OBJECTIVE: 1. To make-famous the bacteria abilities to metabolize multiprofit substrates and purpose issues coercionmed. 2. To behold the development of opposed bacteria cast in regulate of structures and its morphology installed on opposed chemical import applied. 3. To behold physiological and immunological properties utilized by opposed cast of bacteria. INTRODUCTION: Bacteria biochemical ordealing can designate the signs and quantity in regulates of colonization coercionming units of bacteria offer in a illustration of opposed chemical. The ordealing could be focused on a peculiar sign of bacteria, medical bacteria or a unreserved dispose of environmental bacteria.
Gone bacteria are offer in virtually any environment, it’s material to be apparent why the ordealing is life manufactured. The more peculiar the ordealing is the improve and the easier it is to declare the upshots. Quantity and signs of bacteria that should be a motive coercion matter insists upon separate factors, including the sign of bacteria offer and the sign of illustrations. Escherichia coli are individual of the deep cast of bacteria succor in the inferior entrails of mammals. E. coli can be root in the intestinal credit of warm-blooded voluptuouss. The influence of E. coli in foundations is consideruddy to be an parade of fecal dirt.
Staphylococcus organisms are systematically root in the environment. Separate cast of Staphylococcus are root on the husk, entrails, nasal passages, absence of wonder. of warm-blooded voluptuouss. Some cast of Staphylococcus, detailly Staphylococcus aureus can be pathogenic are capoperative of causing ailment. Pseudomonas aeruginosa is widely reserved in contaminate, impart and plants. It survives in glowing tubs, whirlpools, continuity lens discontinuance, sinks and paradeers. It can motive a estimate of opportunistic infections including infections of the husk, exterior ear canal and of the intention.
Nitrifying bacteria recycle radical nitrogenous materials from ammonium (the purposepoint coercion the analysis of proteins) to nitrates. Their influence can parade that the impart may entertain been defiled by nitrogen-rich radicals from accounts such as concerned septic tanks, sewage systems, industrial and imperilled wane sites and is undergoing an aerobic coercionm of deprivation. The influence of denitrifying bacteria can parade that the impart has been defiled by nitrogen-rich radicals from accounts such as concerned septic tanks, sewage systems, industrial and imperilled wane sites. MATERIALS: 1. Nutrient broth cultivations of Escherichia coli . Nutrient broth cultivations of Serratia marcescens 3. Nutrient broth cultivations of Salmonella typhimurium 4. Nutrient broth cultivations of Bacillus subtilis 5. Nutrient broth cultivations of Klebsiella spp. 6. Nutrient broth cultivations of Streptococcus spp. 7. Nutrient broth cultivations of Staphylococcus aurieus 8. Nutrient broth cultivations of Proteus vulgaris 9. Nutrient broth cultivations of Pseudomonas fluorescens 10. Parafilm tape 11. Inoculating loops 12. Gloves 13. Incubator 14. Nutrient agar dish 15. Nutrient agar slants 16. Ruefulness agar dishs 17. Gelatine agar dishs 18. 2 tubes Clark’s-Lub average (MR-VP average) 19. Tryptindividual broth 20. 3 Kigler’ slant 21. 5 tubes nitrate broth ( 0. 1% KNO3) 22. 5 urea broth 23. Tube embraceing 10ml of barren not attributefficacious attributefficacious attributefficacious attributoperative attributoperative attributoperative attributoperative attributoperative attributoperative attributoperative attributableoperative 24. Glucose broths with Durham tubes and phenol ruddy indicator 25. Lactose broths with Durham tubes and phenol ruddy indicator 26. Sucrose broths with Durham tubes and phenol ruddy indicator 27. Gram’s iodine 28. Kovac’s indol reconstituency 29. Mercuric chloride discontinuance 30. KOH-creatine discontinuance or 40% KOH 31. F&R reconstituency 32. Nessler’s reconstituency PROCEDURE: A. CARBOHYDRATE METABOLISM 1. Fermentation of sugars Materials: 1. Glucose broths with Durham tubes and phenol ruddy indicator 2.
Lactose broths with Durham tubes and phenol ruddy indicator 3. Sucrose broths with Durham tubes and phenol ruddy indicator 4. 18 hour nutrient broth cultivations of E. coli and S. typhimurium Procedure: 1) The slight bottles of opposed sugars were inoculated with a loopfuls of E. coli and Salmonella spp. 2) The tubes were labelled and brew at 37oC coercion 24 hours 3) All contemplations were commemorative coercion influence of harsh or fume coercionmation. 2. Hydrolysis of ruefulness Materials: 1. Ruefulness agar dishs 2. Broth agar cultivations of B. subtilis and E. coli Procedure: 1) Ruefulness dish was streaked with E. coli in coercion sections and numerous coercion B. ubtilis bacteria in other ruefulness dish. 2) The dishs were securuddy with parafilm, labelled and inoculated at 37oC coercion 24 hours. The coercionthcoming day 1) The dishs were ordealed coercion ruefulness hydrolysis by flooding the pates with Gram’s iodine. 2) The dishs were examined and the colonies that paradeed apparent uncolouruddy zones in dissimilarity with the blue-colored-sombre elucidation of the ruefulness-iodine close were not attributefficacious attributefficacious attributefficacious attributoperative attributoperative attributoperative attributoperative attributoperative attributoperative attributoperative attributableed. 3) The degree of the zones of hydrolysis paraded either the ruddydish colour zones were seen. 4) All upshots and contemplations were commemorative. B. PROTEIN AND AMINO ACID METABOLIM 1. Indole ordeal Materials: 1. Broth cultivations of B. ubtilis, E. coli, and S. typhimurium 2. 3 tubes of tryptindividual broth 3. Kovac’s indole ordeal reconstituency Procedures: 1) The peptindividual impart was inoculated with a loopfuls of the ordeal organism. 2) The tube was labelled and brewd coercion 24 hours. The coercionthcoming day 1) The tubes were pretended with a lacking drops of Kovac’s indole reconstituency (dimethylaminobenzaldehyde) 2) The ruddy or sombre tinge parades the influence of indole. 4. Hydrogen sulphide Materials: 1. Broth cultivations of B. subtilis, E. coli, and S. typhimurium 2. 3 Kigler’s slant Procedures: 1) The Kigler’s slant was inoculated with a loopfuls of the ordeal organism by the pierce process. ) The tube was labelled and brewd coercion 24 hours. The coercionthcoming day 3) The Kigler’ slant was beholdd coercion coercionmation of H2S where the sombre dive parallel the course of development in the Kigler’s slants paraded the H2S entertain been executed. 4) The contemplations were commemorative. 3. Gelatine hydrolysis ordeal Materials: 1. Broth cultivations of B. subtilis, E. coli, and S. typhimurium 2. Gelatine agar dishs 3. Mercuric chloride discontinuance Procedures: 3) The gelatine agar dishs were inoculated with a loopfuls of the ordeal organism with a unique streak at the capital of the dishs. ) The dishs were securuddy with parafilm, labelled and brewd coercion 24 hours. The coercionthcoming day 5) The dishs were flooded with mercuric chloride discontinuance. 6) The average befit turbid in regions that quiescent embrace gelatine and apparent regions where gelatine has been hydrolysed. C. VOGES-PROSKAUER TEST Materials: 1. Broth cultivations of E. coli, and Klebsiella spp. 2. 2 tubes of Clark-Lub’s average (MR-VP average) 3. KOH-creatine discontinuance Procedures: 1) The tubes of Clark-Lub’s average (MR-VP average) were inoculated with a loopfuls of the ordeal organism. 2) The tubes were labelled and brewd coercion 24 hours.
The coercionthcoming day 1) The tubes were ordealed with Voges-Proskauer ordeal. 2) The 0. 5ml of KOH-creatine solutuin was addd. 3) The tube was shaked powerfully coercion 30 seconds. 4) The ruddy or pink tinge parades the influence of acetoin. D. CATALASE TEST Materials: 1. Broth cultivations of Streptococcus spp. and Staphylococcus aureus. 2. Nutrient agar slant Procedures: 1) The nutrient agar slant was inoculated with a loopfuls of the ordeal organism. 2) The tube was labelled and brewd coercion 24 hours. The coercionthcoming day 1) The tubes were ordealed with catalase ordeal by adding separate drops of a 5% discontinuance of hydrogen peroxide. ) The powerful bubbling parades the influence of oxygen. E. NITRATE REDUCTION TEST Materials: 1. Broth cultivations of E. coli, Proteus vugaris, Serratia marcescens, Pseudomonas fluorescens. 2. 5 tubes embraceing nitrate broth (0. 1% KNO3) 3. Nitrate ordeal reconstituency Procedures: 1) The nitrate broth was inoculated with a loopfuls of the ordeal organism. 2) The tube was labelled and brewd coercion 24 hours. The coercionthcoming day 1) The tubes were ordealed with 1ml of Follet and Ratcliff’s (F&R reagent) 2) The odispose or brown tinge parades the influence of nitrate. 3) The listless of nitrate parades that: a.
There has been no nitrate ruddyuction b. The ruddyuction has proceeded raise that nitrate stage. 4) The listless of odispose or brown tinge were raise ordealed with slight equality of cadmium to the tube. If nitrate quiescent offer, it accomplish be catalytically veer to nitrate which accomplish then reacts with the F&R reconstituency in the tube. 5) In the listless of a confident nitrate upshot, the trashs f H2 fume was beholdd in the Durhams tube OR 6) The illustrations were ordealed with 1ml of Nessler’s reagent. The brown or odispose tinge parades the influence of ammonia. F. UREASE TEST Materials: 1. Broth cultivations of E. coli, P. vugaris, S. arcescens, P. fluorescens. 2. 5 urea broth with indicator Procedures: 1) The urea broth was inoculated with a loopfuls of the ordeal organism. 2) The tube was labelled and brewd coercion 24 hours. The coercionthcoming day 1) The urease-confident organism executed in solemn ruddy/purple tingeation of the average subjoined incubation. 2) All contemplations were commemorative. RESULTS AND OBSERVATION: Ordeal| Contemplation(Subjoined 24 hours incubation)| Description| A. Carbohydrate Ordeal 1. Fermentation of ruefulnessDurham tubes and phenol-ruddy indicator. 2. Hydrolysis of ruefulness| Glucose: Lactose: Sucrose: Ruefulness agar dishs:B. ubtilisE. coli| * Confident upsfervent coercion E. coli as tube adapt yellow * Confident upsfervent coercion S. typhimium as tube adapt yellow * Confident upsfervent coercion E. coli as tube adapt yellow * No fume executed by S. typhimium bemotive the tube adapts ruddy. * No fume executed by E. coli bemotive the tube is subordinately ruddy. * Confident upsfervent coercion S. typhimium as tube adapt yellow * Confident zindividual of apparenting. * Disclaiming zindividual of apparenting. | B. Protein And Amino Harsh Metabolism 1. Indole ordeal 2. Hydrogen disulphide 3. Gelatine hydrolysis ordeal| Tryptindividual broth:B. subtilisE. coli. S. typhimuriumKigler’s slant:B. subtilisE. oli. S. typhimuriumGelatine agar dishs:B. subtilisE. coli. S. typhimurium| * Disclaiming Indole ordeals no tinge veer. * Flashing fuschia at the interface is confident ordeal coercion Indole. * Disclaiming Indole ordeals no tinge veer. * Sombre dive coercionm parades confident sulphur ruddyuction. * Disclaiming reaction. * Confident reforce coercionming the sombre dive. * Confident hydrolysis of gelatine into amino harsh to be representationd as nutrients/gelatinase. * Disclaiming hydrolysis of gelatine. * Disclaiming hydrolysis of gelatine| C. Voges- Proskaeur’s Ordeal| MR-VP average:E. coli. Klebsiella spp. | * Disclaiming upshots of E. oli * Confident upshots Klebsiella spp. | D. Catalase Ordeal| Nutrient agar slant:S. aureusStreptococcus spp. | S. aureus * Confident catalase reforce bemotive offer of trashsStreptococcus spp. * Disclaiming catalase reforce no trashs offer. | E. Nitrate Ruddyuction Ordeal| Nitrate broth:E. coliP. vulgarisS. marcescensP. fluorenscens| * No tinge veer subjoined denitrification of ammonia. * No tinge veer subjoined denitrification of ammonia. * Adapts ruddy. Confident nitrate ordeal parades nitrate ruddyuctase offer. * Adapts ruddy beside disclaiming catalase ordeal. | F. Urease Ordeal| Urea broth:E. coliP. vulgarisS. marcescensP. luorenscens| * Disclaiming urease ordeal bemotive the tube ruddyeep purple. * P. vulgaris parade confident urease ordeal from yellow to pinkish. * S. marcescens parade disclaiming urease ordeal bemotive the tinge ruddyeep purple. * P. fluorenscens parade disclaiming urease ordeal bemotive the tinge ruddyeep purple. | DISCUSSION: Biochemical ordeals of bacteria oobjectively to ordeal the metabolism of carbohydrate and akin issues of opposed bacteria cast, ordeal peculiar breakdown of issues through tinge veers and fume executed. As-courteous that, the coercionce of bacteria utilizes a peculiar import and the metabolism of protein and amino harsh by bacteria.
A. CARBOHYDRATE TEST Carbohydrate is an radical mixture that consists of singly carbon, hydrogen and oxygen which is basically the main carbon account of most organisms. Peculiar carbohydrate can be fermented by organism that incorporated in a average pliant ruddy or harsh with fume. Pinkish ruddy tinge parades confident upshots where harshic gratified coercionmed in the tube bemotive carbon dioxide realised if fermentation supervene. Disclaiming catabolism of carbohydrate parades by yellow to colourless of Durham’s tube as the discontinuance ruddyeep alkacourse in the listless of carbon dioxide fume.
Fume coercionmation can be seen as trashs in Durham’s tube. Central carbohydrate metabolism or the breakdown of sugars into slighter mixtures accompanied by the coercionmation of ATP and ruddyuction of coenzymes, follows individual of separate track. Carbohydrate utilization and fermentation accomplish be assessed by growing cells outside shaking (aeration) in defined resources embraceing a unique carbohydrate. Harsh issues of sugar fermentation accomplish motive a bulky tinge veer in the pH indicator embodyd in the average.
Sugar fermentation does not attributefficacious attributefficacious attributefficacious attributoperative attributoperative attributoperative attributoperative attributoperative attributoperative attributoperative attributoperative attributoperative profit alkacourse issue, nevertheless non-fermentative hydrolysis of amino harshs in the peptone, offer in most fermentation resources, may bestow an alkacourse reaction, which accomplish too motive a tinge veer in the pH indicator. Fume coercionmation, H2 in detail, can be designated by placing a slight, inverted Durham tube in the ordeal average. If fume is executed, it is stratagemped in the Durham tube and can be seen as a trash. Hydrogen sulfide (H2S) is executed by bacterial anaerobic deprivation of the brace sulfur-containing amino harshs, cysteine and methionine.
Hydrogen sulfide is released as a by-issue when carbon and nitrogen atoms in the amino harshs are consumed as nutrients by the cells. Under anaerobic provisions the sulfhydryl (-SH) class on cysteine is ruddyuced by cysteine desulfurase. Ferrous ammonium sulfate-indicator. H2S reacts with ferrous sulfate coercionming the sombre dive Sodium thiosulfate is ruddyuced to sulphite/thiosulfate The Kligler’s Iron ordeal is representationd to discover easydom of H2S fume by bacteria growing on an extravagance of these sulfur-containing amino harshs. The agar embraces haughty levels of peptones or accounts of cysteine and methionine and ferrous sulfate as an indicator.
When H2S is executed, the ferrous ion reacts with it to bestow ferrous sulfide, an insoluble sombre dive. In ruefulness hydrolysis ordeal Iodine must be on the dish to visualize the zindividual of apparenting extreme the bacteria. This zindividual parades ruefulness was docile down to dextrins, maltose, and glucose. B. PROTEIN AND AMINO ACID METABOLIM Indole ordeal measures the coercionce of bacteria to rive indole from tryptophan monad beside in regulate of biochemistry, Indole ordeal is individual of the metabolic deprivation issues of the amino harsh tryophan.
Bacteria that hold the enzyme trytophanase are capoperative of hydrolysing and deaminating tryptophan with the coercionmation of Indole, pyruvic harsh and ammonia. Confident reforce paradeed by E. coli, P. vulgaris and disclaiming upshots beholdd in Klebsiella and Salmonella from contemplation in the Indole ordeal. Development of fuchsia ruddy tinge at the interface of the reconstituency and the broth among seconds subjoined adding the reconstituency is speaking of the influence of Indole and is a confident ordeal. Kovac’s reconstituency discovers if tryptophan has been hydrolyzed to indol or tryptophanase.
Gelatin is the protein extraneous from the voluptuous protein collagen, has been representationd as a cubicifying constituency in foundation coercion a covet date as-courteous nutrient gelatine as an existing sign of cubic development average. Individual drift is that numerous bacteria entertain the coercionce to hydrolyze or liquefy the gelatin. This gelatin liquefforce coercionce coercionms the foundation coercion this ordeal. C. VOGES-PROSKAUER TEST The coercionmation of acetoin by bacteria is perprofit through Voges Proskauer Ordeal to designate the coercionce of the organisms to profit unavowed purpose issue acetyl methyl carbinol (acetoin) from glucose fermentation.
Disclaiming upshots gained from E. coli meanwhile confident reforce bestows by. Changing of tinge to ruddy pinkish tinge at the exterior of the average paraded confident upshots and yellow tinge at the exterior of the average parade disclaiming reaction. The KOH reconstituency should not attributefficacious attributefficacious attributefficacious attributoperative attributoperative attributoperative attributoperative attributoperative attributoperative attributoperative attributoperative attributoperative be extravaganceively pretended to the illustration bemotive extravagance KOH may mask diluted VP confident reactions. The MR ordeal accomplish be confident coercion organisms that entertain adequate tracks coercion partial harsh fermentation. The Voges-Proskauer (VP) ordeal designates whether a peculiar unavowed metabolic embodyd, acetoin, has been executed instead of harsh from glucose.
Acetoin is the conclusive embodyd in the besideanediol track, which is a beggarly fermentation track in B. subtilis. The ordeals are complementary in the understanding that repeatedly a bacterium accomplish bestow a confident reforce coercion individual ordeal and a disclaiming reforce coercion the other. The three likely patterns of upshots where the acetoin fermentation track, discovesanguine by the VP ordeal, brace monads of pyruvate confine and brace monads of CO2 are released. The 4 carbon embodyd that is coercionmed, acetoin, embraces a carbonyl class. The acetoin acts as a regulateinal electron acceptor with the carbonyl class life ruddyuced to a hydroxyl class.
The ruddyuced issue, besideanediol, is excreted by the bacteria and acetoin is oxidized to diacetyl by alkacourse -naphthol, which coercionms a ruddy close with creatinine. D. CATALASE TEST Catalase is offer in most cytochrome embraceing aerobic and facultative anaerobic bacteria save Streptococcus spp. Hydrogen peroxide coercionms as individual of the oxidative purpose issue of aerobic carbohydrate metabolism. If hydrogen peroxide undisputed accumulating in the bacterial cells it befits deadly to the bacteria. Catalases succor in converting H2O2 to impart and oxygen.
In the catalase ordeal manufactured, Streptococcus spp bestows disclaiming reforce as coercion S. aureus, the confident reforce supervenered. Individual of the by-products of oxidation-contrforce in the influence of O2 during aerobic respiration is hydrogen peroxide (H2O2). This mixture is haughtyly reactive and must be cheap in the cytoplasm of the cell pliant it. It can be in-particular damaging to monads of DNA. Most aerobes synthesize the enzyme catalase, which breaks down H2O2 into impart and oxygen. The O2 fume is verified by the coercionmation of trashs from a tight cell suspension.
The ordeal coercion catalase is pure and usually very true. It is a main process of make-famousing among Staphylococcus (catalase confident), Streptococcus (catalase disclaiming), and Enterococcus (catalase disclaiming), although some strains of Enterococcus faecalis may be confident. Catalase coercionmation is generally associated with aerobic organisms, gone H2O2 is a toxic by-issue of aerobic development, beside not attributefficacious attributefficacious attributefficacious attributoperative attributoperative attributoperative attributoperative attributoperative attributoperative attributoperative attributoperative attributoperative frequently. E. NITRATE REDUCTION TEST Nitrate ruddyuction ordeal basically ordeal the coercionce of organism to ruddyuce the nitrate to nitrites of easy nitrogen fume.
In regulate to designate either the bacteria can ruddyuce nitrate, the ordeal organism is inoculated into nitrate ruddyuction broth, monstrous average that embraces bulky equalitys of nitrate (KNO3). Subjoined incubation, reconstituency pretended concurrently reacts with nitrite and adapt to ruddy tinge, indicating a confident nitrate ruddyuction. If there is no tinge veer at this tramp, nitrite is listless. If the nitrate is unmoderate and tpurpose in its first coercionm, this would be a disclaiming nitrate ruddyuction upshot. Nevertheless it is likely that the nitrate was ruddyuced to nitrite beside has been raise ruddyuced to ammonia or nitrogen fume.
This would be commemorative as confident nitrate ruddyuction upshot. Under anaerobic provisions, some bacteria are operative to representation nitrate (NO3-) as an exterior regulateinal electron acceptor. This bark of metabolism is equally to the representation of oxygen as a regulateinal electron acceptor by aerobic organisms and is named anaerobic respiration. Nitrate is an oxidized mixture and there are separate tramps likely in its ruddyuction. The moderate tramp is the ruddyuction of nitrate (NO3-) to nitrite (NO2-). Separate likely issues can be made from raise ruddyuction of nitrite. Likely ruddyuced purpose issues embody the coercionthcoming N2, NH3 (ammonia), N2O (nitrous oxide).
Bacteria alter in their coercionce to perprofit these reactions, a representationful distinction coercion identification. A average that accomplish aid development must be representationd and the cells must be confirmed anaerobically. Development in the influence of oxygen accomplish wane or segregate nitrate ruddyuction. There are numerous likely purpose issues of nitrate ruddyuction such as nitrite, nitrogen fume (N2), nitrous oxides, ammonia, and hydroxylamine. The unlikelihood of nitrate or the air of the purpose issues. The ordeal relies on the coercionmation of nitrous harsh from the nitrite. This, in adapt, reacts with the iodide in the reconstituency to profit iodine.
The iodine then reacts with the ruefulness in the reconstituency to profit a blue-colored-colosanguine tinge. Gone some of the likely issues of NO3- ruddyuction are fumeeous, a Durham tube is casually inverted in the cultivation tube to stratagem fumees. This life the contingency, it is material to pre-ordeal the average to secure no discoveroperative nitrite is offer at the rise, and, in the contingency of a disclaiming ordeal, to ruddyuce any nitrate to nitrite to designate whether the nitrite was too ruddyuced. If nitrite is executed, it reacts with hemoglobin to bestow a flashing ruddy tinge, instead of the sombre ruddy tinge of hemoglobin.
It is this reforce that is binding coercion the tinge of meats, such as glowing dogs, which are preserved with sodium nitrite. The class agar ordeal has the utility of no tinge veer supervenering if the nitrite is raise ruddyuced. F. UREASE TEST Urease ordeal deeply haughtylighted to designate the coercionce of the organism to rive urea coercionming 2 monads of ammonia by the cece of the enzyme Urease with upshoting alkalinity. Disclaiming reforce paraden by E. coli meanwhile Klebsiella spp. parades confident upshot. Extra premunition needed bemotive twain the urease ordeal average deppurpose upon the parade of alkalinity that not attributefficacious attributefficacious attributefficacious attributoperative attributoperative attributoperative attributoperative attributoperative attributoperative attributoperative attributoperative attributoperative peculiar coercion urease.
Moreover the protein hydrolysis may upsfervent I alkalinity hereafter mendacious confident may be seen in Pseudomonas. The mendacious positivity can be segregated by manage ordeal using the selfselfsame average outside urea as monition. Urea is a nitrogenous wane issue of voluptuouss. Some bacteria can cleaved it to profit carbon dioxide and ammonia. The ammonia is a nitrogen account coercion amino harsh biobody as courteous as coercion body of other nitrogen-containing monads in the cell. The urease ordeal was bequeathed to make-famous Proteus cast from other enterics.
The average feeling here is bufferuddy sufficient so that diluted urease profitrs answer disclaiming. The coercionmation of ammonia raises the pH of the average. The indicator phenol ruddy is offer in the broth. Phenol ruddy is ochreous-colored-flame-yellow at pH adown than 6. 8, and adapts flashing pinkish-ruddy at pH haughtyer than 8. 1. Hereafter, a confident urea ordeal is denoted by the veer of average tinge from yellow to pinkish ruddy. CONCLUSION: Installed on the laboratory, opposed bacteria cast entertain opposed abilities to metabolize multiprofit substrates and purpose issues coercionmed were operative to be beholdd and make-famoused.
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