit gram-positive bacteria and retard the growth of many coliforms
  • Lactose, sucrose (saccharose), and salicin are the carbohydrates from which acids are produced; in the presence of acids, acid fuchsin react with bromthymol blue producing a yellow color
  • Sodium thiosulfate is the source of sulfur, and H2S is detected by ferric ammonium citrate forming black-centered colonies
• Fermentation of xylose, sucrose and/or lactose by organisms on XLD

  1. Produces large amounts of acids

  2. Results in yellow colonies with yellow conversion of the medium

  3. Typical of Escherichia coli, Klebsiella, and Enterobacter
• Salmonella growing on XLD agar

  1. Shows red colonies surrounded by pink halo

  2. Fermentation of xylose

  3. Decarboxylates lysine in the medium causing an alkaline pH

  4. Blackening of the colonies, indicating H2S production
• Non-lactose fermenting, H2S producing Proteus or Edwardsiella on XLD

  1. Produce red colonies with black centers

  2. Lack the pink halo because they do not decarboxylate lysine
• Shigella species on XLD


  1. H2S-negative and do not ferment xylose, sucrose or lactose

  2. Alter the pH to alkaline and produce red colonies
• Hektoen Enteric Agar. Uninoculated.
• Bismuth Sulfite Agar (BSA)


  • Highly selective medium used for isolating Salmonella species, particularly Salmonella Typhi
  • Bismuth sulfite and brilliant green are complementary in inhibiting G+ bacteria and coliforms, while allowing Salmonella to grow luxuriantly
  • Sulfur from bismuth sulfite forms H2S which, in the presence of ferrous sulfate, giving positive cultures the characteristic brown to black color with metallic sheen
• Brilliant Green Agar (BGA)


  • Highly selective medium for the isolation of Salmonella other than Salmonella Typhi/Salmonella Paratyphi
  • Brilliant green dye inhibits G+bacteria and a majority of G- baciili
  • Phenol red serves as pH indicator in the fermentation of lactose and/or sucrose in the medium
  • Salmonella species (other than S. Typhi/Paratyphi) produce white to red colonies surrounded by red zones in the medium. S. Typhi/Paratyphi shows no growth to trace growth on BGA
• For fecal specimens or rectal swabs


  1. Inoculate into an enrichment broth (Selenite broth or GN broth)

  2. Subculture into BAM and one medium from each of the groups of selective media

  3. Incubate at 35 ± 2°C for 18-24 hours. If negative after 24 hours, reincubate an additional 24 hours. Select suspicious colonies for definitive biochemical or serologic testing
• For non-fecal specimens


  1. Directly inoculate into a combination of BAM, and MAC or EMB

  2. Incubate at 35 ± 2°C for 18-24 hours. If negative after 24 hours, reincubate an additional 24 hours. Select suspicious colonies for definitive biochemical or serologic testing
• Yersinia enterocolitica


  • Most common Yersinia species isolated from humans acquired from contaminated food
  • Specimen: Feces
  • Enrichment: Cold enrichment of feces by incubating cultures at 4 oC for 1-3 weeks in buffered saline enhances recovery
• Subculture Yersinia enterocolitica


  1. On MAC: lactose-negative colonies, flat, colorless, or pale pink, 1-2 mm diameter

  2. On CIN (Cefsulodin-Irgasan-novobiocin) Agar: Fermentation of mannitol in the presence of neutral red results in a characteristic "bull's-eye" colony, colorless with red center. Selective inhibition of G+ and G- bacteria is obtained by means of crystal violet, sodium desoxycholate and Irgasan (triclosan). Supplementation with cefsulodin and novobiocin improves inhibition of normal enteric bacteria
• Triple Sugar Iron Agar (TSI)

  • Nutritionally very rich, lacks inhibitors to permit growth of all but the most fastidious bacterial species
  • Two reaction chambers: upper slant is aerobic, lower butt is anaerobic
  • Glucose, lactose, and sucrose are evenly distributed throughout both the slant and butt
  • Phenol red indicator is yellow below a pH of 6.8
  • Presence of cracks in the medium or the "pulling away" of the medium from the walls of the test tube indicates gas (H2 + CO2) production
  • Sodium thiosulfate is the source of sulfur atoms used for hydrogen sulfide production, which reacts with iron salts to produce an insoluble black precipitate (ferrous sulfide)
• A/A


  Acid slant / Acid butt. The organism ferments glucose, and lactose and/or sucrose producing large amounts of acids
• K/A


  Alkaline slant / Acid butt. Glucose-fermenting organism that cannot utilize lactose and sucrose produces only a small quantity of acid from the 0.1% concentration of glucose in the medium
• K/K


  Alkaline slant / Alkaline butt. Indicates a lack of acid production and failure of the test organism to ferment any of the sugars present
• IMViC Reactions


  1. Indole Test

  2. Methyl Red (MR) Test

  3. Voges-Proskauer (VP) Test

  4. Citrate Utilization Test
• The "i" in the acronym IMViC does NOT stand for any test. It is added for phonetic purposes only.
• Kligler Iron Agar (KIA)


  • Similar formula to TSI, except it lacks sucrose and the H2S indicator is ferric ammonium citrate instead of ferrous sulfate
• When inoculating a series of tubes of differential culture media with an unknown organism, it is important that the citrate medium be streaked first to prevent carryover of proteins or carbohydrates from the other media.
• Indole test


  Bacteria that possess the enzyme tryptophanase are capable of cleaving tryptophan in tryptone, thereby producing indole, pyruvic acid, and ammonia. Indole can be detected in tryptophan test medium by observing the development of a red color after adding a solution containing p-dimethylaminobenzaldehyde (e.g., Ehrlich's or Kovac's reagent)
• Methyl red (MR) test

  Principle: Bacteria that produce and maintain a low pH (below 4.4) in glucose-phosphate medium turn the methyl red indicator red
• When inoculating a series of tubes of differential culture media with an unknown organism, it is important that the citrate medium be streaked first to prevent carryover of proteins or carbohydrates from the other media
• Indole test


  1. Bacteria that possess the enzyme tryptophanase are capable of cleaving tryptophan in tryptone, thereby producing indole, pyruvic acid, and ammonia

  2. Indole can be detected in tryptophan test medium by observing the development of a red color after adding a solution containing p-dimethylaminobenzaldehyde (e.g., Ehrlich's or Kovac's reagent)
• Ehrlich's Reagent


  1. Dimethylaminobenzaldehyde 2 g, Absolute ethyl alcohol 190 ml, Concentrated HCl 40 ml
• Kovac's Reagent


  1. Dimethylaminobenzaldehyde 10 g, Pure amyl or isoamyl alcohol 150 ml, Concentrated HCl 50 ml
• Because indole is soluble in organic compounds, xylene or chloroform should be added to the test medium before adding Ehrlich's reagent. This extraction step is less critical for Kovac's reagent because amyl alcohol is used for the diluent (ethyl alcohol is used with Ehrlich's reagent)
• Methyl red (MR) test

  Bacteria that metabolize pyruvate formed from the fermentation of glucose via the mixed acid fermentation pathway produce sufficient strong acid to maintain a pH below 4.4, the acid color breakpoint of the methyl red indicator to change into a red color