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Medical Microbiology - Bi431 Gram (+) and Acid-Fast Bacteria Quick Outline of Activities I. Survey Set-Up (Monday) 1. Body - Staphylococcus, Streptococcus 2. Grocery - Listeria 3. Throat -Corynebacterium II. Gram (+) Cocci Controls (Wednesday) 1. Coagulase Testing - Staphylococcus 2. Tests Using Control Staphylococcus 3. Tests Using Control Streptococcus III. Special Staining (Wednesday) 1. Spore-Staining Bacillus 2. Acid-Fast Staining Mycobacterium I. SURVEY SET-UP - PERSONAL AND GROCERY Anything that says ANAEROBIC must be put into the anaerobic chamber together; no mistakes or redo's once chamber is activated! 1. Body Surveys For Staphlyococcus and Streptococcus Mannitol-Salt - Skin, Aerobic Growth Each person labels 2 Mannitol-Salt plates - inner nostril, between toes For each plate, use a sterile Q-tip to lightly swab respective area and apply to plate as directed Incubate at 37°C until next session - AEROBICALLY Blood Agar - Throat, Anaerobic Growth Each person labels 1 Blood Agar plate - throat (have your partner sample, see diagram above) Use long Dacron swab to sample throat and apply to plate as directed SET ASIDE UNTIL ALL ANAEROBIC PLATES DONE - LEARN ABOUT ANAEROBIC JARS 2. Grocery Surveys for Listeria No available controls; enrichment depends on how dirty the local/fancy cheeses areā¦ Cold Enrichment Technique As pairs, use both streaking and spreading techniques to inoculate 2 blood agar plates Incubate AEROBICALLY in refrigerator for at least 1 week, checking for growth and/or hemolysis 3. Body Surveys For Corynebacterium Serum Tellurite Agar - Throat Survey Each person labels 1 Serum Tellurite plate - throat (have your partner sample, see diagram above) Use long Dacron swab to sample throat and apply to plate as directed Incubate at 37°C until next session - AEROBICALLY I. GRAM (+) COCCI CONTROLS Anything that says ANAEROBIC must be put into the anaerobic chamber together; no mistakes or redo's once chamber is activated! 1. Immune/Defense-Based Tests (Complete All Wednesday!) Controls You Will Be Provided With (Use Liquid Cultures): S. aureus, epidermidis, saprophyticus Coagulase Tube Test Label 3 sterile test tubes - all Staphylococcus controls above Aseptically add 0.5 ml rabbit plasma (which contains prothrombin/fibrinogen target) to each tube Aseptically add 0.5 ml each test culture to the rabbit plasma - DO NOT SHAKE! Incubate in 37°C water observing after 1 hour - you may need to come in within 24 hours to examine When examining, DO NOT SHAKE! If clotting not obvious, gently slant to look for fibers. 2. Staphylococcus ID Testing (Setup Wednesday; BRIEF follow-up Monday) Controls You Will Be Provided With (Use Plate Cultures): S. aureus, epidermidis, saprophyticus Mannitol-Salt Each pair divides 1 Mannitol-Salt plate into thirds; label according to cultures above Using a loop, make a small squiggle of each culture into each respective sector Incubate at 37°C until next session - AEROBICALLY Blood Agar Each pair divides 1 Blood Agar plate into thirds; label according to cultures above Using a loop, make a small squiggle of each culture into each respective sector SET ASIDE UNTIL ALL ANAEROBIC PLATES DONE - LEARN ABOUT ANAEROBIC JARS 3. Streptococcus ID Testing (Setup Wednesday; BRIEF follow-up Monday) Controls You Will Be Provided With (Use Plate Cultures): S. pyogenes, & pneumoniae, Enterococcus Blood Agar Each pair divides 1 Blood Agar plates into thirds; label according to cultures above Using a loop, make a small squiggle of each culture into each respective sector SET ASIDE UNTIL ALL ANAEROBIC PLATES DONE - LEARN ABOUT ANAEROBIC JARS MacConkey Agar Each pair divides 1 MacConkey plates into thirds; label according to cultures above Using a loop, make a small squiggle of each culture into each respective sector Incubate at 37°C until next session - AEROBICALLY Bacitracin Sensitivity Testing Each pair divides 1 Blood Agar plates into thirds; label according to cultures above Using a loop, squiggle each culture into each respective sector - cover as much area as possible Using tweezers, aseptically place a bacitracin disk in the center of each squiggle Tap down disk to secure and incubate at 37°C until next session - AEROBICALLY Optochin testing is performed in a similar manner, also distinguishes different species. Novobiocin testing is also similar, used to distinguish Staphylococcus. III. SPECIAL STAINING Complete ALL Wednesday 1. Bacillus Spore Staining Control You Will Be Provided With (Use Plate Culture): B. cereus Spore Staining Each pair will prepare heat-fixed smears using controls; use provided steam rack/chamber Cover smear with a piece of filter paper (not just a Kimwipe) and flood with carbol fuchsin Place slides on rack in the steam area and fully steam for 7-10 minutes - watching carefully If carbol fuchsin diminishes, add more - but remember adding it cold will add to proper steaming time Remove paper/stain and decolorize with 95% ethanol for a few seconds; rinse with water and dry Counterstain with methylene blue for 1 minute; wash with water and air dry Examine under oil immersion/100X; spores will be red, the rest of the cell blue 2. Mycobacterium Acid Fast Staining Control You Will Be Provided With (Use Slant Culture): M. smegmatis Mycobacterium Acid-Fast Stain - should prepare with mix or acid fast/non-acid fast Each person will prepare a heat-fixed smear using control; use provided steam rack/chamber Cover smear with a piece of filter paper (not just a Kimwipe) and flood with carbol fuchsin Place slides on rack in the steam area and fully steam for 5 minutes - watching carefully If carbol fuchsin diminishes, add more - but remember adding it cold will add to proper steaming time Remove paper/stain and GENTLY wash off excess stain with distilled water Gently flush with Acid Alcohol until all freely removable stain is gone - typically 5-10 seconds Flood with Acid Alcohol, waiting until no more color comes off - between 30 seconds to 2 minutes Gently wash with distilled water, counterstain with methylene blue for 1 minute, gently rinse/dry again Examine under oil immersion/100X; acid-fast organisms will be pink; non-acid fast will be blue Acid alcohol steps vary by thickness of smears so be careful during your smear preparation to make as even a sample as possible; Acid alcohol = 97 ml 95% alcohol plus 3 ml concentrated HCl PAIR IMAGE PORTFOLIO ASSIGNMENT COAGULASE Name agent 1 - Interpretation Name agent 2 - Interpretation Name agent 3 - Interpretation Picture of Tube Picture of Tube Picture of Tube STAPHYLOCOCCUSAND STREPTOCOCCUS BODY SURVEY - EACH PERSON Pair 1 Name - Nose Interpretation Picture of Plate Pair 1 Name - Toes Interpretation Picture of Plate Pair 1 Name - Throat Interpretation Picture of Plate STAPHYLOCOCCUS PLATING EXERICSES - CONTROLS MS Phenotypes - Interpretation Picture of Plate BA Phenotypes - Interpretation Picture of Plate STREPTOCOCCUS PLATING EXERICSES - CONTROLS BA Anaerobic - Interpretation Picture of Plate MacConkey - Interpretation Picture of Plate Bacitracin Testing - Interpretation Picture of Plate GROCERY LISTERIA SURVEY Item 1 - Interpretation Picture of Plate Item 2 - Interpretation Picture of Plate CORYNEBACTERIUM BODY SURVEY Pair 1 Name - Interpretation Picture of Plate Pair 2 Name - Interpretation Picture of Plate MICROSCOPY - SPORE AND ACID-FAST STAINING Spore Stain - Interpretation Digital Image Acid-Fast Stain - Interpretation Digital Image INDIVIDUAL ASSIGNMENT QUESTIONS Each person will type out answers and turn in Word documents - due end of Bacteria Unit. (1) Media/Growth Conditions Review - maximum length 3 pages Use text (physiology and culture detection sections) to explain the purpose of the following, including how they worked and why we chose them for each target microbe/groups of microbes: Mannitol-Salt, Blood Agar (aerobic, anaerobic, refrigerator), Serum Tellurite. It is also a good idea to focus on ingredients and use vocabulary like selective and differential as appropriate. (2) Streptococcus Method Assignment - maximum length 1 page (excluding attachment) Searching either Fisher or VWR Scientific websites, find a commercially available rapid immunological (that means Ab-based) test kit that distinguishes Streptococcus groups. Explain how the kit works, how long it takes, what antigen (Ag) it detects, and the cost per sample. Please attach a copy of the product description to your report. (3) Staphylococcus Research Article Summary - maximum length 2 pages (excluding attachment) Choose any Staphylococcus disease relevant to lab or text chapters - whatever is interesting to you. Searching either J. of Clinical Microbiology or Infection and Immunity, locate a primary research paper about this genus. Summarize the goals, methods, and findings of this article. Please attach a copy of the entire article to your report. (4) Listeria MMWR Question - maximum length 0.5 page (excluding attachment) Use the CDC/MMWR website to find a CURRENT (within 1 YEAR) epidemiology report about Listeria outbreaks in the US. Briefly summarize the data, emphasizing the reservoirs of infection and general characteristics of the victims. Please attach a copy of the entire article to your report. (5) Mycobacterium tuberculosis EID Question - maximum length 1 page (excluding attachment) Use the CDC/EID website to find a CURRENT (within 1 YEAR) epidemiology article about Mycobacterium tuberculosis outside the US. Briefly summarize, emphasizing features of the outbreak (where, when, morbidity/mortality), how patients were identified and diagnosed, and specific factors that seemed unique to this country. Please attach a copy of the entire article to your report. (6) B. anthracis Method Assignment - maximum length 0.5 page (excluding attachment) Searching anywhere on-line, find a commercially available rapid test that identifies anthrax in samples in situ (i.e. something portable that you can take into the field and use), preferably something developed in the aftermath of the post-911 scare. Briefly summarize how this product works. Please attach a copy of the product description to your report.