Bo I] (ne ve ed rath ith Sod Nog Nec THOIDOTOISONDIN Int Rod None PresStarch Solution: Composition per 200ml. Sir oth Preparation of Starch Solution: Aid stash to distilled’deianized water and bring volume to 200m]. Mix thoroughly, Gently heat while stirring: and bring to biding Preparation of Medium: Combine 30m. of heart infusion broth, 2n of Beameresal Purple o- lution, 200.0. of dishlledideionized water, and 210ml, of starch solution. Mis theroughly: Disiib- ite into tubes oF asks. Autoclave for 15 min at 13 psi pressure. 121°C. Pour into sterile Petri dishes or leave in bes Use: For the cultivation of Corynebacterium spe sles. Starch Hydrolysis Agar Composition per liter Beefheart, infusion from 500g, Slab starel 200g ‘Agar 150g ‘ryptose 10g Naci $08, p74 so2a28¢ Preparation af Medium: Add componcnis te sdstlled deionized water and bring volun to LOL, Mix thoroughly: Gently heat and bring to boiling Distribute inte tubes or flasks. toslave for 15 at 1S psi pressure 121°C, Pour into sterile Petr dish= cer of leave in tubes, User For the sultvation anu liffereetiation ofa vat- ‘ty of microorganisms based on amylase production. ‘Aller incubation, starch hydrolysis re determined by Ihe addition of Grams o Lugot"s iodine satution. Or- ‘ganisms that produceamylase appear as colonics su rounded by a clear zome. Starch Medium Camposition ps lit Agar 150g Soluble starch, 100g, ‘nari Farieina Starch Mineral Salt Agar Composition per iter Agar Starch, solute. aco, (NEL) 805 .. KHPO,... Mg80,7HL0........ Nal PHT 25020 25C Preparation af Medium: Adi components tw distilled deionized water and bring volume to 1.0 Mix thosoughly, Gently heat and bring to healing Distribute ite tubes or Masks. Autoclave for 15 min 81S psi pressure-121°C, Pour into sterile Petri dish ex ar leave in tubes ‘User For the cultivation and maitisnance of Srep- foverucilium species. and Thermowonospora for Starch Nitrate Medium (DSMZ Medium 886) Composition per liter RuCl 100.0% Agar 2008 Stacch cacy, KNO,. Maso, 058 Trace salt soletion 10mt. PIT 1 0290 25°C ‘Trace Salt Solution: ‘Composition per 100.0ml.: FeSO, 7H,0. Og MaCiy-#H,0 ole x80;-71,0 lg Preparation of Trace Salt Solution: Add coms Ponetits to distilledilsromized water ard being. val- ‘ume to 100-0ml., Mix thoroughlyXTBOOK OF 3 v Me ON “AL CHARACTERISTICS OF BACTERIA aveen KarLoghattows Figure’48: Kinds of fagelaion seen in hacen lure grow nin glucose phosphatet th outer wlucose medium), Appearance ron while no change in ‘original colour ( negative result The testis usod to differentiate between bacirial genera, via. Ecol! (MR x5) from Enterobacter (MR —v Yersunta sp. (MR +e) from other Gram negative non-cnteric bacteria CMR -46) 2. Indole production: This test demonsuates the production of indole from thy! carbinol H,CO.COCH,), Ths daeets! reats with peptone to give red ealour. This es (0 Textbook of Mlerobil is performed by addition of 0.6 ml ofa 5% (0.2m of 40% KOH to | ml of glacose-phosphate culture both of the organism “The postive result is indicated by development of pink colour that darkens to ‘inlcs time, The testis wed to diferente betwen bacterial sohacter «NP positive) from F cal (VP negative). Opposite results| 4. Citrate irate as the sole source of carbon with resulting a green when acidic {pH- 6. oF low) and blue when al ‘This dye is used asa pl souree of carbon, Development of colony on Kose ‘or Simmon’s citrate agar plate indicates that the test organism is able to wie ciate as a souroe of ‘arbon, The growth is accompanied with arise in pH to change the colour ofthe medium from green to deep blue These four tests are routinely performed for identification of enteric bacteria ‘of the Bacterium 10 utilize ity Bromthy mat bi i line (pH 7.6 and higher). ing citrate asthe sole oF supa is feshly prepared by mixing equal im SN acetic acid just before use. 0.1 broth, Appoarance of red color indicates positive re of nitrate reductase, Brths showing a negative wi on shoul be Fuhr tested by adding a pinch of zinc. The appearance ofa red volour indicates that mitate is resent and hence as not been reduced by the organism. IFthere is no change inthe colour of solution then it can be inferred thatthe organism has reduced the nitrate through nitrite to nitrogen pas and theres ive, This testis used in ‘demiicaion of Enterobacicriaceac members which usualy givea positive result 7, Ammonia production: Production of aramonia can be confirmed by production oF brown colour on addition ofa Few drops of Nessler’s regen 0 the bacterial culture grown on peplone water 1, Urease test: Urea ig reduced to sammonia in presence of urease envyme ‘This test employs Christenscn's urease agar medium an form of a slant. The appearance of puple-pink colour indicates form ‘of ammonia,‘CHAPTER II Biochemical Tests for Identification of Bacteria 1 hacteris microscopically. Staining provides reaction, and presence o scopic abscrvation gives capsules and endospores. Beyond that, howevr. the genus and species of x particu s based an the Aifferent species of bac que series of nucleotide bases), wwof which the organism is capable. This EVEL AVEC ICD LTO) _ cither exoensymes or endoenzymes, Exoenzymes are othe surrounding environment inorder to break down larger nutrient mole the bacterium, Once inside the organism. some of the mut (0 yield cnergy for driving various cellular functions, while others are used to form ‘building blocks for the F components. These later reactions are catalyzed by ‘endoeney mes located wi Nau Crate STARCH HYDROLYSIS K.L. GARG ari py tech ic pat KG. MUKER, ‘Sie data bok aac Some bacteriaare capable of mst first hydrolyze or break don branched polymer ofthe simple sugar, glucose. ‘This osc molceules hooked together to form a long chain. Sach asa source of carbobydrats. but in erdex to do ths, they 3 starch that ‘The busterium secretes80 Laboratory Manual of Food Microbiologs —, Sen Myosi ostNe Se bys gave Fg Hts Sancho ‘an enosnymie, which hy rolszes the starch by breaking the bonds betwssn the glucose molecules This enzyme i called diastase. For starch hydrolysis, test inoculate a starch plate with the organism tobe tested Incubate at optimum temperature Frat least 48 hours. Flood the plate with iodine and observe the resulls, Blue colour incicaes no hydralvsis, while clear zone indicates hy dol ss, PROTEIN HYDROLYSIS Proicins are made up of various amino avd linked together in lang chain by means of peptide bonds. Many bacteria ean hydrolyze a variety of proteins into peptides (chort chains of amino ‘acids and cventually ino individual amino acids. They can then use these amino acids tosymfcsize their own peocins and other cellular molecules or 49 obiain energy. The hy dtolysis of protcin is termed proteolysis and the eneyme involved 3 alle protease ‘Make a single szeak lnc with the es organism on the skim mitk agar plate and incubate for 3 days, feaseinis hydrolyze, there willbe a clear zone around the bctena! grt, I ass i ot Inydrolyzed, the agar will remain white and opaque.ABOJOIGOIOII/\ onsoubeiq evs was shownto be more sensitive than routine Blood agar ‘media in detecting S. aureus when i was used ay pat of the primary specimen plating protocol Chromogenic agar ‘media have also been exploted as api mod for sil taneously idenifsing 5 urews and determining metic resiatange by inorporaing oxi or petit ino the media, The Catalase Test, Staphylococs and microooeei ae ifferentite fom the strepicoce,enterococe. and steprcoceuvlike” bacteria by the catalase test. This test detects the presence of eyto- stome oxidase enzyses(Chiet 12-1). The eae tx fe performed with 3% hydrogen perosie (H.0:) om a ass Side, Immediate and vigorous babbling indisats conversion ‘of the H:0: to water and oxygen gas (Color Plate 12-16), © YoOg)xe| PLE Selly 10|09 S,UBLUSUO} Several methods are avilable for differentiating. Mi crococeus nd Supslocaccus species, the two cals postive genera mon (requetly seen inthe incl labor tory. from other catalase-postive, gram-posive cocci ‘Some require special media and prolonged ineebaton, while ‘ters are commercially availble and provide resus within 18 1924 ours or fess. Table 12-1 Tiss the test methods and results for Micrococcus, Macrococcus, and Stopocercit species, Although Macracoceus species ae primal equine iolates eactons for these specie ae inelded foe pdmary ecopton of macrococcal neat recovered i aboratiies that provide services to veterinary practices FERMENTATION OF GLUCOSE ‘This ts is performed in a manner similar tothe oxi- ation-fermentation (OF) texts for nonfermentaive gram [iti 1 Pena Charo elon teens Meee and apace] _DESCRIPTIONTREACTION FOR Micrococcus and CHARACTERISTIC Related Species” Macrococeus Colony se Tika 7325 um 6-15 ua Cony erie Comex Low come, domed sinc, ow comer rem ete Very stow Slow Stout pid Aci potato fom slic - + ‘der arse cons ysowaphia R s s Pradustion of acid from gy - NA : ‘cl serbicaly inthe presence 104 per ceyroaia Fraroine suscep R s 8 100 wo frztnone disk) Bacircin usepibilty (00 s R R ‘unt To A) Modis ide ft + 4 ’ At Supe rte oe ofS nt ot vit GBo) se: For the direst of tii bail on ad prodct MRVP Broth with Sodivm Choride (Methy! RedeVoges-Proskauer Broth with NaC) (BAM M104 Medium 1) Composition ps her Dale pepo water poder 2 sao, Soe Source: Rule! peptone lr pret x vb fo RD Dg Preparation of Medium ‘cpus to dle doen ‘tia bg elie 1 Mic thorugh iste ino es ‘Vet Fo the ietition f lap ids pp.bado id MRVP Broth with Sodium Chloride (Methyl Red-Voges-Proskauer Broth with (BAM M104 Mediues 2) eee so nie, im ic dgewofeasnn ast Uae Forte dietiaton oC alpibe ve ap, se on sh jo yooqnueHy Use: Fa the deen fhe rio 9. MRVP Median (Mtethyt Red Voges: Proska Composition er iter Pepe $0 Source: Thc di ieaaisble aa pemnl pouder fm Ona Preparation of Meum: (i conposcs to ds dena v MS Ager Compeclden er Ber pow Preparation of Mediums e tate andeing velco Ms oo el an! hing poses 121°C Poirot dss rn ie. User Forth avai Ral seri MS 1 Agar \ iss Preparation of Medium: Ass there Oct ea edb 1 frat seat Mi.Starch Solutio Composition per 20 0m Sac Preparation of Starch Solution: idiloddtecized water aed ting lure to 200m. Mx thoroughly: Gey sat while siring it ang beng Preparation of Medium: Combine Om. of he fsion beh, 2 of Eromersol Ppl so- len, 200 Om. of sitll deonized water, and 200m, of stare solution. Mix thovougly. Dist ‘ein tes or Rasks: Autolave fr 18 min 315 Pt pressure 121°C. Pour into see Pet dishes or Teave in tabs Use: For the cultivation of Corehactertn spe Starch Hydrolysis Agar Composition per liter Best her, infson fom, 590.05 Solute aac, 2008 ‘gat 188 Typiose 008 Nucl S08 pT 0383s Preparation of Medium: Ald compass to abled deionized water and bing volume to 10 Mix thoroughly” Gem heal and ring 1 boing Disuibute mo whe or aks, Autoclave for 15min a1 1S prese 121°C Pour nose Pt ihe ‘sor leave tides Use: Forth evan and diferetaion of ati «yf msrooranisns based on armase proton ‘Aller incubation. starch bya determined by the adltionof Gram rl-agl iodine solution Or ‘nisms thn produc mae appears oni sur Founded by alear one Starch Medium Composition per liter Agar 1508 aria aici Starch Mineral Salt Agar ‘Composition porter Aer nop Starch solute Wag 200, 208 etl) 80, 205 Po, 0p MeS0; 7110. op mct 10g p72 028126 Preparation of Medium: dd components ill deorizell water and being volume to OL, ‘Mix throughly. Geatly beat and beng. boing, Distrib ito tubes or Masks. Astslave fer 155 tS ps presse 121°C. Pour into see Pet diab ‘ior lene in kes, ‘Uses For the extivtion aa maintenance of Sup tevertelum species and Thermemanespara for Starch Nitrate Medium (DSMZ Medium 856) ‘Composition por lite: Nucl 100.08 Aor 2005 Such 300, aco, 305 EXO, 2.0 KIO, oe Maso, 058 “race st ston 1. p71 02825 “Trace Salt Solution: ‘Composition per 100 Oa FeSO,7H1.0. oe MaCiyt.0 Ole 25900; 704.0 on [Preparation of Trace Salt Solution: 8 com- ‘ponenls to dstiled/dtenived water and bring vl