Exercise 2 1.Examine bacteria microscopically by preparing a gram stain. Streptococcus pyogenes Streptococcus agalactiae Streptococcus pneumoniae Enterococcus faecalis 2.Catalase Test--Perform the slide test by emulsifying some visible growth material in a drop of hydrogen peroxide and note that all streptococci are catalase-negative(no rapid ebullition). 3.Rapid Enterococcus Identification--The bile esculin(BE)test is the most accurate way for identifying Group D streptococci because they are all BE-positive and will cause a blackening of the agar medium after overnight incubation.All other streptococci are negative for this reaction.An enterococcus/Group A screen will be available for the rapid identification of Enterococcus faecalis from other streptococci.This specific medium contains esculin and also PYR(pyroglutamylbeta-naphthylamide)which will differentiate enterococci from other Group D streptococci. a)Using a sterile lo obtain a visible,he avy ino mn an stab into the medium in an area near one side next to the glass.Incubate the tube at 35C for 30 minutes to 4 hours b)The esculin hydrolysis usually occurs in 30 minutes with a heavy inoculum and is indicated by development of a dark brown color.The PYR reaction is detected by adding 2 drops of the PYR reagent and observing a cherry red color within 2 minutes.No color change,light orange or light pink-orange is considered negative. Positive and Negative Reactions of BE-PYR strep om other het tc streptoc cci by eaction The usually requires the inoculation of bile esculin agar and tube of broth containing6.5%NaCl d)After overnight incubation,the black discoloration of bile esculin identifies Group D streptococci(E.faecalis,S.bovis,etc.)and growth in the presence of 6.5%NaCl (salt tolerance test)differentiates enterococci which are salt tolerant from other Group D streptococci(S.bovis)which will not grow in this broth. Group A Groups GrOUD D GrOuD D B.CF.G Enterococcus Non-Enterococcus Esculin 十 十 PYR x 、 5.Rapid Streptococeal Grouping Most streptococcal species possess group specific antigens which are usually carbohydrate structural components of the cell wall.These antigens can be extracted and identified by homologous antisera,and the bacterium can be assigned to one of the various Lancefield groups.The principal use of the test is to rapidly identify strepto reported with one hour extraction from r wound swabs). Extraction and Test Procedure: a.To a small glass tube,2 drops of reagent I and 2 drops of reagent 2 are added to generate file://CJ/WINDOWS/Desktop/clown/Microb Lab Web 2001/Exercise2/exercise2.htm (3 of 10)[8/31/0024:30:02 PM] 1. Examine bacteria microscopically by preparing a gram stain. Streptococcus pyogenes Streptococcus agalactiae Streptococcus pneumoniae Enterococcus faecalis 2. Catalase Test -- Perform the slide test by emulsifying some visible growth material in a drop of hydrogen peroxide and note that all streptococci are catalase-negative (no rapid ebullition). 3. Rapid Enterococcus Identification -- The bile esculin (BE) test is the most accurate way for identifying Group D streptococci because they are all BE-positive and will cause a blackening of the agar medium after overnight incubation. All other streptococci are negative for this reaction. An enterococcus/Group A screen will be available for the rapid identification of Enterococcus faecalis from other streptococci. This specific medium contains esculin and also PYR (pyroglutamylbeta- naphthylamide) which will differentiate enterococci from other Group D streptococci. a) Using a sterile loop, obtain a visible, heavy inoculum and stab into the medium in an area near one side next to the glass. Incubate the tube at 35°C for 30 minutes to 4 hours. b) The esculin hydrolysis usually occurs in 30 minutes with a heavy inoculum and is indicated by development of a dark brown color. The PYR reaction is detected by adding 2 drops of the PYR reagent and observing a cherry red color within 2 minutes. No color change, light orange or light pink-orange is considered negative. Positive and Negative Reactions of BE-PYR c) From the foregoing table, you should realize that the screen test can also differentiate Group A streptococci from other beta-hemolytic streptococci by the PYR reaction. The identification of Enterococcus faecalis usually requires the inoculation of bile esculin agar and tube of broth containing 6.5% NaCl. d) After overnight incubation, the black discoloration of bile esculin identifies Group D streptococci (E. faecalis, S. bovis, etc.) and growth in the presence of 6.5% NaCl (salt tolerance test) differentiates enterococci which are salt tolerant from other Group D streptococci (S. bovis) which will not grow in this broth. Group A Groups B,C,F,G Group D Enterococcus Group D Non-Enterococcus Esculin - - + + PYR + - - - 5. Rapid Streptococcal Grouping Most streptococcal species possess group specific antigens which are usually carbohydrate structural components of the cell wall. These antigens can be extracted and identified by homologous antisera, and the bacterium can be assigned to one of the various Lancefield groups. The principal use of the test is to rapidly identify streptococci growing on agar plates (but satisfactory results have been reported with one hour extraction from blood cultures and from throat, skin, or wound swabs). Extraction and Test Procedure: a. To a small glass tube, 2 drops of reagent 1 and 2 drops of reagent 2 are added to generate Exercise 2 file:///C|/WINDOWS/Desktop/clown/Microb Lab Web 2001/Exercise2/exercise2.htm (3 of 10) [8/31/2002 4:30:02 PM]