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Enterobacteriaceae 
Biochemical reactions 
By 
Dr. Nabil El Aila 
Assistant Professor of Molecular Microbiology 
Medical T...
Lactose Fermentation 
• MacConkey Agar contains bile salts and crystal violet, both 
inhibitory to Gram-positive bacteria ...
Results of Lactose Fermentation 
Dr. Nabil El Aila 
Diagnostic Microbiology
IMViC Test 
• Indole, Methyl Red, Voges-Prosakaur, Citrate 
(IMViC) Tests: 
– The following four tests comprise a series o...
IMViC: Indole test 
 Principle 
 Certain microorganisms can metabolize 
tryptophan by tryptophanase 
 The enzymatic degrad...
IMViC: Indole test 
 Method: 
 Inoculate the test organism into tryptophane 
broth 
 Incubate at 37°C for 24 hours 
 After...
IMViC: Indole test 
 Result: 
 A bright pink color in the top 
layer indicates the presence of 
indole 
 The absence of co...
IMViC test 
Methyl Red-Voges Proskauer (MR-VP) Tests 
• Different bacteria convert dextrose and glucose to pyruvate 
using...
IMViC test 
Methyl Red-Voges Proskauer (MR-VP) Tests 
Glucose 
Principle 
Acidic pathway 
Mixed acids 
 pH less than 4.4 
...
IMViC: Methyl red 
Principle: 
• Methyl red test is used to identify enteric bacteria based on 
their pattern of glucose m...
IMViC: Methyl red 
• Method: 
• Inoculate 10ml portion of the MR-VP medium and incubate at 
37°C for 2-5 days. 
• After in...
IMViC: Methyl red 
• Results: 
– Positive test: acids + methyl red = red solution 
– Negative test: neutral end products +...
IMViC: VOGES-PROSKAUER TEST 
Principle: 
• It is used to identify enteric bacteria based on their pattern of 
glucose meta...
IMViC: VOGES-PROSKAUER TEST 
• Method: 
• Inoculate medium and incubate at 37°C for 48 hours. 
• After incubation, transfe...
IMViC: VOGES-PROSKAUER TEST 
• Results: 
– Positive test: acetoin + alpha-naphthol + KOH = red color 
– Negative test: alp...
IMViC: CITRATE TEST 
Principle: 
Citrate Na2CO3 
Alkaline,↑pH 
Pyruvate CO2 + Na + H2O 
Simmone’s Citrate media 
Contains ...
IMViC: CITRATE TEST 
• Principle: 
• Citrate is an organic molecule that can be utilized by bacteria 
that produce the enz...
IMViC: CITRATE TEST 
• Results: 
– Positive test: Growth and color changes to blue 
– Negative test: No growth and color r...
Urease Test 
Principle 
• Urea agar contains urea and phenol red 
• Urease is an enzyme that catalyzes the conversion of u...
Urease Test 
• Result 
• Positive test: production of alkaline 
end products = pinkish red color 
• Negative test: No colo...
Motility Test 
• Principle: 
• Motility Test Media is a semi-solid agar designed to 
demonstrate motility by diffusion. 
•...
Motility Test 
• Expected results: 
– Positive test: Growth spread away from the line of 
inoculation = motile 
– Negative...
H2S Production 
• Principle: 
• Bacteria use enzyme cysteine desulfurase to hydrolyze the amino 
acid cysteine, forming hy...
H2S Production 
• Expected Results: 
– Positive Test: H2S production = Black 
– Negative Test: No H2S production = No blac...
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Enterobacteriacea i biochemical reaction بكتريا عملي

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بكتريا عملي

Publicada em: Saúde e medicina
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Enterobacteriacea i biochemical reaction بكتريا عملي

  1. 1. Enterobacteriaceae Biochemical reactions By Dr. Nabil El Aila Assistant Professor of Molecular Microbiology Medical Technology Department Al -Aqsa University Dr. Nabil El Aila DiagnosticMicrobiology
  2. 2. Lactose Fermentation • MacConkey Agar contains bile salts and crystal violet, both inhibitory to Gram-positive bacteria and selects Gram-negative bacteria, such as E. Coli. • It also differentiates lactose-fermenting bacteria, such as E. Coli from non-lactose fermenting bacteria. • Media and Reagent: MacConkey Agar and neutral red dye • Method: Streak MAC plate and incubate at 37°C for 2 days. • Expected results: – Positive test: Lactose fermentation = Growth and color change to pink – Negative test: No lactose fermentation = May or may not grow and no color change Dr. Nabil El Aila Diagnostic Microbiology
  3. 3. Results of Lactose Fermentation Dr. Nabil El Aila Diagnostic Microbiology
  4. 4. IMViC Test • Indole, Methyl Red, Voges-Prosakaur, Citrate (IMViC) Tests: – The following four tests comprise a series of important determinations that are collectively called the IMViC series of reactions – The IMViC series of reactions allows for the differentiation of the various members of Enterobacteriaceae. Dr. Nabil El Aila Diagnostic Microbiology
  5. 5. IMViC: Indole test Principle Certain microorganisms can metabolize tryptophan by tryptophanase The enzymatic degradation leads to the formation of pyruvic acid, indole and ammonia The presence of indole is detected by addition of Kovac's reagent. Tryptophane amino acids Tryptophanase Indole + Pyurvic acid + NH3 Kovac’s Reagent Red color in upper Dr. Nabil El Aila organic layer` Diagnostic Microbiology
  6. 6. IMViC: Indole test Method: Inoculate the test organism into tryptophane broth Incubate at 37°C for 24 hours After incubation interval, add 1 ml Kovacs reagent which contain 4 (p) – dimethylamino benzaldehyde, shake the tube gently and read immediately Dr. Nabil El Aila DiagnosticMicrobiology
  7. 7. IMViC: Indole test Result: A bright pink color in the top layer indicates the presence of indole The absence of color means that indole was not produced i.e. indole is negative Significance: Used in the differentiation of genera and species. e.g. E. coli (+) from Klebsiella (-). Positive test e.g. E. coli Negative test e.g. Klebsiella Dr. Nabil El Aila DiagnosticMicrobiology
  8. 8. IMViC test Methyl Red-Voges Proskauer (MR-VP) Tests • Different bacteria convert dextrose and glucose to pyruvate using different metabolic pathways. • Some of these pathways produce unstable acidic products which quickly convert to neutral compounds. • Some organisms use the butylene glycol pathway, which produces neutral end products, including acetoin and 2,3- butanediol. • Other organisms use the mixed acid pathway, which produces acidic end products such as lactic, acetic, and formic acid. These acidic end products are stable and will remain acidic.
  9. 9. IMViC test Methyl Red-Voges Proskauer (MR-VP) Tests Glucose Principle Acidic pathway Mixed acids pH less than 4.4 Methyl Red indicator Red color MR positive E. coli Or Neutral pathway Acety methyl carbinol (ACETOIN) Barrit’s A (a-naphthol) Barrit;s B (40% KOH) Pink color VP positive Klebsiella
  10. 10. IMViC: Methyl red Principle: • Methyl red test is used to identify enteric bacteria based on their pattern of glucose metabolism. • If they use mixed acid pathway and produce acidic products, then they are called methyl-red-positive. • If they use butylene glycol pathway and produce neutral end products, then they are called methyl-red-negative Dr. Nabil El Aila Diagnostic Microbiology
  11. 11. IMViC: Methyl red • Method: • Inoculate 10ml portion of the MR-VP medium and incubate at 37°C for 2-5 days. • After incubation, transfer 2.5 ml of inoculate to another tube and add five drops of methyl red. • Roll between the palms of hands to disperse methyl red. Dr. Nabil El Aila Diagnostic Microbiology
  12. 12. IMViC: Methyl red • Results: – Positive test: acids + methyl red = red solution – Negative test: neutral end products + methyl red = yellow color • Significance: This test is used to differentiate Enterobacteriacaceae species espcially E. coli and E. aerogens Dr. Nabil El Aila Diagnostic Microbiology
  13. 13. IMViC: VOGES-PROSKAUER TEST Principle: • It is used to identify enteric bacteria based on their pattern of glucose metabolism. • The enterics that produce neutral end-products, such as acetoin are detected by VP test. • Its presence is used as indicator of 2,3 butylene glycol Fermentation • The detection of acetoin in alkaline pH is accomplished by alpha-Naphthol reagent. Dr. Nabil El Aila Diagnostic Microbiology
  14. 14. IMViC: VOGES-PROSKAUER TEST • Method: • Inoculate medium and incubate at 37°C for 48 hours. • After incubation, transfer 2.5 ml of inoculate to another tube and add six drops of Barritt’s Reagent A (contains alpha-naphthol) and two drops of Barritt’s Reagent B(contains KOH). • Gently mix and let it sit for 10-15 minutes to allow time for color development. Dr. Nabil El Aila Diagnostic Microbiology
  15. 15. IMViC: VOGES-PROSKAUER TEST • Results: – Positive test: acetoin + alpha-naphthol + KOH = red color – Negative test: alpha-naphthol +KOH = copper color • Significance: This test is used to differentiate Enterobacteriacaceae species espcially E. coli and E. aerogens Dr. Nabil El Aila Diagnostic Microbiology
  16. 16. IMViC: CITRATE TEST Principle: Citrate Na2CO3 Alkaline,↑pH Pyruvate CO2 + Na + H2O Simmone’s Citrate media Contains Citrate as a sole of C source Bromothymol blue Positive test Dr. Nabil El Aila Diagnostic Microbiology
  17. 17. IMViC: CITRATE TEST • Principle: • Citrate is an organic molecule that can be utilized by bacteria that produce the enzyme citrase. • Citrase is produced by some bacteria such as E. aerogenes but not by others like E. Coli • Method: • Inoculate the test organism onto a slant containing Simmon Citrate agar. • Simmon’s Citrate Agar contains sodium citrate (carbon source), pH indicator—bromthymol blue. • Incubate at 37°C for 24 hours. Dr. Nabil El Aila Diagnostic Microbiology
  18. 18. IMViC: CITRATE TEST • Results: – Positive test: Growth and color changes to blue – Negative test: No growth and color remains green • Significance: • This test is used to help differentiate species of the family Enterobacteriaceae. • It is selective for bacteria that has the ability to consume citrate as its sole source of carbon Dr. Nabil El Aila Positive Negative Diagnostic Microbiology
  19. 19. Urease Test Principle • Urea agar contains urea and phenol red • Urease is an enzyme that catalyzes the conversion of urea to CO2 and NH3 • Ammonia combines with water to produce ammonium hydroxide, a strong base which ↑ pH of the medium. • ↑ in the pH causes phenol red r to turn a deep pink. This is indicative of a positive reaction for urease Urea Urease CO2 + NH3 H2O NH4 OH ↑ in pH Phenol Red Pink Positive test Method Streak a urea agar tube with the organism incubate at 37°C for 24 h
  20. 20. Urease Test • Result • Positive test: production of alkaline end products = pinkish red color • Negative test: No color change • Significance: • Differentiate salmonella and shigella which are urease negative from urease positive Non pathogens. • Proteus, klebsiella and some citrobacter species are urease positive • Helicobater pylori is also Urease positive Negative test Positive test Dr. Nabil El Aila DiagnosticMicrobiology
  21. 21. Motility Test • Principle: • Motility Test Media is a semi-solid agar designed to demonstrate motility by diffusion. • This is not a biochemical test, but it can distinguish bacteria. It determines presence of flagella. • Method: • Inoculate a semi-solid nutrient medium by stabbing 2 cm into the center of the medium • Inoculate at 37C° for 24-48 hours. Dr. Nabil El Aila DiagnosticMicrobiology
  22. 22. Motility Test • Expected results: – Positive test: Growth spread away from the line of inoculation = motile – Negative test: Growth only occurred at the line of inoculation = Non-motile • Significance: • This test is used for the differentiation of microorganisms on the basis of motility. Dr. Nabil El Aila Diagnostic Microbiology
  23. 23. H2S Production • Principle: • Bacteria use enzyme cysteine desulfurase to hydrolyze the amino acid cysteine, forming hydrogen sulfide as end-product. • To test for hydrogen sulfide production, a medium with a sulfur-containing compound and iron salts is inoculated and incubated. If the sulfur is reduced and hydrogen sulfide is produced, it will combine with the iron salt to form a visible black ferric sulfide (FeS) in the tube • Media and Reagent: SIM tube (sulphide, Indole and Motility) with cysteine and ferrous sulfate (detects H2S) • Method: Inoculate the media and incubate at 37°C for 24-48 hours. Dr. Nabil El Aila Diagnostic Microbiology
  24. 24. H2S Production • Expected Results: – Positive Test: H2S production = Black – Negative Test: No H2S production = No blackening of medium • Significance: • This test is used to determine the ability to reduce sulfur into H2S. • Differentiate species of the family Enterobacteriaceae. • Identifying unknown organisms such as certain Proteus and salomenella Dr. Nabil El Aila Negative Positive Diagnostic Microbiology

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