UNIVERSITY OF PORT HARCOURT FACULTY OF PHARMACEUTICAL SCIENCES DEPARTMENT OF PHARMACY AN ASSIGNMENT ON BIOCHEMICAL TEST PRESENTED BY NAME: NWACHUKWU ONYEDIKACHI R

MAT NO: U2009/4725253 COURSE: PHARMACEUTICAL MICROBIOLOGY CODE: PMB 341.1

COURSE LECTURER: PHARM A. AWANYE

APRIL 2013

INTRODUCTION Biochemical testing is used to identify bacteria and yeast because different species produce different enzymes. Such biochemical test are designed to detect the presence of enzymes. Examples of biochemical tests are: 1. Oxidase test 2. Indole test 3. Methylated test 4. Galactosidase test 5. Citrate test 6. Urease test 7. Production of hydrogen sulphide (H2S) 8. Agglutination of trivalent serum 9. Dnase test 10. Coagulase test GALACTOSIDASE TEST Lactose utilization requires a couple of enzymes, one of which is betagalactosidase. In this test, you use a molecular decoy called ONPG (Orthonitrophenyl--D-galactopyranoside that will turn to a yellow colour in the presence of this enzyme. ONPG is an analog of lactose that the enzyme can break down to produce a yellow coloured end-product O-nitrophenol. Since this enzyme is made only in the presence of the lactose substrate, you need to be sure to grow this organism on media high in lactose from lactose broth on TSIA agar to name a couple. Note, the test is generally for gram bacteria and also to identify some staphylococcus species

OBJECTIVE Identify the ability of the bacterium to make the enzyme beta-galactosidase . Materials needed are: - ONPG Disc - 0.85- 0.99 Nad

- 1ml pipettes and pi-pumps - Sterile small tubes and cups

PROCEDURE 1. First, your organism needs to be growing in any medium with lactose (to induce the production of galactosidase enzyme) 2. Pipette 0.5ml of the saline into a sterile tube. 3. Inoculate with the bacterium and add the ONPG disc in a sterile manner (for cups dipped in alcohol and flame) to the tube. 4. Incubate at 3C for 4 hours. There is so little fluid in the tube, it will dehydrate if you do not read it in a few hours (alternatively you can cover it with paraffin) RESULT Fluid and the disc will turn any shade of yellow if positive for galactosidase enzyme. TEST ORGANISM CitrobacterFreyndii EnterobacterGerogenes Escherichia Coli Proteus Vulgaris Salmonella Serotype Arizone Salmonella Serotype Typhirium ONPG HYDROLYSIS + + + +

OXIDASE TEST
H 3C CH3 N N CH3

CH3

H2N

DMPD
N

H 3C

CH3

TMPD

INTRODUCTION
Oxidase test can be defined as a test carried out in microbiology to determine whether a bacterium produces certain cytochrome c oxidase. This test uses disks that are impregnated with either of the reagents;-N,N,N,NTetramethyl-p-phenylenediamine(TMPD) or N,N/diamethylphenylenediamine(TMPD). These reagents are redox indicators. They give dark blue colour to Maroon colour when being oxidized and colourless when being reduced. Bacteria which are oxidase positive possess cytochrome oxidase or indolepheno l oxidase. These both catalyse the transport of electron from donor compounds(NADPH) to electron acceptors (usually oxygen).TMPD dihydrochloride acts as an artificial electron donor for the enzyme oxidase.

CLASSIFICATIONS Strains are found to be either oxidised positive or oxidase. OXIDASE POSITIVE BACTERIA Bacteria which are oxidase positive contain cytochrome c oxidase. These bacteria use oxygen for their energy production with an electron transfer chain. Examples of oxidase-positive bacteria are: a. b. c. d. The pseudomonadaceae Neisseria Gonorrhoeae Legionella pneumophila Gram-negative, spiral curved rods-Helicobacter phylori,Vibro cholera and Campylobacter jejuni.

OXIDASE-NEGATIVE BACTERIA Bacteria in this case do not also use oxygen for their energy production with an electron transfer chain. Examples are: a. Enterobacteria b. Escherichia coli COLOR CHANGE 1. An oxidase positive test will give purple colour through Maroon and then black within 10-30 seconds. 2:An oxidase negative test will give no colour change at all. PROCEDURES -Moisten filter paper with the substrate 9%tetramethyl-pphenylenediaminedihydrochloride or select a commercially available paper impregnated with the substrate. -Use a platinum wire to remove a small portion of a bacterial colony from the agar surface and rub the sample on the filter paper. -Observe inoculated areas of the paper or disk for colour change to deep blue or purple within 10seconds. INDOLE TEST The indole test can be defined as a biochemical test carried out on bacterial species to determine the organisms ability to split indole from

the amino acid called tryptophan. This division is performed by a chain of a number of different ultra cellular enzymes referred to as trytophanase. CLASSIFICATION Strains are found to be either indole-positive bacteria or indolenegative bacteria INDOLE-POSITIVE BACTERIA Bacteria that test positive for clearing indole from tryptophan includes a. Aeromona punctata b. Bacillus Alvei c. Aeromonas Hydrophilia d. Pasteurella Multocida e. Streptococcus Faecalis etc INDOLE-NEGATIVE BACTERIA Bacteria which test negative with the indole test includes: a. Pasteurella Ureae b. Most Bacillus sp c. Bordetella sp d. Pseudomonas sp COLOUR CHANGES - A positive test gives a red or red-violet colour in the surface alcohol layer of the broth. - A negativ e test gives a yellow colour. A variable result can also occur, showing an orange colour as a result, due to the presence of Skatole also known as Methyl Indole or Methylated Indole. PROCEDURE A pure bacteria culture was grown in sterile tryptophan or peptone broth and allowed to incubate for 24-48 hours at 32C.After incubation, 5 drops of Kovac's reagent which contains isoamyl alcohol paradimethyl aminobenzaldehyde and concetrated hydrochloric acid is then added to the broth culture. A red colour indicates an indole positive bacteria and a yellow colour indicates an indole-negative test.

METHYL RED TEST Methyl Red with the molecular formula C15H15N3O2 and also with the molecular structure below is an indicator dye that turns red in acidic solutions. It is an azodye and a dark-red crystalline powder. It gives red in pH under 4-4 yellow in pH over 0-2 and orange between, with a PKA of 5.1 Methyl Red is a test in microbiology used to identify bacteria producing stable acids by mechanisms of mixed acid fermentation of glucose. It is also a test carried out to identify enteric bacteria based on their pattern of glucose mechanism. All enteric bacteria will produce pyruvic acid from glucose metabolism. CLASSIFICATION Enteric bacteria may be found to be either methyl red positive or negative. METHYL RED POSITIVE The enteric bacteria that are found here makes use of the mixed acid pathway to metabolize pyruvic acid to other acids like lactic, acetic, and formic acids. These bacteria are referred to as methyl Red-positive bacteria. Examples are: a. Escherichia coli b. Proteus vulgaris METHYL RED NEGETIVE The bacteria make use of the butylenes glycol pathway to metabolize pyruvic acid to neutral end-products. They are referred to a methyl Red negative bacteria. Examples are a. Serratia marcescens b:Enterobacter aerogenes RESULTS OR COLOUR CHANGES The Enteric bacteria that tend to metabolise pyruvic acid to other acids lower the pH of the medium to 4.2 thereby giving out a red colour which is positive to the test. While those that metabolize pyruvic acid to

neutral end products tend to lower the pH of the medium to 6.0,there by giving out a yellow colour which is negative to the test. PROCEDURE An isolate is inoculated into a tube with sterile transfer loop. The tube is incubated at 35c for 2-3 days. After incubation,2.5ml of the medium is between palms of the hands to disperse the methyl red. CITRASE TEST This type of test is used to detect an organism ability to use citrate as its sole source of carbon and energy. Bacteria are incubated in a medium containing sodium citrate and a pH indicator like bromothymol blue. The medium alone contains inorganic ammonium salts used as the sole source of nitrogen. CITRATE POSITIVE BACTERIA There will be growth on the medium and the colour change from green to blue. Examples are: a. Frateuria aurantia b. Escherichia coli CITRATE NEGETIVE BACTERIA No growth is observed or takes place on the medium and no colour changes. PROCEDURE Bacteria colonies are picked up using a loop and inoculated into Simmon's citrate agar and incubated overnight at 3 c for up to days. The medium changes colour from green to blue. UREASE TEST This is a rapid test for the diagnosis of H.pylori. It is also a test to detect the ability of H.pylori to secrete enzymes which catalyses the conversion of urea to ammonium and bicarbonate. This test is performed at the of gastroscopy.

PROCEDURE A biopsy of mucosa is taken from the antrum of the stomach and placed into the medium containing urea and phenol red. The urease that is being produced by H.pylori hydrolysis urea to ammonia and colour changes of the specimen from yellow to red are being observed. The specimen that gives a colour change of red is positive to the test while the specimen that gives a yellow colour is negative to test. COAGULASE TEST Coagulate test is a protein that can or be produced by several micro organism that enable the conversion of fibrinogen to fibrin. Coagulase is also produced by Yersinia pestis. Coagulase test is carried out to differentiate Staphylococcus aureaus from coagulase negative staphylococci. S.aureus always produce to forms of coagulase which are either bound coagulase or free coagulase. Bound coagulase, otherwise known as ''clumping factors'' csn also be detected by doing a tube coagulase test. SLIDE TEST Two drops of saline are put on to the slide which is labelled test(t) and control(c).The two saline are emulsified with the test organism by using a wire loop. Place a drop of plasma on the inoculated saline drop corresponding to the test an mix well with wooden applicator stick. Rock the slide gently for losses. RESULT Macroscopic clumping would be observed in the plasma within 10 seconds and no change in the saline drop, if the test is positive If the test is negative,no clumping will be observed TUBE TEST This test uses rabbit plasma that has been inoculated with a staphylococcus colony (i.e gram positive cocci which is catalase positive)the tube is then incubated at 3 C for one hour thirty minutes.if negative then continue incubation up to 18hours. RESULT

- the serum will coagulate resulting in a clot if the test is positive. - if negetive,the plasma remains liquid.The negative result may be S.epidemides. example of coagulase positive are; a:Staphylococcus aureus b:Staphylococcus delphini c:Staphylococcus hyicus example coagulase negetive are; a:S.caprace b:S.epidermis c:S.warneri d:S.hominis DNASE TEST This principle is a test use to determine the ability of an organism to hydrolyze DNA.The medium is pale pale green because of the DNAmethyl green complex.If the organism growing on the medium hydrolyses DNA,the green colour fades and the colony is sorrounded by a colourless zone. METHOD - inoculate the DNASE agar with the organism to be tested and streak eus for isolation . - incubate aerobically at 35c for 3-24 hours. RESULT Positive when DNA is hydrolyzed and combined with highly polymerized DNA at a pH of 7.5,turning the medium coloured around the test organism. Negetive if there is no degradation of DNA,the medium remains green. QUALITY CONTROL positive-staphylococcus aureus Negetive-staphylococcus epidermis PRODUCTION OF HYDROGEN SULPHIDE Principle this is used to detdct hydrogen sulphide librated as aresult of the degradation of sulphur containing amino acid METHOD

- Inoculate two sim tubes,one with E.coli and the other with proteus vulgaris.Use an inoculating needle and stab the agar with a single in and out motion. - Examine each sim tube for the presence of a black colour which indicates the production of hydrogen sulphide which combines with the peptonised iron in the sim medium and represents a positive test.the absence of black is a negetive test. EXPECTED RESULT Proteus vulgaris is positive with E.coli AGGLUTINATION This is a suspension of the specific antibody mixed with the insoluble antigen, such as red blood cells,bacteria or fungi.In agglutination reactions, however relatively large insoluble particles are involved rather than soluble molecules.Streptococcus species are used. EXPECTED RESULT - Readily visible clump will form if the test is positive - No clumping if result is negetive