Transcript Salmonella
Salmonella • Salmonella is a genus of rod-shaped, Gram-negative, non-spore forming, predominantly motile enterobacteria with diameters around 0.7 to 1.5 µm, lengths from 2 to 5 µm. • Salmonella are closely related to the Escherichia genus and are found worldwide in warm- and cold-blooded animals, in humans, and in nonliving habitats. They cause illnesses in humans and many animals, such as typhoid fever, paratyphoid fever, and the foodborne illness salmonellosis. • Salmonella is named after Daniel E. Salmon, an American veterinary, not the salmon fish. • A distinction is made between enteritis salmonella an typhoid/paratyphoid, whereby the latter because of a special virulence factor and a capsule protein (virulence antigen) can cause serious illness, such as Salmonella enterica subsp. enterica Serovar Typhi, or Salmonella typhi). • Enteritis Salmonella (e.g., Salmonella enterica subsp. enterica Serovar Enteritidis, for short Salmonella enteritidis or Salmonella typhimurium) cause diarrhoea. • The genus has 2 species: Salmonella enterica and Salmonella bongori. S. enterica has seven subspecies consistently delineated by sequence variation. The majority of disease causing serovars are from subspecies I which includes the serovars Typhimurium and Typhi. • 培養 (enrichment) – General enrichment – Selective enrichment • 劃線分離 (streaking isolation) – Streak on selective agar to obtain well-isolated single colonies • 確定 (confirmation) – Colonies showing typical characteristics on selective agar are confirmed with biochemical, serological, or molecular biological methods. Materials and methods • Bacteria: Salmonella enterica subsp. enterica ser. Typhimurium 鼠傷寒桿菌 • Food sample: chicken meat • Medium: as following Meats, meat substitutes, meat by-products, animal substances, glandular products, and meals (fish, meat, bone). 1. Aseptically weigh 25 g sample into sterile blending container. Add 225 ml sterile lactose broth and blend 2 min. Aseptically transfer homogenized mixture to sterile wide-mouth, screwcap jar (500 ml). 2. Two jars for whole class. (二血清瓶/一班) 3. One jar is inoculated with S. Typhimurium (one mL with 10-5 of overnight culture) (group 4 to 11) 4. One jar is inoculated with S. Typhimurium just meat sample (one mL with 10-7 of overnight culture) (group 1 to 4) 5. Loosen jar caps 1/4 turn and incubate sample mixtures 24 ± 2 h at 35°C. Selective enrichment 1. Transfer 1 ml mixture to 10 ml selenite cystine (SC) broth 2. Transfer 1 ml mixture to 10 ml tetrathionate Broth Base (TT) broth . Vortex. 3. Each group should have four tubes. Two SC and two TT. 4. Loosen jar caps 1/4 turn and incubate SC and TT broths 24 ± 2 h at 35°C. • Mix (vortex, if tube) and streak incubated TT and SC broth on bismuth sulfite (BS) agar, xylose lysine desoxycholate (XLD) agar, and Hektoen enteric (HE) agar. • Each group should have 4 plates (two XLD and HE). Two for SC and two for TT broth. • Incubate plates 24 ± 2 h at 35°C. 劃線分離 streaking isolation • Hektoen enteric (HE) agar. Blue-green to blue colonies with or without black centers. Many cultures of Salmonella may produce colonies with large, glossy black centers or may appear as almost completely black colonies. • Xylose lysine desoxycholate (XLD) agar. Pink colonies with or without black centers. Many cultures of Salmonella may produce colonies with large, glossy black centers or may appear as almost completely black colonies. 確定 (confirmation) 1. Pick up colonies with typical characteristics and streak and stab TSI and LIA slants. 2. Four tubes for each group; two for XLD and two for HE plates. We will do TSI first, then LIA. 3. Incubate at 35°C for 24 ± 2 h. Cap tubes loosely to maintain aerobic conditions while incubating slants to prevent excessive H2S production. 4. Salmonella in culture typically produces alkaline (red) slant and acid (yellow) butt, with or without production of H2S (blackening of agar) in TSI. In LIA, Salmonella typically produces alkaline (purple) reaction in butt of tube. confirmation 1. Pick up TSI and LIA slants with typical characteristics and streak onto TSA plates. 2. Two plates for each group. One for TSI and one for LIA. 3. Incubate at 35°C for 24 ± 2 h. • • • • • Difco™ Lactose Broth Per Liter Beef Extract ..................... 3.0 g Peptone ........................... 5.0 g Lactose ….......................... 5.0 g 值日生配製 500 mL (2 個500 mL血清瓶; 225mL/瓶). • The peptone and beef extract provide essential nutrients for bacterial metabolism. Lactose provides a source of fermentable carbohydrate for coliform organisms. Selenite Cystine Broth • • • • • Pancreatic Digest of Casein ........ 5.0 g Lactose ....................................... 4.0 g Sodium Phosphate ….................. 10.0 g Sodium Selenite …........................ 4.0 g L-Cystine ...................................... 0.01 g Selenite Cystine Broth • • • • Peptone provides amino acids and other nitrogenous substances. Lactose provides a source of energy Sodium phosphate buffers the medium to maintain the pH. Sodium selenite inhibits gram-positive bacteria and suppresses the growth of most gram-negative enterics other than Salmonella. • L-cystine is incorporated to improve the recovery of Salmonella. • 值日生 • Suspend 4.6 g of the powder in 200 mL of purified water in a 500-mL flask. • Heat to boiling. Avoid overheating. DO NOT AUTOCLAVE. • 分裝至 20 試管，每管有10 mL • Use immediately. Tetrathionate Broth Base • • • • • Proteose Peptone ........................... 2.5 g Pancreatic Digest of Casein ............. 2.5 g Oxgall …............................................ 1.0 g Sodium Thiosulfate …...................... 30.0 g Calcium Carbonate .......................... 10.0 g • Peptones provide nitrogen, vitamins, amino acides and carbon. Oxgall inhibits grampositive microorganisms. • Tetrathionate, which is formed in the medium by the addition of the iodineiodide solution, inhibits the normal intestinal flora of fecal specimens. • Calcium carbonate neutralizes and absorbs toxic metabolites. Tetrathionate Broth with iodine • 值日生 • Suspend 18.3 g of the powder in 200 mL of purified water, mix, and heat to boiling. DO NOT AUTOCLAVE. • Iodine-Potassium Iodide (I2-KI) solution – – – • potassium iodide 2 g resublimed Iodine 1.25 g sterile distilled water, 10 ml add 8 mL I2-KI solution solution to 200 mL base. Resuspend precipitate by gentle agitation and aseptically dispense 10 ml portions into sterile test tubes. Do not heat medium after addition of I2-KI and dye solutions.分裝至 20 試管，每管有10 mL • Use immediately. Xylose Lysine Desoxycholate Agar (XLD) • • • • • • • • • • • Xylose ........................................................................ 3.75 g L-Lysine ...................................................................... 5.0 g Lactose ...................................................................... 7.5 g Saccharose ................................................................. 7.5 g Sodium Chloride ........................................................ 5.0 g Yeast Extract .............................................................. 3.0 g Phenol Red ................................................................ 0.08 g Sodium Desoxycholate ............................................... 2.5 g Sodium Thiosulfate .................................................... 6.8 g Ferric Ammonium Citrate ........................................... 0.8 g Agar ......................................................................... 15.0 g • Lysine is included to enable the Salmonella group to be differentiated from the nonpathogens since, without lysine, salmonellae rapidly would ferment the xylose and be indistinguishable from nonpathogenic species. • After the salmonellae exhaust the supply of xylose, the lysine is attacked via the enzyme, lysine decarboxylase, with reversion to an alkaline pH which mimics the Shigella reaction. • To add to the differentiating ability of the formulation, an H2S indicator system, consisting of sodium thiosulfate and ferric ammonium citrate, is included for the visualization of H2S produced, resulting in the formation of colonies with black centers. The nonpathogenic H2S producers do not have decarboxylate lysine; therefore, the acid reaction produced by them prevents the blackening of the colonies. • XLD Agar is both a selective and differential medium. It utilizes sodium desoxycholate as the selective agent and, therefore, it is inhibitory to gram-positive microorganisms. Hektoen Enteric (HE) Agar Hektoen Enteric Agar • • • • • • • • • • • • Proteose Peptone ............................................. 12.0 g Yeast Extract ….................................................... 3.0 g Bile Salts No. 3 .................................................... 9.0 g Lactose .............................................................. 12.0 g Saccharose ........................................................ 12.0 g Salicin .................................................................. 2.0 g Sodium Chloride .................................................. 5.0 g Sodium Thiosulfate .............................................. 5.0 g Ferric Ammonium Citrate .................................... 1.5 g Agar .................................................................... 14.0 g Bromthymol Blue ............................................... 65.0 mg Acid Fuchsin ............................................................ 0.1 g • This medium contains three carbohydrates, lactose, sucrose and salicin, for optimal differentiation of enteric pathogens by the color of the colonies. High lactose concentration is used to aid the visualization of enteric pathogens and minimize the problem of delayed lactose fermentation. • Ferric ammonium citrate and sodium thiosulfate in the medium enable the detection of hydrogen sulfide production. • The indicator system, consisting of acid fuchsin and bromthymol blue. Triple sugar iron TSI • • • • • • • • • • • • • Approximate Formula* Per Liter Beef Extract ................................................................ 3.0 g Yeast Extract .............................................................. 3.0 g Pancreatic Digest of Casein ...................................... 15.0 g Proteose Peptone No. 3 ............................................. 5.0 g Dextrose ..................................................................... 1.0 g Lactose ..................................................................... 10.0 g Sucrose .................................................................... 10.0 g Ferrous Sulfate ........................................................... 0.2 g Sodium Chloride ........................................................ 5.0 g Sodium Thiosulfate .................................................... 0.3 g Agar ......................................................................... 12.0 g Phenol Red ............................................................... 24.0 mg Lysine Iron Agar • • • • • • • • Peptone ................................. 5.0 g Yeast Extract …....................... 3.0 g Dextrose ................................ 1.0 g L-Lysine HCl ............................10.0 g Ferric Ammonium Citrate ...... 0.5 g Sodium Thiosulfate …............. 0.04 g Bromcresol Purple ................. 0.02 g Agar .......................................15.0 g • Dextrose serves as a source of fermentable carbohydrate. The pH indicator, bromcresol purple, is changed to a yellow color at or below pH 5.2 and is purple at or above pH 6.8. • Ferricammonium citrate and sodium thiosulfate are indicators of hydrogen sulfide formation. • Lysine is the substrate for use in detecting the enzymes, lysine decarboxylase and lysine deaminase. Uninoculated Tube Proteus mirabilis ATCC™ 25933 Salmonella typhimurium ATCC™ 14028 Schedule • • • • • 10/27 (Mon) lactose broth incubation 10/29 (Wed) SC and TT broth 10/30 (Thu) XLD and HE plates 11/3 (Mon) TSI, LIA slants 11/4 (Tue) Observe results Salmonella • XLD : 紅色菌落，黑中心 (red colony, black center) • HE: 綠色菌落，黑中心 (green colony, black center) • TSI: 斜面為紅色，底部為黑色 (surface: red; bottom: black) • LIA: 斜面為紫色，底部為黑色 (surface: purple; bottom: black) • Gram stain : 陰 Servoars confirmation • Antiserum test – Surface antigen • DNA test – PCR – Multiplex PCR – Pulsed Field Gel Electrophoresis (PFGE, standard method for US CDC)