Lab 21 - Pathogens, Snyder, Blood, MSA, Coagulase PDF

Lab 21 - Pathogens using special media to identify pathogens  Lab 21 – using special media to identify pathogens o Selective and differential agar – what are they? o Snyder test – sampling the mouth for acidic fermentation products o Hemolysins – culturing bacteria on blood agar to differentia...

Lab 21 - Pathogens using special media to identify pathogens  Lab 21 – using special media to identify pathogens o Selective and differential agar – what are they? o Snyder test – sampling the mouth for acidic fermentation products o Hemolysins – culturing bacteria on blood agar to differentiate for hemolysis o MSA (Mannitol Salt Agar) – demo of MSA results o Coagulase test – demo of the coagulase test o SS (Salmonella/Shigella) agar - demo of SS agar results This weeks GOAL… 1. Learn about and successfully execute the Snyder test. What does it test for?, what are the components and how does it work? Be able to interpret the results. 2. Learn about hemolytic bacteria and hemolysis. What are hemolytic bacteria and why are they pathogenic? Describe and differentiate the 3 categories of hemolysis. Describe a blood agar plate; its components and be able to interpret results. This weeks GOAL… 3. Learn about the Coagulase test What pathogenic species does the coagulase test help identify? What is coagulase and how does it increase the pathogenicity of the bacteria that produces it? Describe how the coagulase test works and be able to interpret the results. 4. Learn about MSA (Mannitol Salt Agar) and SS agar. Describe the pathogenic species each agar is designed to help identify. Describe the selective and differential components for MSA and SS agar. Describe what each selects for and how specifically it selects against unwanted types. Describe the differential aspect how each works to differentiate the selected species. Be able to interpret the results  These are ALL Selective and/or Differential media Selective – encourages growth of some bacteria types / inhibits growth of other types Differential – differentiates between types of bacteria growing on agar Enrichment media: Selective Media Types: enhances growth of desired bacteria; other bacteria grow Antibiotics (+/- resistance) but desired bacteria faster Nutrients (+/- nutritional ability) Salt (+/- halophiles) Selective media pH (+/- acidophiles or alkaliphiles) suppresses unwanted microbes and encourage desired microbes Oxygen (+/- obligate aerobes/anaerobes) contain inhibitors to suppress growth of certain types of bacteria Differential media allows distinguishing of colonies (species) of different microbes on the same plate usually pH indicator induced color change MSA a selective and differential agar  Some media have both selective and differential characteristics Differential and Selective Media examples: Eosin Methylene Blue Agar (EMB) – selects for gram - / differentiates for lactose metabolism Mannitol Salt Agar (MSA) - selects for gram + / differentiates for mannitol metabolism EMB - Eosin Methylene Blue MSA - Mannitol Salt Agar o selects for Gm- (dye toxic to Gm+) o selects for (gm+) halophiles o differentiates lactose fermenters o differentiates for mannitol fermentation lactose fermentation causes dye to mannitol fermentation produces acid precipitate on colonies lowers pH turning agar yellow pink, purple or iridescent green colonies G- / lactose fermenter mannitol fermenter non-mannitol fermenter  Lab 21 – using special media to identify pathogens o Selective and differential agar – what are they? o Snyder test – sampling the mouth for acidic fermentation products o Hemolysins – culturing bacteria on blood agar to differentiate for hemolysis o MSA (Mannitol Salt Agar) – demo of MSA results o Coagulase test – demo of the coagulase test o SS (Salmonella/Shigella) agar - demo of SS agar results  Lab 21 - Activity 1: Snyder test used to measure susceptibility to dental caries (cavities!) a selective and differential agar selective – selects for acidiphiles. Low pH (4.8) inhibits mesophile gowth differential – differentiates for amount of acidic producing bacteria by rate of color change Snyder agar o ~90% glucose / ~10% agar o uninoculated agar = green o bacteria ferment glucose producing acid o lowers pH further – agar turns yellow o HOW FAST turns yellow = susceptibility to dental caries (cavities) o no color change – no dental caries o 24 hr. - high susceptibility o 48 hr. - moderate susceptibility Snyder test results - rate of change determines susceptibility o 72 hr. – low susceptibility  Activity 1 - Snyder test procedure (per pair) 2 Synder agar tubes (keep in 45c bath) 2 paper cups, 2 sterile test tubes and 2 sterile dropper pipettes Procedure: 1. collect saliva in a paper cup 2. transfer saliva into a sterile test tube 3. vortex vigorously! 4. using a dropper pipette… transfer 0.2ml of sample to Snyder tube 5. swirl and tap tube to mix 6. hold tube under cold running water to set agar Snyder test results - rate of change determines susceptibility 7. dry tube, label and place in rack to be incubated  Lab 21 – using special media to identify pathogens o Selective and differential agar – what are they? o Snyder test – sampling the mouth for acidic fermentation products o Hemolysins – culturing bacteria on blood agar to differentiate for hemolysis o MSA (Mannitol Salt Agar) – demo of MSA results o Coagulase test – demo of the coagulase test o SS (Salmonella/Shigella) agar - demo of SS agar results  Lab 21 - Activity 2: hemolysins hemolysins = exotoxins that lyse red blood cells (RBC’s) increases pathogenicity of pathogen some Staphylococci and Streptococci are hemolytic hemolytic bacteria cultured on blood agar plate Blood agar hemolysis – the “lysing” of RBC’s o differential agar o 3 categories of RBC lysis o contains (sheep/goat) red blood cells (RBC’s) Beta – complete lysis; pathogenic notice it’s cloudy! Alpha – partial lysis; potentially pathogenic o if bacteria secrete hemolysins – RBC’s are lysed media becomes clear Gamma – no lysis; non-pathogenic Blood Agar Different degrees of hemolysis (RBC lysis): Beta – complete lysis Alpha – partial lysis Gamma – no lysis  Question: Classify patient sample #6 as alpha, beta or gamma hemolysis 6  Activity 2 - hemolysis test procedure (individual?) 1 blood agar plate 2 sterile tongue depressors; 2 sterile cotton swabs (1 tube); 2 pair vinyl gloves Procedure: 1. divide plate in ½ and label each half with each students initials. 2. put on gloves and sample partners throat place tongue depressor on center of tongue and gently press down tongue swab back of throat behind uvula don’t touch swab to any other part of mouth 3. zig-zag swab appropriate section of plate 4. repeat other half of plate with partner 5. inoculate at 37c see DEMO Plate be sure to evaluate the results *DO NOT OPEN PLATE ONCE INOCULATED!  Lab 21 – using special media to identify pathogens o Selective and differential agar – what are they? o Snyder test – sampling the mouth for acidic fermentation products o Hemolysins – culturing bacteria on blood agar to differentiate for hemolysis o MSA (Mannitol Salt Agar) – demo of MSA results o Coagulase test – demo of the coagulase test o SS (Salmonella/Shigella) agar - demo of SS agar results  Lab 21 - Activity 3: MSA demo MSA agar – selective & differential used to differentiate pathogenic Staph. aureus from non-pathogenic, resident skin bacteria Staph. aureus – mannitol fermenter acidic mannitol fermentation turns agar yellow Gram positive Gram positive non-mannitol fermenter mannitol fermenter MSA (Mannitol Salt Agar): o selective - selects for Gram positive (Staph)(halophiles) – salt toxic to gram negative o differential - differentiates for mannitol fermenters contains mannitol and protein (peptides) acidic mannitol fermentation products cause pH indicator in agar to turn yellow colonies with red agar= no mannitol fermentation: S. epidermidis, M. luteus etc… colonies with yellow agar possibly S. aureus  MSA (Mannitol Salt Agar) – further testing of MSA growth Catalase test (differentiate Staph. from other Gram positive) – Staph is catalase positive drip hydrogen peroxide - catalase produces oxygen bubbles (H2O2 to oxygen and water) Coagulase test (differentiate Staph. aureus from other mannitol fermenters – S. aureus coag. positive Coagulase – activates prothrombin to thrombin. Thrombin activates fibrinogen to fibrin – clot. Staph. aureus has coagulase – hides in blood clot from phagocytes positive results solidifies serum inoculate positive negative catalase coagulase mannitol fermenter positive coagulase non-mannitol fermenter negative catalase  Lab 21 – using special media to identify pathogens o Selective and differential agar – what are they? o Snyder test – sampling the mouth for acidic fermentation products o Hemolysins – culturing bacteria on blood agar to differentiate for hemolysis o MSA (Mannitol Salt Agar) – demo of MSA results o Coagulase test – demo of the coagulase test o SS (Salmonella/Shigella) agar - demo of SS agar results Lab 21 - Activity 4: SS agar demo SS (shigella/salmonella) agar o a selective & differential used to differentiate pathogenic Shigella, Salmonella typhimiurium and E. Coli Salmonella – can metabolize AA cysteine E. Coli – can metabolize lactose Shigella – can not metabolize either  Lab 21 - Activity 4: SS agar demo MSA (Mannitol Salt Agar): o selective - selects for enteric bacteria– bile salts toxic to other bacteria o differential - differentiates for cytstiene and lactose fermenters contains lactose and cysteine protein (peptides) acidic lactose fermentation products cause pH indicator in agar to precipitate purple colonies = E. coli cysteine fermentation produces hydrogen sulfide – H2S reacts with iron in media producing iron sulfide and black colonies = Salmonella typhimiurium white colonies negative for lactose and cysteine = Shigella  Question: A swab was taken from a piece of raw meat tested with SS agar This picture is the result. Explain what you see.