microbiology

• The iodine should remain on the skin for 1 min

  because instant antisepsis does not occur when

  cleansing the skin for a blood culture.
• C. diphtheriae must be recovered from the deep

  layers of the pseudomembrane that forms in the

  nasopharyngeal area. A flexible calcium alginate

  nasopharyngeal swab is the best choice for collecting

  a specimen from the posterior nares and pharynx.
• Specimens for culture of N. gonorrhoeae are best if

  plated immediately or transported in a medium

  containing activated charcoal to absorb inhibitory

  substances that hinder their recovery.
• Hektoen agar selectively isolates pathogenic

  coliforms, especially Salmonella and Shigella.

  MacConkey agar differentiates lactose fermenters

  from nonfermenters. CNA agar contains antibiotics

  that prohibit growth of gram-negative coliforms but

  not gram-positive cocci. Campy agar contains the

  antibiotics cephalothin, trimethoprim, vancomycin,

  polymyxin B, and amphotericin B to prevent growth

  of Enterobacteriaceae, Pseudomonas spp., and fungi.
• CNA agar inhibits the growth of gram-negative

  bacteria and is used to isolate gram-positive cocci

  from specimens. This medium is especially useful

  for stool and wound cultures because these may

  contain large numbers of gram-negative rods.
• MTM, New York City, and Martin–Lewis agars

  contain blood factors needed to support the growth

  of N. gonorrhoeae as well as antibiotics that prevent

  growth of normal genital flora. Cultures must be

  incubated in 3%–7% CO2 at 35°C. Cultures should

  be held a minimum of 48 hours before being

  considered negative.
• Chocolate agar provides X factor (hemin) and

  V factor (NAD) required for the growth of

  Haemophilus spp. Thayer–Martin medium is a

  chocolate agar containing the antibiotics that

  permit isolation of N. gonorrhoeae in specimens

  containing large numbers of gram-negative

  bacteria, including commensal Neisseria spp.
• DCA inhibits gram-positive organisms. N. gonorrhoeae

  and Neisseria meningitidis are too fastidious to grow

  on DCA.
• XLD agar


  Selective for gram-negative coliforms
• XLD agar


  • High concentration (0.25%) of deoxycholate, which inhibits gram-positive bacteria
  • Differential for Shigella and Salmonella spp.
• Fermentation in XLD agar

  1. Xylose, lactose, and sucrose fermented by intestinal coliforms producing yellow colonies

  2. Shigella does not ferment and produces red (or clear) colonies

  3. Salmonella spp. ferment xylose but also decarboxylate lysine in the medium, causing production of ammonia, first appearing yellow but becoming red

  4. Some Salmonella produce hydrogen sulfide (H2S) from sodium thiosulfate and therefore appear as red colonies with black centers
• Fastidious organisms such as Neisseria and

  Haemophilus frequently isolated from the CSF of

  patients with bacterial meningitis are preserved by

  placing the fluid in 3%–7% CO2 at 35°C–37°C (or at

  room temperature for no longer than 30 min), if the

  specimen cannot be cultured immediately.
• Anaerobic culture media can be prereduced before

  sterilization and addition of cysteine or other thiol compounds.
• CNA agar for gram-positive cocci and MacConkey

  agar for gram-negative bacilli. the primary isolation medium for Haemophilus is chocolate agar.
• β-Lactamase


  An enzyme produced by bacteria that are resistant to penicillin and cephalosporin
• Detection of β-Lactamase production

  1. Use one of these drugs as a substrate

  2. Penicillin is hydrolyzed by β-lactamase into acidic products

  3. Acidic products detected as a color change by a pH indicator
• Iodometric method


  • Disk containing a penicillin–starch substrate turns blue when a drop of iodine is added
  • A loop of β-lactamase–positive organisms applied to the center of the blue spot will reduce the iodine to iodide, causing the area to clear
• The breakpoint refers to an antimicrobial

  concentration in the serum associated with

  optimal therapy using the customary dosing

  schedule. An organism is susceptible if the MIC

  is at or below the breakpoint.
• A thin film of growth appearing in the zone area of

  inhibition around the susceptibility disk should be

  ignored when swarming Proteus or other organisms

  are encountered. Discontinuous, poor growth or tiny

  colonies near the end of the zone should also be

  ignored.
• The aminoglycoside antibiotics act by inhibiting protein synthesis. They have low bacterial resistance but must

  be monitored at high doses they can cause ototoxicity and nephrotoxicity. e.g. amikacin, gentamicin, tobramycin,

  kanamycin, streptomycin, and spectinomycin. Administered intravenously or intramuscularly (poorly absorbed in GIT)
• The oxidase positive test result rules out the

  members of the Enterobacteriaceae family. Colonies

  of Aeromonas hydrophilia and Plesiomonas spp.

  might be mistaken for Vibrio spp. since all three

  grow as clear colonies on MacConkey agar, are beta

  hemolytic on blood agar, and are oxidase positive.
• E. coli 0157:H7 ferments lactose, and therefore,

  appears as dark pink colonies on MacConkey agar. To

  differentiate E. coli 0157:H7 from normal fecal flora,

  MacConkey agar with sorbitol is used. E. coli 0157:H7

  does not ferment sorbitol, and usually are colorless

  colonies.
• The ONPG test detects β-galactosidase activity

  and is most useful in distinguishing late lactose

  fermenters from lactose nonfermenters. Some

  strains of E. coli are slow lactose fermenters and

  may be confused with Shigella spp., which do not

  ferment lactose. E. coli are ONPG positive while

  Shigella spp. are ONPG negative.
• Acetoin or carbinol, an end product of glucose

  fermentation, is converted to diacetyl after the

  addition of the VP reagents (α-naphthol and 40%

  potassium hydroxide [KOH]). Diacetyl is seen as a

  red- to pink-colored complex.
• At which pH does the methyl red (MR) test

  become positive? 4.5
• The Simmons citrate test determines if an organism

  can utilize citrate as the sole source of carbon. The

  medium turns blue, indicating the presence of

  alkaline products such as carbonate. Tubes are

  incubated a minimum of 24 hours at 35°C with a

  loose cap before reading.
• The test for urease production is based on the ability

  of the colonies to hydrolyze urea in Stuart broth or

  Christensen agar to form CO2 and ammonia. These

  products form ammonium carbonate, resulting in

  alkalinization. This turns the pH indicator (phenol red)

  pink at pH 8.0.
• The indole test detects the conversion of

  tryptophan (present in the media) to indole by

  the enzyme tryptophanase. Indole is detected

  by the reaction with the aldehyde group of

  p-dimethylaminobenzaldehyde (the active reagent

  in Kovac’s and Ehrlich’s reagents) in acid, forming a

  red complex.
• Specific decarboxylases split dibasic amino acids

  (lysine, arginine, and ornithine), forming alkaline

  amines. These products turn the pH indicators in the

  medium (cresol red and bromcresol purple) from

  yellow to purple.
• LIA is used as an aid for the identification of

  Salmonella species. It contains phenylalanine, lysine,

  glucose, thiosulfate, ferric ammonium citrate, and

  bromcresol purple. Salmonella produce H2S from

  thiosulfate. This reduces ferric ammonium citrate,

  forming ferrous sulfate and causing the butt to

  blacken. Salmonella also decarboxylate lysine to

  produce alkaline amines, giving the slant its purple

  color and differentiating it from Citrobacter spp.,

  which are lysine decarboxylase negative.
• Putrescine is the amine product of the

  decarboxylation of ornithine.
• The malonate test determines whether an organism

  can utilize sodium malonate as the sole source of

  carbon. Malonate is broken down, forming alkaline

  metabolites that raise the pH of the broth above 7.6.

  This causes bromthymol blue to turn from green to

  deep blue (Prussian blue). E. coli, Shigella, and most

  Salmonella are malonate negative, whereas

  Enterobacter and Salmonella (formerly Arizona)

  subgroups 2, 3a, and 3b are positive. Proteus,

  Providencia, Serratia, and Yersinia are also malonate

  negative.
• Serological confirmation of Shigella isolates is based

  upon O antigen typing. If a suspected Shigella spp.

  is serologically typed with polyvalent sera before it

  has been correctly identified biochemically, a

  false-positive confirmation may occur with an isolate

  that is E. coli (i.e., anaerogenic non–gas-producing,

  lactose-negative or delayed, and nonmotile

  strains). These strains were formerly known as

  the Alkalescens-Dispar serotype.
• The Shigella spp. are lactose nonfermenters that

  for the most part are biochemically inert and are

  classified into serogroups A, B, C, and D as a result

  of their biochemical similarity. S. sonnei is the

  species most often isolated from diarrhea cases in

  the United States. It is more active biochemically

  than the other species owing to ornithine

  decarboxylase and β-galactosidase activity. These

  enzymes, found in most strains of S. sonnei,

  distinguish it from other Shigella species.
• E. coli, positive for lactose, indole, and ONPG are

  usually motile. Shigella species do not ferment

  lactose or produce indole, lack β-galactosidase,

  and are nonmotile.
• Shigella spp. and Klebsiella spp. are for the most

  part nonmotile. Yersinia can be motile at 22°C but

  is nonmotile at 36°C. Other members of the

  Enterobacteriaceae that have been isolated from

  human specimens and are usually nonmotile

  include Leminorella, Rahnella, and Tatumella
• Shigella spp. and Campylobacter spp. are both causes

  of diarrhea, abdominal pain, fever, and sometimes

  vomiting. Blood is present in the stools of patients

  infected with Shigella. Young children may also

  exhibit bloody stools when infected with

  Campylobacter.
• Which group of tests, along with colonial

  morphology on primary media, aids most in the

  rapid identification of the Enterobacteriaceae? Indole, oxidase, MacConkey, and blood agar

  plates
• Morganella and Proteus spp. are motile, produce

  urease, and deaminate phenylalanine
• E. sakazakii is called a yellow-pigmented. Best differentiated from E. cloacae by sorbitol fermentation (95% positive for E. cloacae)*also malonate and urease. Both species are usually motile and arginine dihydrolase positive.