Practical 7 Biochemical properties testing 1 part

Primary innoculation on cultivation media according to the sample : basical media (blood agar) and 1 selective or diagnostic medium – shorter diagnostic process Blood agar + chocolate agar (Respiratory tract,CNS – for Haemophilus sp) + McConkey (stool,urine, clinical materials (G-rods) Primary – 1st day isolation – basical orientation according to morphology of colonies, Gramm staining - decission for next procedures - orientation quick tests – (catalase, cytochromoxidase, bile solution testi, slide coagulase) Subcultures - multiplication of one colony and performance of differential test based on biochemical properties

Cultivation media - basical media – microbes without special nutrition requirements, multiplication – (bouillon, meat extract, pepton broth) -enriched media – basical media + enrichment factor (hemoglobin, fildes extract, yeast extract, chemically defined enrichment factors) -diagnostic media – detection of some biochemical properties of bacteriaí – sugar medium for biochemical activity of feremtation of ´glycides,- decaroxylation of aminoacides, production of some molecules during metabolism (H2S). These media contain the indicators. If bacterium is metabolising the structure in the medium acid is formed and pH changes – decrease – and the color indicator change also – visualisation of chemical reaction -selective – for growth only of selected bacterium (medium with special growth factors or inhibitors of growth (egg media, ATB media, Kauffman medium Campylobacter medium, Legionella, mycoplasma medium selektively diagnostic – base + inhibitors + substrate of metabolism + indicator Endo, MacConkey, DC agare – G-paličky Lactose negative or positive -media for storage, transport, ATB susceptibility testing

Tests for biochemical properties and metabolic activity testing Aim – final identification Method – subcultivation on the series of testing diagnostic meida Liquid media – with chemical structure – substrate and indicator, solid media with biochemical – metabolic substrate and indicator and diagnostic disc with substrate, micromethods – liquid media with substrate and indicator in microwells Algorithm – of chosen procedures

Group of biochemical tests aligned so that they allow numeric identification based on statistical probability of the result of one test In the positive result the well is attributed the chifer according to the position in the triplet.( 1 2 or 4) Addition of chifers in triplet gives the number and each result of the triplet gives a subsequent one position of the code that is the combination of numbers of tested triplets. This code is corresponding to one bacteria

Demonstration G+cocci: Staphylococcus aureus a Staphylococcus epidermidis on the selective-diagnostic medium Salt mannit: selectively NaCl allowed growing of staphylococci that tolerate it.while others do not., mannitol is the diagnostic substrate utilised by St. aureus which metabolised it, formed acid that makes the medium becomming acid and change the pH and indicator color. St. aureus changes the original red color to yellow, St. epidermidis is growing on the mediu, tolerates salt without changing the pH and indicator color - not utilising manitl

G-rods Hajn glucose succrose lactose maltose manit indol urea/motility H2S Proteus vulgaris * - + +/+ Proteus mirabilis /+ Salmonella typhi /+ Escherichia coli /+ Shigella /+

Yeast – zymogram, fermentation of glycides – biochemical properties – diagnostic medium: glc,sach,mal,lak,raf +/oranžová, -/modrozelená ––––––––––––––– Helicobacter pylorí fermentation of urea, Helicobacter has urease activity that hydrolyse urea (making so a good environment– NH4 – for surviving in acidic stomach

Surface of tube medium – aerobe environment, lactose negative bacteria do not ferment, it is alkaline, red Lower part - in anaerobe environment, enterobacteria ferments glycids – acidic – yellow or black H2S. 1 negative control 2 Ps.aeruginosa : net fermenting - red 3 Shigella sonnei: H2S - negat.,gas – negat., TSI – acid/alcalic red/yellow 4 Salmonella typhi: H2S – pozit., gas–negat., TSI – acidic/alcalic red/yellow 5 Escherichia coli: H2S – negat.,gas -posit., TSI – acid/acidic red/red 6 Proteus mirabilis: H2S – posit, gas - negat., TSI – acid /acid red/red

Klin Combined diagnostic medium solidified in bent position of Petri dishes, in formed of triangle to 1/3 of the dishes. Endo medium is added too. The medium is thickly innoculated together with some ticks. The cover glass is put - on the surface - for the detection of gas. Fermentation of glc, urea, thiosulfate is detected. Bacteria that hydrolyses urea forme NH4 what change pH and the color of green indicator to blue. Fermentation of glc changes the color of the triangle to yellow, formation of gas that is seen under the cover glass. Producers of H2S turn the medium black. Escherichia coli glc +, gas +, H2S- Citrobacter glc+, gas +, H2S+ Proteus gas-, H2S+

MikroLaTest Identification of Klebsiella pneumoniae – Enterotest + / /+

Detection of catalase and oxidase activity Catalase: - enzyme, hydrolysing H2O2 – toxic for the cell and formation of molecular oxygen. Moraxella catarrhalis –cat.negat. H2O2 hydrolysis, bubbles - Staphylococcus sp. Oxidase: - enzyme active in end phases of metabolism of strict aerobes Pseudomonas aeruginosa – colonies turn black after drop of dimetylparaphenylendiamine is added– (Neisseria gonorrhoe)