Más contenido relacionado

Similar a biochemical reactions(gnb)-4.pptx(20)


biochemical reactions(gnb)-4.pptx

  2. IDENTIFICATION Of BACTERIA: Identification of bacteria can be done by several methods such as: 1. Conventional methods for culture and identification 2. Automated culture techniques 3. Serology 4. Molecular methods
  3. FOR GRAM-NEGATIVE BACILLI: The following are the common biochemical tests done routinely: • Indole test • Methyl red test (MR) • Voges Proskauer (VP) • Citrate utilization test • Urea hydrolysis test • Triple sugar iron test (TSI). • Sugar fermentation test • Oxidation fermentation test (OF) • Nitrate reduction test • Decarboxylase test • Phenyl pyruvic acid test (PPA)
  4. Catalase Principle Catalase is an enzyme that decomposes hydrogen peroxide (H2O2) into water and oxygen. 2H2O2 → 2H2O + O2 (gas bubbles) Reagents A. Hydrogen peroxide 3% B. Glass rod Procedure:- Add 1ml of 3% H2O2 directly to an 18-24h inoculated pure agar slant culture and observe for immediate effervensens and record the results.
  5. Results The rapid and sustained appearance of bubbles or effervescence constitutes a positive test. Quality Control Positive control: Staphylococcus aureus ATCC 25923 Negative control: Streptococcus species ATCC 19615 . CATALASE POSITIVE CATALASE NEGATIVE Staphylococci Streptococci Listeria monocytogenes Enterococci Corynebacteria Lactobacillus Micrococcus Gardnerella vaginalis Gonococci Ekienella corrodens Enterobacteriaceae Kingella kingae Actinomycetes
  6. Cytochrome OxidaseTest Principle It detects the presence of cytochrome oxidase enzyme in bacteria, which catalyzes the oxidation of reduced cytochrome by atmospheric oxygen. REAGENTS Tetramethyl-p-phenylenediamine dihydrochloride, 1% Dimethyl-p-phenylenediamine dihydrochloride, 1% PROCEDURE two methods: (1) the direct plate technique (2) Modified oxidase test
  7. RESULT Bacterial colonies having cytochrome oxidase activity develop a deep blue color at the inoculation site within 10 seconds Quality Control Positive control: Pseudomonas aeruginosa ATCC 27853 Negative control: Escherichia coli ATCC 25922 OXIDASE POSITIVE OXIDASE NEGATIVE Neisseria Enterobacteriaceae Vibrio Staphylococcus Campylobacter Acinetobacter spp Pseudomonas Bordetella Aeromonas
  8. INDOLE TEST: Principle It detects the ability of certain bacteria to produce an enzyme tryptophanase that breaks down amino acid tryptophan present in the medium into indole. Tryptophan Indole + Pyruvic acid + Ammonia Indole + p- di methyl amino benzaldehyde RED coloured product REAGENT:-Kovac’s reagent Ehrlich’s reagent Procedure Inoculate tryptophan broth with the test organism and incubate at 35°C for 18–24 hours. At the end of this time, add 5 drops of reagent down the inner wall of the tube
  9. . Results The development of a bright fuchsia red color at the interface of the reagent and the broth within seconds after adding the reagent is indicative of the presence Of indole and is a positive test. Quality Control Positive control: Escherichia coli ATCC 25922 Negative control: Klebsiella pneumoniae ATCC 70063
  10. INDOLE POSITIVE INDOLE NEGATIVE E.Coli K. Pnemoniae K.Oxytoca Shigella species Citrobacter koseri Salmonella species Edwardsiella tarda Citrobacter freundii Proteus vulgaris Enterobacter aerogenes Morganella morganii Enterobacter cloacae Providencia Hafnia Proteus mirabilis Pseudomonas aeruginosa
  11. METHYL RED (MR) TEST:  Principle This test detects the production of sufficient acid during fermentation of glucose by bacteria and sustained maintenance of a pH below 4.5.  Organisms that can maintain the low pH after prolonged incubation (48- 72 hrs ) overcoming the pH buffering system of the medium can be called methyl red positive. Methyl red : pH pH 6.0 yellow 4.4 red COMPONENTS  METHYL RED TEST BROTH- GLUCOSE PHOSPHATE PEPTONE WATER  Peptone : 5 g  K2HPO4 : 5 G (dipotassium phosphate)  Water : 5 L  Glucose (10%) : 50 mL
  12. Methyl red indicator solution : Methyl red : 0.1 g Ethanol : 300 ml Distilled water : 200 ml . Procedure The test organism is inoculated in glucose phosphate broth and incubated at 37°C for 48-72hrs Then add five drops of 0.04% solution of methyl red, mix well and read the results immediately Results The development of a stable red color in the surface of the medium indicates sufficient acid production to lower the pH to 4.4 and constitutes a positive test
  13. Quality Control Positive control: Escherichia coli ATCC 25922 Negative control: Klebsiella pneumoniae ATCC70063 MR POSITIVE MR NEGATIVE E. Coli K. Pnemoniae K. Ozaenae Enterobacter spp K. Rinoscleromatis K. Ornitholytica Salmonella species Proteus mirabilis
  14. VOGES-PROSKAUER (VP) TEST ORACETOIN PRODUCTION TEST:  Principle  This test depends on the production of acetyl methyl carbinol (acetoin) from pyruvic acid in the media. In the presence of alkali and atmospheric oxygen, acetoin is oxidised to diacetyl which reacts with a-naphthol to give red colour  MEDIA :- methyl red–Voges-Proskauer (MR/VP) broth  Reagent 1 : α-Naphthol 5 g Absolute ethyl alcohol 100 mL
  15. . Reagent 2 Potassium hydroxide 40 g Distilled water 100 mL Procedure • Inoculate a tube of MR/VP broth with a pure culture of the test organism. Incubate for 24 hours at 35°C. • At the end of this time, aliquot 1 mL of broth to a clean test tube. • Add 0.6 mL of 5% α naphthol, followed by 0.2 mL of 40% KOH. • It is essential that the reagents be added in this order. • Shake the tube gently to expose the medium to atmospheric oxygen and allow the tube to remain undisturbed for 10–15 minutes
  16. VP POSITIVE VP NEGATIVE K. Pnemoniae E. Coli Serratia Salmonella Vibrio cholera Proteus mirabilis Aeromonas sobria Yersinia Results A positive test is represented by the development of a red color 15 minutes or more after addition of the reagents. Quality Control Positive control: Klebsiella pneumoniae ATCC70063 Negative control: Escherichia coli ATCC25922
  17. Citrate Utilization Test Principle To identify organisms capable of using sodium citrate as the sole carbon source & inorganic ammonium salts as the sole nitrogen source  Citrate sodium bicarbonate + ammonia Bromothymol blue (yellow) bromothymol blue (Blue) Ph-6.9 ph- 7.6 MEDIA :- Simmon’s citrate medium  Koser’s citrate medium (liquid medium)
  18.  Procedure  A well-isolated colony is picked from the surface of a primarily isolation medium and inoculated as a single streak on the slant surface of the citrate agar tube. The tube is incubated at 35°C for 24–48 hours Results A positive test is represented by the development of a deep blue color within 24–48 hours A positive test may also be read without a blue color if there is visible colony growth along the inoculation streak line In koser’s citrate medium presence of turbidity is noted due to growth of bacteria.
  19. Quality Control Positive control— Klebsiella pneumoniae ATCC70063 Negative control—Escherichia coli ATCC 25922 CITRATE POSITIVE CITRATE NEGATIVE Salmonella paratyphi E. Coli Citrobacter Shigella Klebsiella Hafnia Enterobacter Yersinia Serratia
  20. UREA HYDROLYSIS TEST  Principle To determine the ability of organism to split urea into two molecule of ammonia by action of enzyme urease, ammonia reactes in solution to form ammonium carbonate with resulting alkalinity And increase in ph of medium. Indicator:- Phenol red Phenol red PH-6.8 PH-8.4 MEDIA:-Stuart’s urea broth Christensen’s urea agar
  21. Procedure  The broth medium is inoculated with a loopful of a pure culture of the test organism The surface of the agar slant is streaked with the test organism and both incubated at 35°C for 18–24hours.  Results Organisms that hydrolyze urea rapidly may produce positive reactions by change in color original yellow color changes to pink in alkaline medium. .
  22. Quality Control Positive control— Klebsiella pneumoniae ATCC70063 Negative control—Escherichia coli ATCC 25922
  23. TRIPLE SUGAR IRON (TSI) AGAR TEST:  Principle  TSI detects three properties of bacteria, which includes fermentation of sugars to produce acid and/or gas and production of H2S  Media:-  TSI medium contains three sugars—glucose, sucrose and lactose in the ratio of 1:10:10 parts.  Phenol red  Ferrous sulphate Procedure:- with the help of inoculating wire which contain isolate of pure colony, stab 3-5 mm and then streak on TSI agar , and incubate at 37c for 18-24hrs.
  24.  Result:- Reaction in TSI Example Acidic slant/acidic butt  > 2 sugars fermented 2.lactose/sucrose A/A, gas production, no H2S Escherichia coli Klebsiella pneumoniae Alkaline slant/acidic butt Only glucose fermenter group K/A no gas, no H2S shigella K/A, no gas,H2S produced(small amount) Salmonella Typhi Proteus mirabilis K/A, gas produced, H2S produce(abundant) Salmonella paratyphi B K/A, gas produced, no H2S Salmonella paratyphi A Alkaline slant/alkaline butt Non-fermenters group K/K, no gas, no H2S Pseudomonas, Acinetobacter
  25. QUALITY CONTROL : A/A + gas production : E.coli ATCC 25922 K/A +/- gas production, H2S + : Salmonella enterica sub sp.enterica serovar typhimurium ATCC 14028 K/K : P. aeruginosa ATCC 27853 K/A, H2S + : Proteus mirabilis ATCC 12453 K/A : Shigella flexneri ATCC 12022
  26. CARBOHYDRATE (SUGAR) FERMENTATION TEST: Principle:- It detects the ability of an organism to ferment a specific carbohydrate (sugar) incorporated in a medium producing acid with/without gas. Glucose, lactose, sucrose, mannitol, maltose and xylose are widely used for sugar fermentation. Media:- broth containing specific sugar 1% with Andrade’s indicator Procedure:- Test organism is inoculated in sugar medium and incubated at 37C for 18-24hr. Interpretation:- Positive:- pinkish red Negative:- yellow
  27. Quality controle:- Glucose Positive control with gas- Escherichia coli ATCC25922 without gas- shigella flexneriATCC12022 Negative control: Alcaligenes faecalis ATCC8750 Lactose Positive control -Escherichia coliATCC25922 Negative control – proteus vulgaris ATCC6380 Glucose and lactose fermenters:-E.coli Klebsiella spp • Glucose and mannitol fermenters:-Salmonella sp
  28. OXIDATION-FERMENTATION TEST (OF TEST):  PRINCIPAL  Hugh and Leifson OF test differentiates between fermenters and non-fermenters (that utilize sugars oxidatively).  MEDIA AND REAGENTS  OF medium differs from ordinary sugar fermentation medium by containing: • Agar (0.3%)—making the medium semisolid that permits the diffusion of acids from the surface to throughout the medium changing the color of the bromothymol blue indicator to yellow • Increased carbohydrate concentration from 0.5% to 1% • Decreased peptone concentration from 1% to 0.2%.
  29. OF Media of Hugh and Leffson media:- Peptone – 2g Sodium chloride -5g D-glucose -10g Bromothymol blue – 0.03g Agar -3g Dipotassium phosphate - 0.30 Distilled water – 1L PH- 7.1 Medium autoclaved in a flask at 121C for 15min Carbohydrate to be added is sterilized separately and added to give a final concentration of 1% Procedure:- Two tubes are required for the OF test, each inoculated with the unknown organism, using a straight needle, stabbing the medium three to four times halfway to the bottom of the tube.
  30.  One tube of each pair is covered with a 1-cm layer of sterile mineral oil or melted paraffin, leaving the other tube open to the air. Incubate both tubes at 35°C and examine daily for several days. RESULT  Open Tube Covered Tubes Metabolism Acid (yellow) Alkaline (green) Oxidative Acid (yellow) Acid (yellow) Fermentative Alkaline (green or blue) Alkaline (green or blue) Nonsaccharolytic
  31. CONTROL E.COLI Psudomonas Alcaligenes QUALITY CONTROL Glucose fermenter: Escherichia coli Glucose oxidizer: Pseudomonas aeruginosa Nonsaccharolytic: Alkaligenes fecalis
  32. NITRATE REDUCTION TEST:  principle  This test detects the presence of an enzyme nitrate reductase in the organism, which reduces nitrate present in the medium (nitrate broth) to nitrite or free nitrogen gas.  REAGENTS  Nitrate Broth or Nitrate Agar (Slant)  Beef extract 3 g  Peptone 5 g  Potassium nitrate (KNO3 ) 1 g  Agar (nitrite-free) 12 g  Distilled water 1 L
  33. Reagent A α-Naphthylamine 5 g Acetic acid (5 N), 30% 1 L Reagent B Sulfanilic acid 8 g Acetic acid (5 N), 30% 1 L Procedure: Inoculate the nitrate medium with a loopful of the test organism isolated in pure culture on agar medium, and incubate at 35°C for 18–24 hours At the end of incubation, add 1 mL each of reagents A and B to the test medium, in that order
  34.  RESULT  The development of a red color within 30 seconds after adding the test reagents indicates the presence of nitrites and represents a positive reaction for nitrate reduction  adding zinc dust indicates the presence of residual nitrates and confirms a true negative reaction.  Free nitrogen gas is detected by using a Durham’s tube QUALITY CONTROL A.Positive control: Escherichia coli ATCC25922 B.Negative control: Acinetobacter baumaniiATCC17978
  35. DECARBOXYLASE TEST;  PRINCIPLE It detects the presence of substrate specific decarboxylase enzyme in the bacteria that break down amino acids, such as lysine, arginine and ornithine to produce alkaline by-products which change the color of the indicator to purple. Each decarboxylase enzyme is specific for an amino acid  Lysine, ornithine, and arginine are the three amino acids routinely tested in the identification of the Enterobacteriaceae.  The specific amine products are as follows: Lysine → Cadaverine Ornithine →Putrescin Arginine →Citrulline
  36. REAGENTS Moller Decarboxylase Broth Base Peptone 5 g Beef extract 5 g Bromcresol purple 0.01 g Cresol red 0.005 g Glucose 0.5 g Pyridoxal 0.005 g Distilled water 1 L Final pH 6.0 Amino Acid Add 10 g of the L (levo) -form of the amino acid (lysine, ornithine, or arginine). Double this amount if the D (dextro) -form is to be used, because only the L-form is active
  37. PROCEDURE inoculate two tubes of Møller decarboxylase medium, one containing the amino acid to be tested and the other to be used as a control tube devoid of amino acid.  RESULT  Conversion of the control tube to a yellow color indicates that the organism is viable and that the pH of the medium has been lowered sufficiently to activate the decarboxylase enzymes. Reversion of the tube containing the amino acid to a blue-purple color indicates a positive test owing to the formation of amines from the decarboxylation reaction  QUALITY CONTROL  Amino Acid Positive Control Negative Control  Lysine Enterobacter aerogenes Enterobacter cloacae  Ornithine Enterobacter cloacae Klebsiella pneumoniae  Arginine Enterobacter cloacae Enterobacter aerogenes
  38. PHENYLALANINE DEAMINASE TEST: PRINCIPLE To determine the ability of an organism to deaminate phenylalanine to phenyl pyruvic acid (PPA) and ammonia. This test is also commonly called as PPA test. MEDIA AND REAGENTS Phenylalanine agar is poured as a slant into a tube DL-Phenylalanine- 2g Yeast extract- 3g (serves as the carbon and nitrogen) Sodium chloride – 5g Disodium phosphate – 1g Agar – 12g Distilled water- 1L PH- 7.3
  39.  Reagents- ferric chloride -12g concentrated HCL-2.5ml distilled water-100ml PROCEDURE  The agar slant of the medium is inoculated with a single colony of the test organism isolated in pure culture of primary plating agar. After incubation at 35°C for 18–24 hours, 4 or 5 drops of the ferric chloride reagent are added directly to the surface of the agar  RESULT  The immediate appearance of an intense green color indicates the presence of phenylpyruvic acid and is positive test
  40. QUALITY CONTROL Positive control: Proteus mirabilis ATCC12453 Negative control: Escherichia coli ATCC25922 PPA positive:- Proteus spp, Morganella spp Providencia spp PPA negative :- All members of enterobacteriaceae.