Biochemical tests for bacterial identification

Basic biochemical tests for identification of most common bacteria along with their principles and methods to perform and quality control for UG & PG Students.

1. Biochemical Tests for Identification of Bacteria Dr. Suprakash Das Assist. Prof.

2. Workflow for Bacteriology Laboratory

3. 1. Patient (D1) 2. Sample Collection & Transport (D1) 3. Direct Smear from Samples and Staining- Gram / Z-N/ Albert (D1)

4. 4. Selection of Appropriate Culture Media (D1) 5. ID- Colony Characteristics (D2) 6. Gram Stain from Colony in culture plate (D2) 6. Selection of Appropriate BIOCHEMICAL TESTS (D2) 7. AST (D2/D3)

5. Tests Used most commonly used for Identification of Gram Positive Cocci Catalase Coagulase- Slide & Tube Hemolysis Sensitivity to Bacitracin/ Furazolidone/ Polymyxin B/Novobiocin PYR Test MR VP Bile Esculin Hydrolysis Bile Solubility DNAse Hippurate/ Ornithin Hydrolysis CAMP test Growth on selective media (e.g. Mannitol salt agar)

6. Catalase Negative Organisms  Streptococcus spp. Enterococcus spp.

7. Coagulase Positive Organism other than S. aureus  S. hycus / S. intermedius

8. Algorithm for Detection of Common Gram Positive Pathogens

9. Tests most commonly used for Identification of Gram Negative Organisms Indole production Catalase Nitrate reduction MR/VP Citrate utilization Urea Hydrolysis Motility TSI reactions O/F Tests Hydrolysis of- Lysin/ Ornithine/ Arginine Sensitivity to Ampicillin/ O-129/ etc. Growth on MAC Oxidase test Pigment Production.

10. Indole Positive Organisms  Aeromonas hydrophila , Aeromonas punctata, Bacillus alvei, Edwardsiella sp., Escherichia coli, Flavobacterium sp., Klebsiella oxytoca , Proteus vulgaris

11. With the exception of a few species,  Salmonella ,  Edwardsiella ,  Citrobacter ,  Klebsiella ,  Enterobacter ,  Serratia , and  Providencia  usually give a positive reaction , and  Escherichia ,  Shigella ,  Morganella , and  Yersinia  give a negative reaction.  Proteus  is a citrate variable.

12. Oxidation and Fermentation of Medium (CDC Method) Purpose :  This test is used to differentiate microorganisms based on the ability to oxidize or ferment specific carbohydrates. Principle :  This test is used to determine whether an organism uses carbohydrate substrates to produce acid by products. Non-fermentative bacteria are routinely tested for their ability to produce acid from six carbohydrates (glucose, xylose, mannitol, lactose, sucrose, and maltose). In addition to the six tubes containing carbohydrates, a control tube containing the OF base without carbohydrate is also inoculated. Triple sugar iron (TSI) agar is also used to determine whether an organism can ferment glucose. OF glucose is used to determine whether an organism ferments or oxidizes glucose.

13. Oxidation and Fermentation of Medium (CDC Method) If no reaction occurs in either the TSI or OF glucose, the organism is considered a non–glucose utilizer. Hugh and Leifson’s formula uses a low peptone-to-carbohydrate ratio and a limiting amount of carbohydrate. The reduced peptone limits the formation of alkaline amines that may mask acid production resulting from oxidative metabolism. Two tubes are required for interpretation of the OF test. Both are inoculated, and one tube is overlaid with mineral oil, producing an anaerobic environment. Production of acid in the overlaid tube results in a color change and is an indication of fermentation. Acid production in the open tube and color change is the result of oxidation. Media: Pancreatic digest of casein (2 g), glycerol (10.0 mL), phenol red (King method) (0.03 g), agar (3 g) per 1000 mL, pH 7.3.

14. Oxidation and Fermentation of Medium (CDC Method) Method  To determine whether acid is produced from carbohydrates, inoculate agar deeps, each containing a single carbohydrate, with bacterial growth from an 18- to 24-hour culture by stabbing a needle four to five times into the medium to a depth of 1 cm. Note: Two tubes of OF dextrose are usually inoculated; one is overlaid with either sterile melted petrolatum or sterile paraffin oil to detect fermentation. Incubate the tubes at 35°C to 37°C in ambient air for up to 7 days. Note: If screwcap tubes are used, loosen the caps during incubation to allow for air exchange. Otherwise, the control tube and tubes containing carbohydrates that are not oxidized might not become alkaline.

15. Oxidation and Fermentation of Medium (CDC Method) Expected Results  Positive: Acid production (A) is indicated by the color indicator changing to yellow in the carbohydrate-containing deep. Weak-positive (Aw): Weak acid formation can be detected by comparing the tube containing the medium with carbohydrate with the inoculated tube containing medium with no carbohydrate. Most bacteria that can grow in the OF base produce an alkaline reaction in the control tube. If the color of the medium in a tube containing carbohydrate remains about the same as it was before the medium was inoculated and if the inoculated medium in the control tube becomes a deeper red (i.e., becomes alkaline), the culture being tested is considered weakly positive, assuming the amount of growth is about the same in both tubes.

16. Oxidation and Fermentation of Medium (CDC Method) Negative: Red or alkaline (K) color in the deep with carbohydrate equal to the color of the inoculated control tube. No change (NC) or neutral (N): There is growth in the media, but neither the carbohydrate-containing medium nor the control base turns alkaline (red). Note: If the organism does not grow at all in the OF medium, mark the reaction as no growth (NG). Limitations  Slow-growing organisms may not produce results for several days. Quality Control  Note: Appropriate organisms depend on which carbohydrate has been added to the basal medium. Glucose is used as an example. Fermenter: Escherichia coli (ATCC25922) Oxidizer: Pseudomonas aeruginosa (ATCC27853)

17. VP Positive bacteria  Viridans  group streptococci (except Streptococcus vestibularis ),  Listeria, Enterobacter, Klebsiella, Serratia marcescens, Hafnia alvei, Vibrio eltor ,  Vibrio alginolyticus

18. Algorithm for Detection of Common Gram Negative Pathogens

19. Identification of Common Pathogens by Colony Characteristics and Biochemical Tests

20. Staphylococcus aureus

21. E. coli

22. I= Indole, M= Methyl Red, Vi= VP Test, C= Citrare ( IMViC Tests) TSI agar= A/A with Gas Urea Hydrolysis

23. I= Indole, M= MR Test, Vi= VP Test, C= Citrate Test ( IMViC Tests) Urea Hydrolysis TSI Agar= A/A with Gas

24. Salmonella  sp. after 24 hours growth on XLD agar. Xylose Lysine (XL) agar is used when trying to culture and isolate Gram-negative enteric bacilli. When XL agar is supplemented with sodium thiosulfate, ferric ammonium citrate, and sodium deoxycholate, it is then termed XLD agar, and is then an even more selective medium than XL alone. The presence of any black colored area indicates the deposition of hydrogen sulfide , (H 2 S) under alkaline conditions.

25. Citrobacter freundii

26. I= Indole, M= MR Test, Vi= VP Test, C= Citrate Test ( IMViC Tests) Urea Hydrolysis TSI Agar= K/A without gas Motility Semisolid Agar

27. Proteus mirabilis On MacConkey Agar -NLF colonies.

28. Proteus mirabilis On Blood Agar showing SWARMING growth.

29. I= Indole, M= MR Test, Vi= VP Test, C= Citrate Test ( IMViC Tests) Urea Hydrolysis TSI Agar= K/A with H 2 S gas production. Motility Semisolid Agar

30. P. aeruginosa showing Production of soluble Pyocyanin pigment on NA

31. 2-3 mm, flat, smooth, non-lactose fermenting colonies with regular margin and Alligator skin like from top view

32. I= Indole, M= MR Test, Vi= VP Test, C= Citrate Test ( IMViC Tests) Urea Hydrolysis TSI Agar= K/K without gas. Motility Semisolid Agar NOTE- Urea Hydrolysis can be variable Oxidase Test Positive

33. Acinetobacter baumannii

34. I= Indole, M= MR Test, Vi= VP Test, C= Citrate Test ( IMViC Tests) Urea Hydrolysis TSI Agar= K/K without gas. Motility Semisolid Agar NOTE- Urea Hydrolysis can be variable Oxidase Test Negative

35. Automated ID & AST

36. Thank You