Faculty of Medicine and Health Sciences Microbiology Lab Second semester 2013-2014

1. Faculty of Medicine and Health Sciences Microbiology Lab Second semester 2013-2014 prepared by: Mohammad Al-Qadi E-mail: m.qadi@najah.edu

2. Bacterial Identification: biochemical tests (1) Lab # 11

3. Introduction -when you work in microbiology you mostly deal with Unknown microbes -Identification of unknown microbes can be achieved by using our knowledge about: *properties and characteristics of media available ( selective, differential, enriched etc.) *Growth characteristics *Staining *Biochemical tests to confirm identification and finalize the result.

4. Introduction Biochemical tests (enzymatic) can be classified into two categories: A) one specific enzyme B) Complete metabolic pathway (several enzymes) 1. Carbohydrate oxidation and fermentation 2. Amino acid degradation 3. Single substrate utilizations Catalase , oxidase , indole ,urease ,PYR, hippurate hydrolysis, etc

5. Catalase -This test is used to identify organisms that produce the enzyme, catalase. -This enzyme detoxifies hydrogen peroxide by breaking it down into water and oxygen gas (bubbles). H2O2 + catalase H2O + O2 -Key identification for Gramm+ vecocci Staphylococci – positive Streptococci – negative

7. Coagulase -This test is used to identify organisms that produce the enzyme, coagulase. -This enzyme converts fibrinogen to fibrin (plasma clot) -Detected by slide (bound) or test tube (free or clumbing factor) method. -Firstly do slide method. If negative confrim using tube method. -In tube method read result after 1-4hoursat 37 C if negative incubate at room temperature over night

8. Coagulase Slide test (bound or clumping factor) Tube test (free coagulase) If negative confirm by tube method check tubes at ½, 1, 2 & 4 hrs and over night • Result • If the organism is has coagulase it will clump the plasma. • If the organism does not have coagulase it will not clump the plasma

9. Oxidase -This test (CytochromeOxidase test) is used to identify organisms that produce the enzyme, Oxidase. -This enzyme Detect by adding few drops of colorless oxidase reagent Colonies turn deep purple in color (positive) • (Oxidasescatalyse electron transport between substrates acting as electron donors in the bacterium and tetramethyl-p-phenylenediamine OR dimethyl-p-phenylenediamine - a redox dye present as the hydrochloride or oxalate salt The dye is reduced to a deep violet-blue colour in the presence of oxidase enzymes)

10. Oxidase It uses disks impregnated with a reagent such as N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) or N,N-Dimethyl-p-phenylenediamine (DMPD), which is also a redox indicator. Note the purple to dark purple color after the colonies have been added to filter paper moistened with oxidase reagent. • Positive control: Pseudomonas aeruginosa • Negative control: Enterobactericia

11. Methyl Red/VogesProskauer (MR/VP) • These tests are used to determine what end products result when the test organism degrades glucose. • MR-VP media contains glucose and peptone (Methyl red & Potassium Hydroxide both used as indicators) . • All enterics oxidize glucose for energy; however the end products vary depending on bacterial enzymes • Inoculate 2 glucose broths with inoculating loop. After24-48 hours of incubation, add a few drops of MR to one tube, and VP reagents to the other tube. • Both tests are used to help differentiate species of the family Enterobacteriaceae. • MR—tests for acid end products from glucose fermentation. • VP—tests for acetoin (Acetyl methyl carbinol) production from glucose fermentation.

12. MR/VP • Reading Results: • MR a positive result is red (indicating pH below 6) and a negative result is yellow (indicating no acid production) • VP a positive result is red after VP reagents are added (indicating the presence of acetoin) and a negative result is no color change. • This colour may take 20 to 30 minutes to develop. E. coli does not produce acetyl methyl carbinol, but Enterobacterand Klebsiellado. Methyl Red: left – and right + VP: left + and right –

13. Good Luck