APPLICATION Detection and isolation of pathogenic intestinal bacteria including Shigella and Salmonella from surfaces, food, or liquid samples.

Transcription

1 HEK/SS Code 5543 COMING SOON! BioPaddles Colony Identification App Hektoen Enteric Agar (HEK) Salmonella Shigella Agar (SS) USE: Detection and isolation of pathogenic intestinal bacteria including Shigella and Salmonella. Side 1: Hektoen Enteric Agar (HEK) (green) (Side 1 is marked with an indented laser line) Side 2: Salmonella/Shigella Agar (SS) (reddish orange; slightly hazy) APPLICATION Detection and isolation of pathogenic intestinal bacteria including Shigella and Salmonella from surfaces, food, or liquid samples. Salmonella contamination is the primary cause of food-borne illness worldwide. Most outbreaks of Salmonella are traced back to dairy, poultry and meat products, but Salmonella can grow on nearly any food. Chicken, eggs and their derivative products are particularly high risk. Salmonellosis continues to be an important public health problem worldwide. Infection with non-typhi Salmonella often causes a mild, self-limiting illness. This infection can result from the consumption of raw, undercooked, or improperly processed foods contaminated with Salmonella spp. Shigellosis, caused by Shigella spp., is an intestinal illness characterized by abdominal pain, fever, and watery diarrhea. When associated with outbreaks, shigellosis is usually transmitted through contaminated food and/or water. PADDLE AGARS Hektoen Enteric Agar (HEK) Medium originally developed by King and Metzger 1 Mixed peptone and yeast extract are rich sources for nitrogenous compounds, vitamins, carbon, sulphur and amino acids. The carbohydrates sucrose, lactose and salicin are the fermentable substrates while bile salts, acid fuchsin, and bromothymol blue inhibit Gram (+) organisms. Sodium chloride contributes to osmotic balance. Ammonium ferric citrate is a source of iron, which is used as indicator for the production of hydrogen sulfide (H 2 S). Sodium thiosulfate is used as the substrate for building hydrogen sulphide which gives, together with iron, a black precipitate (Iron(II) sulphide; FeS). H 2 S-positive colonies have black centers. The indicator bromothymol blue changes the color to yellow and acid fuchsin changes color from yellow to pink when acid is formed. NOTE: Proteus species may resemble salmonellae or shigellae. Further testing must be carried out to confirm the presumptive identification of organisms isolated on this medium. 1 S. King, W.I. Metzger, A new plating medium for the isolation of enteric pathogens. Appl. Microbiol., 16, (1968) Page 1

2 Salmonella Shigella Agar (SS) SS Agar is a modification of the Desoxycholate Citrate Agar described by Leifson. 2 SS Agar is recommended 3 for testing clinical specimens and food testing for the presence of Salmonella spp. and some Shigella spp. Beef extract, enzymatic digests of casein and animal tissue provide sources of nitrogen, carbon, and vitamins required for organism growth. Lactose is the carbohydrate present in SS Agar. Bile salts, sodium citrate and Brilliant Green inhibit Gram (+) bacteria, most coliform bacteria, and inhibit swarming Proteus spp., while allowing Salmonella spp. to grow. Sodium thiosulfate and ferric citrate permit detection of hydrogen sulfide by the production of colonies with black centers. Neutral Red is the ph indicator. Agar and a proprietary polymer are the solidifying agents. NOTE: SS Agar is highly selective and not recommended as the primary isolation of Shigella, some Shigella spp. may be inhibited. CULTURE CONTROLS inoculum (CFU) HEK Agar SS Agar Escherichia coli INHIBITED INHIBITED Enterococcus faecalis INHIBITED INHIBITED Shigella flexneri GROWTH GROWTH Salmonella typhimurium GROWTH GROWTH STORAGE/EXPIRATION Store tightly sealed BioPaddles in a cool, dry location (less than 68 F/20 C). Avoid temperature changes. BioPaddles may be refrigerated, but it is not necessary. Do not freeze. If freezing occurs, thaw (3-6 hours) under refrigeration temperatures (40 F; 4.4 C). Freezing can promote excess water loss and variation in media surface due to crystal formation. The average shelf-life is one year. Refer to expiration date (See: BBE stamped on vial). Discard if paddle agar appears oxidized (darkens from expected color). The expiration date applies to the medium in an intact container when stored as directed. SAMPLING Liquids: Twist to remove paddle from vial. Fill vial to 40 ml fill line with the liquid to be sampled. The 40 ml volume can be used to calculate Total Viable Count (TVC) and/or Total Colony Count (TCC). Replace paddle. Allow a contact time of 15 seconds. Remove the paddle. Empty the vial. Replace the paddle in the vial. Surfaces: Twist to remove paddle from vial. Allow the paddle surface (10 cm 2 ) to come into physical contact with the test surface. Recovery rate is about 50%. To insure an accurate recovery, gently sweep (or touch) the paddle to cover a 20 cm 2 area. Replace paddle in vial. 2 Leifson, E New culture media based on sodium desoxycholate for the isolation of intestinal pathogens and for the enumeration of colon bacilli in milk and water. J. Pathol. Bacteriol 40: McFaddin, J. F Media for isolation-cultivation-identification-maintenance of medical bacteria, Vol. 1. Williams & Wilkins, Baltimore, MD. Page 2

3 INCUBATION Incubate at 35 C ±2 C for hours. Examine, then re-incubate and examine 2 days, then 4 days later. The later incubations and examinations are particularly important for paddles with initial negative growth. NOTE: Proteus species may resemble salmonellae or shigellae. Further testing must be carried out to confirm the presumptive identification of organisms isolated on this medium. COLONY MEASURING Each BioPaddles paddle has molded media attachment points that are 4mm in length (point-to-point). This feature provides a useful guidepost to estimating nearby colony size. IDENTIFICATION ORGANISM ORGANISM HEK SS PHYSIOLOGY Precision Test Strip Available E. coli Lactose (+) Indole (+) ) Enterococcus faecalis Lactose (+) Indole (+) Oxidase (+) Catalase (-) Urease (+) GROWTH COLONY GROWTH COLONY + COMPLETE INHIBITION Salmonorange May have bile ppt PARTIAL - COMPLETE INHIBITION COMPLETE INHIBITION Pink to rosered May have bile ppt Page 3

4 ORGANISM ORGANISM HEK SS PHYSIOLOGY Precision Test Strip Available Proteus spp. Lactose (-) SEE: INDOLE Urease (+) Salmonella ser. Typhimurium Lactose (-) Indole (-) H2S (+) Shigella boydi Lactose (-) Indole (variable) Shigella flexneri Lactose (-) Indole (variable) Shigella dysenteriae Lactose (-) Indole (variable) Catalase (-) LaMotte BioPaddles TECH DOCUMENT GROWTH COLONY GROWTH COLONY Greenishblue ++ Clear, ++ Clear, Colorless or Colorless or Colorless or Page 4

5 ORGANISM HEK SS ORGANISM PHYSIOLOGY Precision Test Strip Available Shigella sonnei Lactose (+) Indole (-) GROWTH COLONY GROWTH COLONY ++ Colorless or ENUMERATION HEK SS Very Light Light Moderate Very Light Light Moderate TVC / TCC TVC/TCC (Total Viable Count/ Total Colony Counts) Colony Counts <1000 Count colonies TVC/TCC Count = Count x 2.5 Colony Counts >1000 Use chart TVC/TCC Count = Count x 2.5 Approximate Colony Count per 100 ml (Based on a 40 ml sample) Page 5

6 Example: Inoculated NUT/TTC paddle showing approximately 1000 CFU/100 ml. DISPOSAL Twist to remove paddle from vial. Fill vial to 40 ml fill line with 1:9 dilution of household bleach (5.25% sodium hypochlorite). Replace paddle in vial. Allow 15 minute contact time. Remove paddle. Discard bleach solution. Replace paddle in vial and dispose. Alternatively, loosen cap and microwave for 30 seconds, autoclave, or incinerate. GLOSSARY : CVEG Convex, Entire, Glossy FED Full, Entire, Dull Catalase Cat (+) contains catalase enzymes that degrade cellular H 2 O 2 Lactose Lac (+) bacteria can ferment available lactose in the medium producing an acid which lowers the ph. Lac (-) are non-fermenting. Indole Biochemical test to determine the ability of an organism to split indole from the amino acid tryptophan. P. vulgaris is indole (+) while P. mirabilis is indole. Oxidase OX (+) contains cytochrome c oxidase. In contact with an indicator turns dark blue if OX (+); colorless if OX (-). Urease UR (+) presence of enzyme urease which hydrolyzes urea into CO 2 & NH 4. β-d-glucoronidase Glu (+) Found in 97% of E. coli strains. The presence of E. coli is determined when both β-d- Glucoronidase and Indole are (+), and the organism is Gram (-). Gram Gram reaction Page 6