Biochemical reactions

Biochemical Reactions - Details about relevant biochemical reactions in analysis of microbes.

1. SAKEENA ASMI T MAHATMA GANDHI UNIVERSITY BIOCHEMICAL METHODS FOR IDENTIFICATION OF BACTERIA

2. To distinguish harmless microbes from pathogenic microbes. Characterize an outbreak of disease and determine the source. Verify the authenticity of pathogenic strain for quality control purposes. Determine appropriate antimicrobial therapy. Basically for the prevention, control and treatment of a disease. WHY IS IDENTIFICATION IMPORTANT?

3. IDENTIFICATION SCHEME SAMPLE (Soil, water, food, etc…) ISOLATION (Spread plate, pour plate, streak plate) STAINING MOTILITY (Hanging drop method) CULTURING (Special media) BIOCHEMICAL REACTIONS MOLECULAR TECHNIQUES

4. Biochemical tests are the tests used for identification of bacteria species based on the differences in the biochemical activities of different bacteria. Differences in carbohydrate metabolism, protein metabolism, fat metabolism, production of certain enzymes, ability to utilize a particular compound etc. Each species of bacteria have a well defined set of metabolic activities different from all other species These biochemical fingerprints are properties controlled by the bacterial enzymes. WHAT ARE BIOCHEMICAL TESTS?

5. Indole test 2. Methyl red test 3. Voges-Proskauer test 4. Citrate utilisation test 5. Carbohydrate fermentation test 6. Triple sugar iron(TSI)test 7. Nitrate reduction test 8. Urease test 9. Oxidase test 10. Catalase test 11. Oxidation-fermentation(O/F)test 12. O-Nitrophenyl galactosidase(ONPG)test IMViC test Intracellular Enzyme Activity

6. Coagulase test Gelatinase test Starch hydrolysis test Lipid hydrolysis test Deoxyribonuclease test(DNase test) Extracellular Enzyme Activity

7. INDOLE TEST To determine the ability of microbe to degrade the amino acid tryptophan. MEDIA : Tryptophan 1% or peptone broth REAGENT : Kovacs’ reagent (Dimethylamine benzaldehyde) PRINCIPLE : Tryptophanase Tryptophan Indole + pyruvic acid + ammonia Indole + Kovacs reagent Rosindole + H 2 O ( Cherry red colour compound) Hcl B utanol POSITIVE : Cherry red coloured ring at the interface of reagent and broth. Eg: E.coli NEGATIVE : No colour change is observed. Eg: Klebsiella

8. SPOT INDOLE To perform this test, a test colony is smeared in a piece of filter paper saturated with Kovac’s reagent. The appearance of red colour indicates a positive test.

9. METHYL RED TEST (MR TEST) To determine the ability of microbes to oxidize glucose with production and stabilization of high content of acid end products. MEDIA : MR broth – peptone, glucose, dipotassium phosphate & distilled water. REAGENT : MR reagent – MR 0.1g in 300ml of 95% ethanol & DW 200ml. PRINCIPLE : Glucose Mixed acids (pH less than 4.4) + Methyl red Red colour POSITIVE : Red colour is observed. Eg: E.coli NEGATIVE : Yellow colour is observed. Eg: Klebsiella

10. VOGES-PROSKAUER TEST (VP TEST) To determine the ability of microbes to produce non acidic or neutral end products. MEDIA : VP broth ( same as MR broth) REAGENT : Barrits A – Alpha naphthol 5 % Barrits B – 40 % Potassium hydroxide PRINCIPLE : Glucose Pyruvate Acetoin 2, 3-butanediol Acetoin + α -napthol (0.6ml) Diacetyl (Pink coloured complex) 40% KOH (0.2ml) POSITIVE : Pink colour is observed. Eg : Klebsiella NEGATIVE : No colour change is observed. Eg: E.coli

11. CITRATE UTILISATION TEST To determine the ability of the microbes to ferment citrate as sole carbon source. MEDIA : Simmons citrate medium INDICATOR : Bromothymol blue PRINCIPLE : Sodium citrate Pyruvic acid + Oxaloacetic acid+ CO 2 Excess sodium from + CO 2 + H 2 O Na 2 CO 3 (pH ) ( green blue ) sodium citrate Citrate permease POSITIVE : Change of colour from green to blue Eg: Klebsiella NEGATIVE : No colour change is observed. Eg: E.coli

12. CARBOHYDRATE FERMENTATION TEST To determine the ability of microbes to ferment specific carbohydrates with the production of acid and/or gas. MEDIA : Nutrient broth with sugars (Glucose, lactose, sucrose, maltose) INDICATOR : Phenol red or Bromocresol purple to detect acid & Durham's tube to detect gas. PRINCIPLE : Carbohydrate Organic acids + CO 2 + H 2 Acid lowers the pH which can be detected by pH indicators. Gas production (CO 2 ) can be detected in Durham’s tube. POSITIVE : Acid only – colour change to yellow (A) Eg: S.aureus Acid & Gas – colour change to yellow & bubble in durham's tube(A/G) Eg: E.coli , Klebsiella NEGATIVE : No colour change is observed. Eg: Pseudomonas fermentation

13. TRIPLE SUGAR IRON TEST (TSI TEST) To differentiate among and between the members of Enterobacteraceae and screen for enteric pathogens based on carbohydrate fermentation and H 2 S production. MEDIA : TSI agar - glucose 0.1%, lactose & sucrose 1% concentration, protein source(peptone), NaCl, Sodium thiosulfate, Ferric ammonium citrate. INDICATOR : Phenol red (pH indicator) PRINCIPLE : Carbohydrates Acid + CO 2 Peptones NH 3 (makes medium alkaline) Phenol red Acid lowers the pH and ammonia increases the pH which can be detected by pH indicators. Gas production (CO 2 ) can be detected by cracks in the medium. Yellow Red acid alkali

14. Bacteria + Sodium thiosulfate H 2 S gas H 2 S gas + Fe3+ FeS (black precipitate) H 2 S can be detected by black precipitate formation in the medium. acid environment

15. Result interpretation: Red slant, red butt, no gas, no H2S - K/K no H 2 S Red slant, yellow butt, no gas, no H2S - K/A no H 2 S Yellow slant, yellow butt, gas, no H2s - A/ A no H 2 S Yellow slant, yellow butt, gas , H2s - A/ A H 2 S Red slant, yellow butt, gas, H2S - K/ A H 2 S Alkaline slant /acidic butt only glucose is fermented Acidic slant / acidic butt glucose, sucrose, lactose all 3 sugars are fermented Bubbles or cracks present gas production Black precipitate present H 2 S production

16. NITRATE REDUCTION TEST To determine the ability of some microbes to reduce nitrate(NO 3 - ) to nitrites(NO 2 - ) or beyond the nitrite stage. MEDIA : Nitrate broth – Potassium nitrate REAGENTS : Alpha-naphthylamine (Reagent A) & Sulfanilic acid (Reagent B) PRINCIPLE : NO 3 - NO 2 - N 2 If nitrite(NO 2 - ) is formed, then NO 2 - Nitrate reductase Other enzymes + reagent A + reagent B Sulfobenzene azo-alpha (colorless) naphthylamine Eg: E.coli, S. aureus (red coloured) If nitrogen gas(N 2 ) is formed Colourless reaction If the organism is non reducer Colourless reaction To differentiate these two cases, add zinc powder. Zinc powder can reduce nitrate to nitrites.

17. If organism reduced nitrate to nitrogen gas, then there are no nitrates present and the addition of zinc dust will have no effect. Hence the test is positive and gives colourless reaction the organism is a REDUCER If nitrates were not reduced then zinc will reduce them to nitrites. Nitrites will react with 2 reagents and give a red colour. Test is negative and organism is NON REDUCER. Eg: N.gonorrhoeae Nitrate broth Zinc powder Nitrogen gas /non reducer Nitrogen gas Reducer Non reducer

18. UREASE TEST To determine the ability of microbes to degrade urea by urease. MEDIA : C hristensen’s Urea agar REAGENTS : Phenol red PRINCIPLE : Urea + 2 H 2 O CO 2 + H 2 O + 2 NH 3 Ammonia increases the pH which can be detected by pH indicator Urease POSITIVE : Pink colour is observed Eg: Proteus NEGATIVE : No colour change Eg: E.coli

19. OXIDASE TEST To determine the ability of microbes to produce oxidase enzyme. REAGENT : 1 % Kovacs reagent (tetra methyl para phenylenediamine dihydrochloride) PRINCIPLE : Cytochrome C oxidase is an enzyme that facilitates transfer of electrons to oxygen in aerobic bacterial respiratory transport system. Here the oxidase reagent substitutes as the electron acceptor. Kovacs reagent Indophenol (colourless oxidase reagent) (purple colour) Cytochrome oxidase POSITIVE : Purple colour is observed Eg: Pseudomonas NEGATIVE : No colour change Eg: E.coli, S. aureus

20. CATALASE TEST To determine the ability of microbes to produce catalase enzyme REAGENT : Hydrogen peroxide PRINCIPLE : During respiration many microbes produce byproducts that are toxic to cells like Hydrogen peroxide(H 2 O 2 ) To neutralize this toxin microbes produce an enzyme CATALASE 2 H 2 O 2 2 H 2 O + 0 2 (gas bubbles) POSITIVE : Presence of effervescence Eg : E.coli, S.aureus etc NEGATIVE : No effervescence Eg: Streptococcus Catalase

21. OXIDATIVE – FERMENTATIVE TEST (OF TEST) To differentiate between oxidative and fermentative bacteria MEDIA : Hugh Leifson’s OF media (high glucose & low peptone) INDICATOR : Bromothymol blue PRINCIPLE : Under anaerobic condition(overlay with a layer of oil), fermentative organisms covert glucose mixed acids. Under aerobic condition, non-fermenting(oxidative) organisms produce small amount of weak acids. The decrease amount of peptones and increase amount of glucose facilitates detection of weak acids. Acid decreases the pH which can be detected by pH indicator which changes the colour from green to yellow. Examples: Oxidative – Pseudomonas aeruginosa Fermentative - E.coli Non saccharolytic - Alcaligens faecalis

22. Open (Aerobic tube) Covered (Anaerobic tube) Metabolism Acid (Yellow) Alkaline (Green) Oxidative Acid (Yellow) Acid (Yellow) Fermentative Alkaline (Green) Alkaline (Green) Non saccharolytic A B C

23. ONPG TEST FOR β -GALACTOSIDASE To determine the presence or absence of the enzyme β -galactosidase MEDIA : ONPG broth PRINCIPLE : β – galactosidase is an enzyme that converts Lactose Galactose + Glucose O – Nitro – β – D – galactopyranoside is structurally similar to lactose except that orthonitro phenol has been substituted for glucose. Onpg(colourless) Galactose + orthonitro phenol(yellow) galactosidase galactosidase POSITIVE : Yellow colour observed Eg: E.coli NEGATIVE : No colour change Eg: Proteus vulgaris

24. COAGULASE TEST This test is used to identify Staphylococcus aureus (positive) from Coagulase negative S.aureus and other microbes. Coagulase is an enzyme that convert fibrinogen in plasma fibrin S.aureus SLIDE TEST Heavy suspension of organism made on glass slide and mixed with a drop of plasma. COAGULASE POSITIVE : Macroscopic clumping in 10 seconds or less in coagulated plasma drop and no clumping in saline. Bound coagulase (SLIDE TEST) Free coagulase (TUBE TEST)

25. COAGULASE NEGATIVE : No clumping in either drop. Negative results should be confirmed with tube coagulase test. TUBE TEST A suspension of organism is suspended and incubated with plasma at 37 C COAGULASE POSITIVE : Clot of any size is observed. Eg: S.aureus COAGULASE NEGATIVE : No clot (plasma remains wholly liquid or shows only a flocculant or ropy precipitate) Eg: S.epidermidis, E.coli

26. GELATINASE TEST To determine the ability of an enzyme to produce the enzyme gelatinase that hydrolyze gelatin MEDIA : Gelatin medium – gelatin, peptone, beef extract PRINCIPLE : Gelatin Polypeptides Amino acids If the organism produce gelatinase, then it will liquefy the medium. gelatinase gelatinase POSITIVE : Partial or complete liquefaction Eg: Aeromonas hydrophila NEGATIVE : No liquefaction Eg: E.coli

27. STARCH HYDROLYSIS TEST To determine the ability of an organism to produce the enzyme amylase and hydrolyze starch. MEDIA : Starch agar – nutrient agar + starch INDICATOR : Iodine solution (Grams iodine) PRINCIPLE : The starch molecule consists of two constituents: Amylose, an unbranched glucose polymer Amylopectin, a large branched polymer Both amylose and amylopectin are rapidly hydrolyzed by certain bacteria using enzymes called α -amylases. Starch Dextrin + Maltose + Glucose (Amylose + Amylopectin) α - amylase

28. POSITIVE : If the organism has produced amylase, will hydrolyze starch and a clear area appears after adding grams iodine. Eg: Bacillus subtilis, B.megaterium NEGATIVE: Absence of clearing after adding grams iodine, no starch hydrolysis. Eg: Staphylococcus epidermidis, S.agalactiae

29. API STRIPS – RAPID TESTS (Analytical Profile Index) Commercial miniaturized biochemical test panels – cover a significant number of clinically important groups of bacteria as well as food and water associated microorganisms. Different test panels are prepared in dehydrated forms which are reconstituted upon use by action of bacterial suspensions. After incubation, positive test results are scored as a 7 digit number (profile). Identity of the bacterium is then easily derived from the database with the relevant cumulative profile code book or software.

30. MOST USED API SYSTEM

31. AUTOMATED IDENTIFICATION SYSTEM Offer greater accuracy and increased speed identification over manual methods. MicroScan Walkaway & Vitek 2 – two very popular automated identification system. Both have used convectional panels with instruments that can read and interpret panel results, perform antibiotic susceptibility tests and print results without human intervention. VITEK - 2

32. MICROSCAN WALKAWAY

33. THANK YOU…