Download Medical Microbiology

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 are…
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.