Identification methods of gram positive and gram negative cocci Presented by: Eunice Lorán, Ruth Cerpa, Katian melendez

Objectives Learn how to perform different methods of detection and differentiation of gram negative and gram positive cocci in the laboratory. Learn the mechanism of action of the differentiation and identification methods used. Interpret the results from a coccus differentiation and identification method.

Hemolysis test

Hemolysis test This test provides information on what hemolytic enzyme a bacterium possesses. The test is performed using blood agar which contains 5% sheep blood. It is used to identify Streptococcus pneumoniae. Alpha-hemolysin partially breaks down the red blood cells and leaves a greenish color behind. Beta-hemolysin breaks down the red blood cells and hemoglobin completely this leaves a clear zone around the bacterial growth.

Hemolysis test procedure Streak culture for isolation on TSA plate with 5% sheep blood. Incubate the plate for 24 hours at 35° C. Observe the results. The hemolysins produced by streptococci perform better in an anaerobic environment.  Because of this, it is standard procedure to streak a blood plate and then stab the loop into the agar to provide an area of lower oxygen concentration where the streptolysins can more effectively break down the blood cells.

Hemolysis test results Alpha-hemolysin s .pneumoniae Beta-hemolysin

Bacitracin test

Bacitracin test Bacitracin is a mixture of cyclic polypeptides produced by Bacillus subtillis. Bacitracin interferes with the dephosphorylation of C 55 – isoprenyl pyrophosphate. Differentiates between β-hemolytic Streptococcus. C55 isoprenyl es una molecula que transporta los building blocks del peptoglicano de l apared celular fuera de la memrana interna de la bacteria. Se usa 0.04 U de bactracina

Bacitracin test procedure Using an inoculating loop, streak two or three suspect colonies of a pure culture onto a blood agar plate. Using heated forceps, place a bacitracin disk in the first quadrant (area of heaviest growth). Incubate the plate for 18 to 24 hours at 35° C RT. Look for zone of inhibition around disk.

Bacitracin test results Inhibition zone Positive: Any zone of inhibition around the disk Streptococcus pyogenes Negative: No zone of inhibition Streptococcus agalactiae

Novobiocin test

Novobiocin test Novobiocin interferes with the unpackaging and repackaging of DNA during DNA replication the bacterial cell cycle. Novobiocin is obtained from the actinomycete Streptomyces niveus. Novobiocin test is a reliable presumptive identification of Staphylococcus Saprophyticus from another coagulase negative Staphylococci spp.

Novobiocin test procedure From a pure culture transfer isolated colonies of aerobic, catalase-positive, coagulase-negative gram-positive coccus aseptically to a sterile plate of nutrient agar. Apply one 5 µg novobiocin disk onto the inoculated agar surface. Incubate plate aerobically for 18 to 24 hours at 35 to 37° C. Measure (in millimeters) the diameter of the zone of inhibition around the novobiocin disk, and record as susceptible or resistant.

Novobiocin test results Resistant – zone size of < 12 mm Sensitive – zone size greater or equal to 16 mm. Staphylococcus saprophyticus – growth < 12mm or uniform growth up to the edge of the disk Staphylococcus epidermidis – Zone of inhibition >16 mm or larger Staphylococcus saprophyticus Staphylococcus epidermidis

D-dimethylaminocinnamaldehyde PYR Hydrolysis Test Differentiates those gram-positive cocci that hydrolyze L-pyrrolidonyl-β-naphthylamide (PYR) from other species. Streptococcus Group A, β-hemolytic (S. pyogenes) Enterococcus spp. Detects the enzymatic activity of pyrrolidonyl aminopeptidase. 1- En ambos casos con resultados positivos para esta prueba. 2- Es la enzima encargada de hidrolizar este compuestos en estas bacterias. 3- El principio de esta prueba se da a traves de discos impregnados con el sustrato de PYR, si las bacterias contienen esta enzima hidrolizaran el compuesto y al anadirle este reagente producira un color rojo. enzyme Red Color PYR β-naphthylamide D-dimethylaminocinnamaldehyde (reagent)

PYR Hydrolysis Test Procedure Moisten a PYR disk with sterile water. Using a sterile loop, rub colonies on the disk. Incubate disk at room temperature for 10 minutes. Add few drops of reagent and observe any color change within 5 minutes. 1- debe ser humedecerlo un poco, no demasiado! 2- Puede ser necesario mas de un loop para cubrir todo el disco y q de resultados. 3- Puede variar la temperatura y el tiempo segun el manufacturer

PYR Hydrolysis Test results Interpretation: Positive Negative Positivo puede indicar Strept Grupo A, B hemolit- S. pyogenes o Enterococos.

Optochin Test Identifies Streptococcus pneumoniae from an α-hemolytic streptococcal culture (Viridans spp). Optochin is a toxic chemical that harms some bacteria. Optochin disks are placed in the culture and are incubated. Susceptibility = zone of inhibition of 14 mm with a 6 mm disk. Resistance = visible growth up to the margin of the disk. 2-Asi q esta prueba nos va a decir si es susceptible o resistente a este quimico. Optochin es toxico para S.penumonia ya que es sensitivo, o sea da positivo 3- Como se mide esta susceptibilidad ps.. 4- zona de inhibicion= no crecimiento de bact Si se ve una zona parcial se debe hacer otras pruebas (como bile solubility) para confirmar su identidad.

Optochin Test Procedure Using a sterile swab, transfer and spread an inoculum of the culture to a SBA plate. Place an optochin-disk at the center of the plate. Incubate at 35 - 37° C for 24 hours in a CO2 incubator. Observe the growth on the surface of the plate. 2- siguiendo las tecnicas asepticas.

Optochin Test results Interpretation: Resistant Susceptible

Bile Esculin Test Differentiates group D streptococci and Enterococcus from other gram-positive, catalase-negative cocci. Two-step test: Growth in the presence of bile. Hydrolysis of esculin to esculetin and glucose. Bile inhibe crecimiento de otras bacterias excepto estos dos. Esculin es un compuesto q algunas bacterias pueden hidrolizar y utilizar la glucosa como energia dejando esculetin en el medio. Algunas bact pueden hidrolizar esculin pero no en presencia de bile. Ferric citrate esta en el medio. Black Color Esculin Esculetin Ferric citrate Glucose

Bile Esculin Procedure Using a sterile loop, inoculate colonies from a blood agar plate to a bile esculin agar plate. Incubate at 35° C for 18 to 24 hours. Observe growth and color change in the medium.

Bile Esculin Results Interpretation: Positive Positive Negative Crecimiento sin color no indica un resultado positivo. Positive Positive Negative

Motility Test Determines the ability of a bacteria to move in a medium. Useful in Enterococcus spp. Different methods are used: The Wet Mount – a bacterial sample is observed under the microscope to identify motility. The Hanging Drop Method – uses a depression slide to observe motility. Culturing Method in a semi-solid medium –inoculates bacteria in a semi-solid medium using an inoculating needle. Se considera util para diferenciar enterococos. 1- Es el mas rapido y simple ya que vamos a… 2- La tecnica es parecida a wet mount pero utiliza una lam con depresion en donde se va a colocar la muestra. 3-de acuerdo al patron de crecimiento en el tubo se podra determinar si es motil o no.

Motility Test Procedure The Wet Mount and the Hanging Drop Method: 2- Se coloca la muestra en el cubreobjeto y se le anade vaselina tanto en la laminilla donde se va a colocar las esquinas del cubreobj o en el cubreobjet antes de anadirle la muestra. Se invierte la laminilla y se pega el cubreobj. Luego rapidamente se vira la laminilla para que no se rompa la gota y utilizando aceite se observa bajo microscopio Es importante diferenciar movimiento real a un artefacto llamado Brownian movement en donde las bacterias son movidas por particulas que chocan con ellas, se debe ver si es un mov multi-direcc para saber que real.

Motility Test procedure Culturing in a semi-solid medium procedure: Apply a colony to the end of an inoculating needle. Insert the needle into the center of the semi-solid medium tube for about one inch. Incubate at 30° C for 24 to 48 hours. Observe growth pattern in the tube.

Motility Test Results Interpretation: Very motile Non-motile Motile

CAMP Test CAMP factor Extracellular protein that along with the β-lysin of Staphylococcus aureus enhances the lysis of red blood cells. CAMP test identifies non-hemolytic group B and β-hemolytic streptococci. Ej. S. agalactiae -Ciertas bacterias producen el factor CAMP (nombre viene de quienes lo descubrieron) -S. agalactiae es un coco gram +, flora normal en algunos humanos… puede causar septicemia mayormente en neonatos

CAMP Test Procedure Inoculate a single streak of β-lysin producing S. aureus into a TSA-sheep blood agar plate. Use an inoculating loop to pick the unknown β-hemolytic streptococcus strain and make a single streak perpendicular to S. aureus. Incubate overnight at 35° C. Read and interpret results. Franja de arriba a abajo en el centro del plato Dejar de 2 a 3mm entre las franjas de S. aureus y el desconocido

CAMP Test Results Bacteria beta hemolítica formará un “arrow” en dirección a S. aureus (aumento de lisis de células rojas) Bacteria no hemolítica o no parte del grupo B Lancefield no formará el “arrow” (S. pyogenes es parte del grupo A de streptococos)

Hippurate Hydrolysis Test Hippurate hydrolase hippurate + H2O   benzoic acid + glycine benzoic acid + ferric chloride Test detects hippurate hydrolyzing bacterias, specially from group B streptococci. Example: S. agalactiae Ferric benzoate -Bacterias del Grupo B contienen la enzima hidrolasa de hipurato -Grupos A, C, F y G no hidrolizan hipurato. Algunas bacterias de los grupos D y bien pocos viridans lo hidrolizan

Hippurate Hydrolysis Test Procedure Inoculate an hippurate broth with one (1) drop of a fresh Todd-Hewitt broth culture. Incubate the broth for up to seven (7) days or until turbid growth is seen at 35° C. Centrifuge the tube of broth to sediment the bacteria. Pipette 0.8 mL of the clear supernatant to a small clear tube. Add 0.2 mL of ferric chloride reagent to the supernatant and mix well. Read and interpret results. Utilizar un cultivo (overnight) incubado a 35°C o una suspension fresca de bacterias (16-20h) para el inoculo. Tambien se puede utilizar un “loopful” de cultivo de un plato de agar sangre

Hippurate Hydrolysis Test Results A heavy precipitate that does not clear within 10 minutes A clear golden-brown liquid

Modified Oxidase Test Detects enzyme – cytochrome oxidase. Modified Oxidase Reagent: 1% tetramethyl-p-phenylenediamine in dimethyl sulfoxide. Utilized for differentiating Micrococcus from Staphylococcus. Micrococci yields a positive result. Staphylococci yields a negative result. -Esta prueba es solamente para cocos gram +, catalasa + -Staphs dan negativo excepto Staphylococcus sciuri.

Modified Oxidase Test Procedure Use bacteria from a culture grown on blood agar for 24 - 36 hours. Transfer a Microdase disk from the stock bottle to a petri dish using sterile forceps. Use an inoculating loop to streak a loopful of bacteria onto the top of the Microdase disk. Read and interpret results. El cultivo a utilizar no debe ser ni muy fresco ni muy viejo pues puede dar resultados erróneos Hay que regresar la botella de los discos a la nevera rápidamente luego de tomar uno

Modified Oxidase Test Results A blue or purple-blue color change within 2 minutes No change in color

6.5% NaCl Tolerance Test This test differentiates between bacteria that tolerate high (6.5%) concentrations of sodium (NaCl) and those that are inhibited by this salt concentration. Enterococcus species = positive (growth, turbid). Group D Streptococcus = negative (no growth, clear). Streptococcus species Viridans group = negative.

6.5% NaCl Tolerance Test Procedure Inoculate one (1) or two (2) drops of an overnight Todd Hewitt broth culture into the 6.5% NaCl broth. Incubate at 37°C in ambient air for a week. Read and interpret the results. Puede utilizarse tambien un “loopful” de agar sangre en plato si no hay el cultivo en caldo Todd Hewitt Tiempo de incubación puede ser más, dependiendo de las características de crecimiento de la cepa siendo probada La mayoría de los enterococos dan resultados positivos a las 24hrs de incubación

6.5% NaCl Tolerance Test Results Positive Negative -En algunas ocasiones, se le añade 0.5% de dextrosa para facilitar la lectura (color violeta). Cambio de color de violeta a amarillo es positivo, pero no necesariamente tiene que cambiar. Si hay turbidez (aunque no cambie de color), es positivo.

6.5% NaCl Tolerance Test Results (A) Enterococcus faecalis: Color change from purple to yellow, indicating fermentation of the dextrose and salt tolerance. (B) Streptococcus bovis: No color change or growth. -En algunas ocasiones, se le añade 0.5% de dextrosa para facilitar la lectura (color violeta). Cambio de color de violeta a amarillo es positivo, pero no necesariamente tiene que cambiar. Si hay turbidez (aunque no cambie de color), es positivo.

References Optochin Susceptibility Test. Retrieved September 2014, from http://www.vumicro.com/vumie/help/VUMICRO/Optochin_Susceptibility.htm Microbugz. Bile Esculin Test. Retrieved September 2014, from http://www.austincc.edu/microbugz/handouts/Bile%20Esculin%20Test%20Handout.pdf Murray, P.R., Baron, E. J., Jorgensen, J.J., Pfaller, M.A., and Yolken, R.H. Manual of Clinical Microbiology, 8th ed Retrieved September 2014, from http://www.cdc.gov/streplab/downloads/general-methods-sections1-2.pdf

References Novobiocin differentation disk. Retrieved September 2014, from https://catalog.hardydiagnostics.com/cp_prod/content/hugo/NovobiocinDiffDisks.htm Bacitracin test: principle, procedure, expected results and quality control. Retrieved September 2014, from http://microbeonline.com/bacitracin-test-principle-procedure-expected-results-and-quality-control/