Isolation of Staph , Strep and Gram – pathogen part II

1. Isolation of Staph, Strep and Gram – pathogen part II LAB # 13 Medgar Evers College Prof. Santos

2. Exercise 70 week 3 • Last week you streaked 2 plates, SM110 and MSA. Today, you will pick isolated colonies from the plates that are characteristic of S. aureus. • Pick colonies that caused the medium to turned yellow because that is indicative of S. aureus. • You will inoculate plates of blood agar and tubes of plasma. 97 % of S.aureus strains can coagulate plasma so we will use this as confirmation. • Tomorrow you will check for beta hemolysis on your blood agar plates.

3. Procedure for coagulation 1- obtain three tubes or as many as needed of plasma and inoculate heavily with your colonies from the nose, fomite and unknown. 2-Place the tubes in a 370 C water bath and check for coagulation after 30 minutes. It may take up to 24 hours for coagulation to occur but if the inoculate was heavy, you should see results faster. 3-While you wait, you might want to do a gram stain on the colonies and confirm it’s a gram positive organism.

5. Beta hemolysis

6. Exercise 71 week 3 • Last week, you inoculated tubes of trypticase soy broth with your alpha or beta strains of Strep. • This week, you will do confirmation test on your strains. Depending if you had beta or alpha, you will perform different tests.

7. Beta hemolysis 1- obtain a blood agar plate and perform the bacitracin, SXT test, and camp test. Streak the blood agar plate as shown in your book and place a disk of bacitracin and SXT. 2- streak S. aureus as shown in your book in the same plate. 3- Inoculate a tube of Hippurate broth. Beta strains will hydrolyze hippurate and produce a positive result indicated by a blue coloration. 4- Tomorrow, if both the Hippurate test and camp test are positive that is indicative of Streptococcus agalactiae, a group B organism.

9. Alpha hemolysis • 1- streak a plate of blood agar, and place a disk of Optochin. • 2- inoculate a tube of TSB • 3- inoculate a tube of Bile esculin agar • 4- Inoculate a tube of 6.5 % NaCl • Tomorrow, if both the NaCl and BE are positive then it’s a group D enterococcus.

10. Exercise 72 week 2 • Examine your plates from last week and look for colonies that are indicative of either Salmonella or Shigella. • Mac Conkey agar, Salmonella and Shigella and other non-lactose fermenting species produce smooth colorless colonies. Coliforms that ferment lactose produce reddish or dark centered colonies. • Hektoen Enteric agar, Salmonella and Shigella colonies are greenish-blue. Some species of Salmonella will have greenish-blue colonies with black centers due to Hydrogen sulfide production. Coliform colonies are salmon to orange and may have a bile precipitate. • Xylose Lysine Desoxycholate agar, most Salmonella colonies are red with a black center; Shigella colonies are red. Coliform colonies are yellow.

11. This week, you will pick colonies of Shigella or Salmonella and streak tubes of Iron Kliger. Keep in mind that both can not ferment lactose. • Tomorrow, you will pick colonies that do not ferment lactose and do the urea and SIM test. • Salmonella is motile + • Shigella is non motile and cant produce Hydrogen sulfide and is urea negative