انت هنا الان : شبكة جامعة بابل > موقع الكلية > نظام التعليم الالكتروني > مشاهدة المحاضرة

Biochemical test

Share |
الكلية كلية التمريض     القسم قسم العلوم الطبية الاساسية     المرحلة 2
أستاذ المادة ميس هادي جبر       22/01/2016 12:04:54
Lab: 5 Biochemical tests used for identification of medical bacteria
Biochemical tests have an important role in the identification of bacteria to classify bacteria and determine the causative agent of diseases.
1-Haemolysis: Some types of pathogenic bacteria are able of producing haemolysin enzyme that lyses Erythrocytes (RBCS). This can be detected in vitro on blood agar plates. There are three types of haemolysis:
A- ?-haemolysis: Complete clear circular zone around the bacterial colonies due to complete lysis of red cells. e.g. Streptococcus pyogenesand Staphylococcus aureus



B- ?-haemolysis: appear as greenish zone around the colonies• due to partial haemolysis of RBCs. e.g. Streptococcus viridians

C- ?-haemolysis: (no haemolysis) no any obvious changes around the colonies e.g.Enterococcus faecalis
2- Mannitol fermentation: This can be detected in vitro on mannitol salt agar plates. Staphylococcus aureus can be ferment the sugar (mannitol) in this media &become yellow, while S. epidermidis cannot ferment the sugar &become white.

3-Pigment production: Some type of bacteria able to produce a characteristic pigments. There are two types of pigments:
Endopigment: Remain bound to the body of the M.O. and doesn t diffuse to the surrounding media e.g. Serratia and Staphylococcus
Exopigment: Soluble which readily diffuse into the surrounding media e.g.Pseudomonas aerogenosa produce four types of pigments Pyocyanin (blue-gree) Pyoveridin (green), Pyorubin (red) and Pyomelanin (black)
4- Motility test: Motility of bacteria can be detected by several methods; used to determine whether an organism is equipped with flagella or not e.g:-
1-Hanging drop technique
2-Stabbing of semisolid medium. -
3-Flagellar stain
Motile bacteria such as Salmonella, Proteus and E coli
5-Catalase production test: Some aerobic bacteria able to produce catalase enzymethat catalyses H2O; (Hydrogen peroxide) and releases O2 and H2O
2O2 + 2H — catalase —> O2 + 2H2O
Procedure: A small amount of bacterial culture to be tested is picked from nutrient agar by stick or glass rod and put it on the surface of a clean slide, where a drop of (3% H2O) was added. Formation of gas bubbles indicates a positive result. A false positive reaction may obtain if the culture medium contain catalase (Blood agar) orif iron loop is used.

6-Coagulase production: Some bacteria produce coagulase enzyme that converts soluble fibrinogen protein to insoluble fibrin protein (coagulation of plasma).Coagulase is a virulence factor of Staphylococcus aureus. The formation of clot around an infection caused by this bacterium will protects it from phagocytosis
A-Boundcoagulase(Slide method)
B-Free coagulase (Tube method)
7-Oxidase test: Use to detect the production of cytochrome oxidase which related to respiratory electron transport chain and it produced by strictly aerobic bacteria e.g.Pseudomonas and Neisseriae.
Procedure: A small area of filter paper is soaked with a freshly prepared 1% oxidase reagent (Tetramethyl-p-pheuyleneDiamineDihydrochloride) bacterial colony to be tested is picked from agar by stick or glass rod and put it on the soaked area. A positive result is indicated by formation of deep purple color due to reduction of this dye by oxidase enzyme.
8-Triple sugar iron (TSI) and Kligler siron agar (KIA)
TSI medium contain(glucose, lactose and sucrose)
KIA contain only (glucose and lactose)
*pH indicator: phenol red (red in alkaline pH and yellow in acidic pH).
*Ferrous sulfate as an indicator of H2S production
These media are used to detect ability of bacteria to ferment these sugars and this aid in the identification and classification of enteric G-ve bacilli) enterobacteriaceae).
Three criteria can be detected:
l- Bacterial ability to produce gas from sugar fermentation. This makes the media to push up or break up.
2-H2S gas production can be detected by the production of black precipitate in the bottom of the media. As H2S react with iron in the media to form black ferrous sulfide in the butt.
3-Ability to ferment sugars that can be detected by color changes from red to yellow. Position of the color change distinguishes the acid production associated with glucose fermentation from the acidic products of lactose or sucrose fermentation. Bacteria that ferment glucose produce acid that turn the color of the pH indicator to yellow inthe butt but not in the slant (result——> K/A). While lactose or sucrose fomenters produce more acid that turn both butt and slant to yellow (result—> A/A).



9-Urease test: This test is used to identify bacteria able of hydrolyzing urea using the enzyme urease to make ammonia and carbon dioxide. The hydrolysis of urea raises the pH to above 7.0 and the pH indicator (phenol red) turns the medium from yellow to red pink.
NH2-CO-NH2 + H2O — urease —>¬¬¬¬¬¬¬¬¬¬¬¬¬¬2NH3 + CO2
Urea ammonia
Ex: of urease producer are Helicobacter pylori and V. cholera ,Klebsiella& Proteus
10-IMVC: These are a group of biochemical test that help in the identification anddifferentiation between enteric G-ve bacilli (enterobacteriaceae).
A-Indole production test: It tests for the bacterial ability to produce indole. Bacteriause an enzyme, tryptophanase to break down the amino acid (tryptophan) to giveindole, ammonia and pyruvic acid.
Tryptophan — Tryptophanase —>¬¬¬¬¬¬¬¬¬¬¬¬¬¬Indole + ammonia + pyruvic acid
Peptone liquid medium containing tryptophan is inoculated the- tested bacteria and incubated at 37 °C for 24 hrs. Few drops of kovac s reagent are added to the bacterial growth. The presence of red rig in the superficial layer of the medium indicate +ve result of indole production e.g. E.coli. Yellow ring indicate —ve result e.g. Klebsiella.
.
B- Methyl red/ Voges-Proskauer tests: Both MR and VP tests are used to determine what end products result when the tested organism degrades glucose (for energy production) and this depend on the type of enzyme that the bacteria have.

MR- used to detect acid as an end product from complete glucose fermentation.
VP- used to detect acetoin (acetyl methyl carbinol) production from partial glucose fermentation.
Glucose phosphate peptone water medium is used for both tests; it s inoculated with the test bacteria, alter incubation at 37 °C for 24hrs.
In MR; 5 drops of methyl red indicator are added. Color changes of the medium to red indicate positive result e.g. E. coli and yellow in negative result e.g. Kebsiella.





ln VP; Vogesproskauer reagent (Barritt reagent) is added to the medium. This reagent is consists of reagent A (5% or-naphtbol) and reagent B (40% KOH). Positive reaction can be detected by developing a pink-burgundy color within 20-30 min. e.g. of +ve result is Enterabacreraerogener and Klebsiella while -ve result as E. coli

C- Citrate utilization: It used to test the ability of bacteria to consume citrate as a sole source of carbon. Simmon’s citrate agar can be used with bromthymol blue as pH indicator.The tubes will be incubated after inoculation by stabbing, +ve result is blue )meaning the bacteria metabolised citrate) e.g. Enterobacter and Klebsiella and –ve result remains green e.g. E coli.



المادة المعروضة اعلاه هي مدخل الى المحاضرة المرفوعة بواسطة استاذ(ة) المادة . وقد تبدو لك غير متكاملة . حيث يضع استاذ المادة في بعض الاحيان فقط الجزء الاول من المحاضرة من اجل الاطلاع على ما ستقوم بتحميله لاحقا . في نظام التعليم الالكتروني نوفر هذه الخدمة لكي نبقيك على اطلاع حول محتوى الملف الذي ستقوم بتحميله .
download lecture file topic