Angeles University Foundation College of Allied Medical Professions Department of Medical Technology S.Y. !"# $ !

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&ntero'acteriaceae (iochemical Testing) Triple Sugar *ron +TS*, and Urease Test

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Introduction The urease test identifies those organisms that are capa'le of hydroly7ing urea to produce ammonia and car'on dio8ide. *t is primarily used to distinguish ureasepositive Proteeae from other Enterobacteriaceae. rease activity is considered to 'e a ma9or criterion for the identification of Proteus species and allo:s for Proteus to 'e distinguished from non-lactose-fermenting mem'ers of the Enterobacteriaceae Triple sugar iron +TS*, agar is a tu'ed differential medium used in determining car'ohydrate fermentation and ; S production. /as from car'ohydrate meta'olism can also 'e detected. (acteria can meta'oli7e car'ohydrates aero'ically +:ith o8ygen, or fementatively +:ithout o8ygen,. TS* differentiates 'acteria 'ased on their fermentation of lactose. glucose and sucrose and on the production of hydrogen sulfide. TS* is most fre<uently used in the identification of the Enterobacteriaceae. although it is useful for other gram-negative 'acteria. Methods and Materials Materials +Urease Test,) Christensen=s urea 'roth Proteus and &. coli 'roth cultures *noculating needle and loop Alcohol lamp

Materials +TS*,) &. coli. Pseudomonas. Salmonella. >le'siella 'roth cultures Triple Sugar *ron Agar *noculating needle and loop Alcohol lamp

T4*P0& SU/A4 *451 T&ST)

U4&AS& T&ST)

Results and Findings 4&SU0TS F54 U4&AS& T&ST) U4&AS& 4&ACT*51 Proteus +?, &. coli +-,

4&SU0TS F54 T4*P0& SU/A4 *451 T&ST) Slant (utt /as ; S

Sugars Fermented

>le'siella A A / +-,
0actose or Sucrose@ /lucose

Pseudomonas > > +-, +-, +-,

Salmonella > A / +?, /lucose

&. coli > A +-, +-, /lucose

Summary and Discussion Urease Reaction Urease, an enzyme that splits urea into alakaline end products

 Urease will split urea molecule in ammonia, carbon dioxide and water. Ammonia reacts in solution to form an alkaline compound, ammonium carbonate, which will result in an increase pH of the medium and a color change in the indicator to pinkred. useful in identification of rapid urease producers, such as roteus and !organella, as well as weak urease producers such as "lebsiella pneumoniae and some species of #nterobacter.

$%& $%& can be considered an initial step in the identification of the #nterobacteriaceae rotein sources ' beef extract, peptone, yeast extract, proteose peptone (arbohydrate sorces ' lactose, glucose ) sucrose indicator ' phenol red indicator of H*% production ' +errous sulfate has two reaction chamber with anaerobic slant portion and an anaerobic deep portion.

Reaction A/@H2S-

CHO Fermented Glucose w/ acid and Gas !actose and /or Sucrose w/ acid and Gas Glucose w/ acid and Gas

Typical Organisms "sc#eric#ia $le%siella "ntero%acter Salmonella 'roteus Citro%acter S#igella 'ro(idencia Serratia 'seudomonas Alcaligenes Citro%acter *reundii

$/@H2S&

$/AH2S-

Glucose w/ acid no gas

$/$H2SA/@H2S&

)one Glucose w/ gas !actos and/or Sucrose w/ acid and Gas

Update

Selective enrichment and production of highly urease active bacteria by non-sterile (open) chemostat culture.
*n general. 'ioprocesses can 'e su'divided into naturally occurring processes. not re<uiring sterility +e.g.. 'eer 're:ing. :ine ma6ing. lactic acid fermentation. or 'iogas digestion, and other processes +e.g.. the production of en7ymes and anti'iotics, that typically re<uire a high level of sterility to avoid contaminant micro'es overgro:ing the production strain. The current paper descri'es the sustaina'le. non-sterile production of an industrial en7yme using activated sludge as inoculum. (y using selective conditions +high p;. high ammonia concentration. and presence of urea, for the target 'acterium. highly active ureolytic 'acteria. physiologically resem'ling Sporosarcina pasteurii :ere reproduci'ly enriched and then continuously produced via chemostat operation of the 'ioreactor. Ahen using a p; of "! and a'out !. M urea in a yeast e8tract-'ased medium. ureolytic 'acteria developed under aero'ic chemostat operation at hydraulic retention times of a'out "! h :ith urease levels of a'out B! Cmol min ml culture. For cost minimi7ation at an industrial scale the costly protein-rich yeast e8tract medium could 'e replaced 'y commercial mil6 po:der or 'y lysed activated sludge. /lutamate. molasses. or glucose-'ased media did not result in the enrichment of ureolytic 'acteria 'y the chemostat. The concentration of intracellular urease :as sufficiently high such that the produced ra: effluent from the reactor could 'e used directly for 'iocementation in the field.

Questions for Research

&8ercise 1o. " -C) Urease Production ". Ahat is the importance of urease testD Urease is an en7yme that splits urea into al6aline and products. The reaction is useful in the identification of rapid urease producers. such as Proteus and Morganella. as :ell as :ea6 urease producers such as >le'siella pneumonia and some species of entero'acter. . 5ther than the mem'ers of the Family &ntero'acteriaceae. to :hat 'acteria is the diagnostic value of urease test very importantD ;elico'acter pylont

&8ercise 1o. " -D ". Ahat are the e8act concentration of the # fermenta'le car'ohydrates present in TS*D /lucose $ !."E +" part, 0actose $ "E +"! parts, Sucrose $ "E +"! parts, *f an organism can reduce sulfur. the ; S gas :hich is produced

. Ahat is the reaction involved in the demonstration of ; S productionD :ill react :ith the *ron and form *ron Sulfide :hich appears as 'lac6 precipitate. *f formed it can any al6aline or acid results. Sulfur reduction acidic environment. *f 'lac6 precipitate is present. some fermentation too6 place.

#. Descri'e the follo:ing TS* reactions as to sugars 'eing o8idi7ed and changes in the color indicator) #." AFA after " hours of incu'ation

Yello: slant and 'utt indicates all sugars :ere fermented +/lucose.

0actose. and Sucrose, #. >FA after % hours of incu'ation 4ed Slant and Yello: (utt@ only glucose is fermented. Yello: slant and 'utt indicates all sugar are fermented +/lucose. #.# AFA after % hours of incu'ation 0actose and Sucrose, References *ntroduction to Diagnostic Micro'iology) A Te8t'oo6 and Aor6'oo6. (y) Delost. M.D. http)FF:e'.e'scohost.comFehostFdetailDsidGHcBd eIJ-fJ!#-%B!d-'%aIJBH#f!KdBJa%E%!sessionmgr" LvidG"LhidG %L'dataG2n1pd/UJMAhvc#Nt'/l MN E#DE#DOd'GaphLA1GJ!"BJ#J"