Review: Effectiveness of Hand Scrubbing



Exercise 27, Urinary Tract Pathogens

1. There are bacteria normally found in urine:

Coagulase-negative Staphylococci, Diptheriods, Enterococci, Proteus,

Hemolytic Streptococci, Yeasts.

2. There are pathogens commonly causing Urinary Tract Infections:

1. Members of the Family, ___________________________, ex. E. coli.

2. Pseudomonas aeruginosa

3. Staphylococcus aureus

3. Steps of Urine Culture

1. __________________________. Clean catch, catheter, baby bag.

• Clean catch: __________________________________ and area cleaned with towelette.

2. ____________________________ or _________________________, because after 2 hours at room temperature the lab rejects the urine. In 2 hours, bacteria can reproduce several times, leading to a false positive. For example, the generation time for _________________________ is about 40 minutes.

3. ______________________________________. This tells you which antibiotics may be good choices to start. Also tells you the shape of the bacteria.

4. Perform _________________________________, and determine if an infection is present.

• An infection is greater than or equal to _____________________ CFUs/mL.

• The loop is calibrated to be 0.001 ml, so a dilution calculation will be made when the plate count is performed.

5. __________________________________________, because bacteria exist in mixed populations.

6. __________________________________________, to know which is the problem organism.

7. Determine ____________________________________________, to be sure the medication will cure the infection.

4. Case study: Baby born with urinary tract reflux to kidneys:

• Page 1. Pediatrician has capability of determining whether a UTI is likely. Blood found, high protein, positive nitrate, moderate leukocytes indicate UTI. Sent out for C & S (culture & sensitivity).

• Page 2. Partial (immediate analysis) results show “GRAM NEGATIVE BACILLI ISOLATED”, greater than 100,000 CFUs/ mL.

• Page 3. FINAL results tell identification of pathogen as Pseudomonas aeruginosa, which was sensitive to all antibiotics tested.

• Page 4. FINAL of UTI #2 was Enterococcus sp. which was resistant to ciprofloxacin, but sensitive to other antibiotics.

• Note that a different set of antibiotics were tested when the UTI was caused by a gram + than a gram – organism.

5. ______________________________ are antibiotics that work against several types of bacteria (i.e. gram + and gram -). Can kill normal flora.

______________________________ kill specific bacteria.

6. Why does the doctor need a list of several antibiotics to choose from?

1.

2.

3.

Exercise 28, Assessing Toilet Paper as a Barrier Against Microbes

1. Our goal is to determine:

1.) If toilet paper protects hands from pathogens, and

2) If typical hand washing is sufficient to clean the pathogen off of our hands.

2. Why do we use E. coli?

3. MacConkey plates: used primarily for the detection of ____________________ bacteria. It is selective and differential.

Review: Assessing Toilet Paper as a Barrier Against Microbes

1. Did toilet paper protect fingers from contamination?

2. Was washing hands effective?

Review: Urinary Tract Pathogens

1. STEPS OF URINE CULTURE

1.

2.

3.

4.

5.

6.

7.

2. If a loopful of sample is 0.001mL, and yields 8 colonies, how many CFUs/ mL is in the original sample?

___8 CFUs___ = ___X___

0.001mL 1mL

0.001X = 80 (1)

X = __8__ = 8,000 CFUs/ mL

0.001

Exercise 26: Enterobacteriaceae ID - The Enterotube

1. The Enterotube

• All “Fake Urines” are members of the Family Enterobactriaceae.

1 – Escherichia coli

2 – Enterobacter aerogenes

3 – Proteus mirabilis

4 – Citrobacter freundii

5 – Klebsiella pneumonia

6 – Enterobacter cloacae

• If the pathogen was NOT an Enterobacteriacae, a different tube would be used.

2. The big picture for Enterotube identification:

1.

2.

3.

4.

5. ____________________________________: if there are several organisms listed for a specific 5-digit number, the book will give extra tests to perform to determine which is the organism is your pathogen.

Exercise 22: Kirby Bauer Test for Antibiotic Sensitivity

1. The agar we will use is the _________________________________. The oatmeal base does not interfere with antibiotics (chemically).

2. You will know the __________________________________, and the disks will tell

you the _______________________________________ you are looking at.

You need both pieces of information to use the Table in Lab Manual.

3. Definitions:

__________________________ (S) = The bacteria is sensitive to the antibiotic.

__________________________ (I) = Intermediate. The bacteria has some resistance, and this antibiotic is not the best choice.

__________________________ (R) = The bacteria is resistant, and this antibiotic will not cure the infection.

MIC, ___________________________________________, the lowest concentration of the antibiotic that will be effective against the pathogen.

• The Kirby Bauer does not give us this information. Clinical labs would use a different testing method using machines that would determine MIC based on color.

4. Main concepts:

This Week Next Week (Tomorrow)

• For a given antibiotic, the zone of inhibition (diameter in mm) varies with the specific bacteria:

Look at penicillin in The Zones of Inhibition in the Kirby Bauer Method of Antimicrobic Sensitivity Testing.

Compare the Z of I for sensitive for Enterococcus sp. (15mm) & that for N. gonorrhoeae (47mm).

• For a specific organism, the Z of I differ for different antibiotics:

For N. gonorrhoeae sensitivity, penicillin is 47mm, but spectinomycin is 18mm.

• Mueller-Hinton II is the standard agar for this test. The oatmeal base does not chemically interfere with the antibiotic.

• If a bacterial colony grows within the zone of inhibition, that specific colony must be taken and growth in broth culture and re-tested. Otherwise, we consider that colony resistant, and that makes this antibiotic not the best choice.

• For a given antibiotic, if an organism is not listed in the Lab Manual Table, then the result is reported “Cannot be determined”.

• Enterobacteriaceae is a FAMILY of bacteria, so there are many that fall in this group, such as all of the 6 unknown urines, including E. coli. On the test, I would tell you, “…which is a member of Enterobacteriaceae”.

Results: Enterotube

See Enterotube Review and Practice Power Point in TEST 3 Folder.

Results: Kirby Bauer

See Kirby Bauer Antibiotic Sensitivity Worksheet.

Exercise 31: Snyder Caries Susceptibility Test

1. _____________________________________ is tooth decay.

2. The overall reaction:

____________ + bacteria (Streptococcus mutans) = __________________ = tooth decay.

3. The Snyder agar contains two unique components:

1. _____________________________________

2. _____________________________________

4. Tooth decay happens in __________________________ mouths, pH 5.5 to pH 4.4.

Exercise 20: The Lysozyme Test

1. __________________________ are antibacterial proteins.

They degrade _____________________________, which weakens the cell wall&

causes bacteria cells to rupture.

2. Lysozymes are found in most human body fluids, such as _______________________,

_______________________ and _______________________. They are also found in

chicken _______________________.

3. In Gram ____________________ bacteria, the protective outer membrane makes these

bacteria cells resistant to lysozymes.

4. Gram __________________ bacteria are more susceptible to lysozymes, unless there

is a high amount of ________________________ acid.

*Teichoic acid limits the effect of lysozymes, so gram positive cells with teichoic acid are resistant to lysozymes.

Review Lysozyme Test

|Bacteria |Zone of Inhibition |Result |

| | | |

|Micrococcus luteus |Egg | |

|Gram + | | |

|No teichoic acid |Spit | |

| | | |

|Staphylococcus aureus |Egg | |

|Gram + | | |

|w/ teichoic acid |Spit | |

| | | |

|E. coli |Egg | |

|Gram – | | |

|Outer membrane |Spit | |

+ = Zone of inhibition present

- = No zone of inhibition

Staph and Strep Main concepts

1. Hemolysis Pattern (Hemo- blood, lysis- break) in blood agar

• Beta hemolysis- Complete breaking of RBC (red blood cells).

Agar looks clear around bacterial growth.

• Alpha hemolysis- Partial breaking of RBC.

Agar looks cloudy around bacterial growth. Often it is green or

brownish.

• Gamma hemolysis- no breaking of RBC.

There is no hemolytic pattern around growth.

2. Serological Tests

• Serological Test- The use of immunological tests to diagnose and treat disease or identify antibodies or antigens. The Agglutination Test is a type of serological test.

• Antibody- A protein molecule secreted by B plasma cells which bind to specific antigens.

• Antigen- A molecule (i.e. protein or sugar) that triggers and immune response (i.e Protein A in Staph aureus, Antigen A in Strep pyogenes).

• Agglutination Test - The clumping of bacteria cells in the presence of an antibody.

3. Agglutination

Staphylococcus sp., Exercises 30 and 34

1. General characteristics of Staphylococci

• Gram positive, cocci

• Pyogenic (puss forming)

• Form clusters of cells, like grapes

2. Staphylococcus aureus

• Hemolysis patterns & Agglutination (above) & Coagulase (below) concepts apply.

• MRSA- resistant to many antibiotics.

• Opportunistic pathogen found on skin.

• Stages of pathogenesis: 1) colonization, 2) local infection, 3) systemic infection, 4) metastasis, 5) toxinosis.

3. Coagulase Test for Staph aureus

• Coagulase – an enzyme which causes plasma* portion of blood to clot. The jelly like substance coats the bacteria cell so the body’s immune system doesn’t “see”it.

• Only Staph aureus has the coagulase enzyme.

• A positive coagulase test is a definitive test for Staph aureus. It is reported as Staph aureus.

• A negative coagulase test is reported as non-Staph aureus. It could be Staph epidermidis or Staph saphrophyticus or a non-staphylococcus bacteria.

• *Blood plasma is the liquid portion of blood with no red or white blood cells.

Streptococci sp., Exercise 32 & Appendix E

1. Characteristics of Streptococci

• Gram positive, cocci

• Pyogenic (puss forming)

• Forms chains of cells

• Hemolysis pattern, Agglutination concepts apply.

2. Classification of Streptococci is based on:

1) Hemolytic pattern AND

2) Serological techniques.

• Beta hemolytic, Alpha hemolytic, and Gamma hemolytic patterns.

• Serological Groups A, B, C, and D. These are antigens A, B, C and D.

o Group A : Strep pyogenes –which is responsible for 25% of upper respiratory infections.

o Group B: Strep agalactiae –frequently found in milk (mastitis in cows) & 3rd trimester women vaginal cultures.

o Group C: we don’t need to know any specific examples

o Group D Strep: Strep bovis –found in the intestinal tract of cows, humans. Cause UTIs, among other illness.

o Group D Enterococcus: Enterococcus faecalis, Enterococcus faecium –These are resistant to penicillin. They are the “E” in VRE. Cause UTIs among other illness.

o Viridans Group: Strep mutans –dental caries.

o Non-Grouped (no reactive antigens on membrane): Strep pneumoniae –frequent cause of bacterial pneumonia with high death rate in elderly.

Information found in Appendix E

See Staph and Strep Summary Table Handout (also in TEST 3 Folder).

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Resistant bacteria colony

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