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Steps of microbial Detection Techniques:
1. Staining - Gram stain, Acid-fast stain, lactophenol cotton blue, Trichrome stain, India ink.
2. Culture and susceptibilities
T/F: Gram stain is used for bacteria only and is the first step in identifying pathogens, which is why it's used to guide antibiotic choice.
T/F: With regard to gram stains: Cell walls either take up the stain (Positive = pink/red), or doesn't (Negative = purple/blue).
- Positive (Thick cell wall, no outer membrane) = purple/blue
(Thin cell wall w/outer membrane) = pink/red
Describe the steps in gram staining.
- 1. Fixation
- 2. Crystal Violet (primary stain)
- 3. Iodine treatment (trapping agent)
- 4. Decolorization (with alcohol or acetone)
- 5. Counterstaining (with safranin)
T/F: Gram stain can be perform only on certain types body fluid or tissue.
- It can be performed on ANY body fluid or tissue biopsy, but certain ones are more common than others.
Gram staining is most commonly performed on what types of body fluid or tissue biopsy?
- Sputum: Mucus, not spit
- Blood: Requires at least 30ml
- Wound: Swab
- Urine: Clean catch is the better than catheterization
- Tissue biopsy: Lung, liver, kidney, bladder
- Sterile body fluids: Pleural fluids, peritoneal fluid, CSF, BAL (bronchial alveolar lavage) or BW (bronchial wash)
What types of information can you obtain from a gram stain?
- Cell wall form (pos or neg)
- Quantity of bacteria
- Other cells in sample (WBC, RBC, eosinophils, inclusion bodies, casts)
How do you determine the success of a gram stain?
Based on quality of sample, so make sure it's a good sample.
- 1. Minimize contamination of the culture from the site. (clean skin before obtaining blood sample, use "clean catch" to limit the amount of bacteria from shit and vag)
- 2. Use the right tube. (Different tubes uses different techniques for specific organisms)
- 3. Know what you're looking for. (Providing the lab with names of organisms would help them prepare or store culture media, because of different growing time/conditions)
Porins (channels across membranes for diffusion) are only found in what type of bacteria?
Why would you want to use an acid-fast stain?
- Because some bacteria is resistant to traditional gram staining. They have a thin layer of peptidoglycan and large amount of my colic acid.
- Largely used for detecting "Mycobacterium" and "Nocardia."
T/F: Acid-fast staining makes acid-fast bacteria resistant to decolorization, retaining the carbol fuchsin (red), while all other cells will decolorized (those cells will be stained with methylene blue)
With regard to acid-fast staining, detection of mycobacteria in sputum requires:
- Only about 5,000 to 10,000 organisms/mL
- Mycobacteria are often present in lower levels = LOW SENSITIVITY
What is lactophenol cotton blue used for? And describe how it works.
- Used to identify fungi.
- The high concentration of PHENOL deactivates lytic cellular enzymes so cells won't lyse.
- An acid dye, COTTON BLUE, is then used to the chitin in cell walls for fungi.
Trichrome stain is also know as what…and why would you use it?
- AKA: Gomori-Wheatley Stain
- Used to detect (MICROSPORIDIA and INTESTINAL PROTOZOA).
What is a disadvantage of trichrome staining?
May miss HELMINTH eggs and larvae and is not reliable for detecting CRYPTOsporidium.
Iron hematoxylin staining is used to stain what? Disadvantage?
- Stains cells, cell inclusions, and nuclei.
- HELMINTH eggs may stain TOO DARK to identify.
India ink and also known as what? And why would you use it?
- AKA: Colloidal Carbon
- Use do detect encapsulated fungi (cryptococcal) such as Cryptococcus neoformans.
- CSF often tested.
- Not as sensitive as cryptococcal antigen!
Organisms being visible as a "halo" is associated with what kind of staining?
India Ink (Colloidal Carbon)
Culture identification steps:
1. Inoculation in wide variety of media types (biochemical reagents and nutritional supp).
2. Incubation or growth phase
3. Interpretation of results (growth? colony type? color change? oxidase, catalase, fermenter?)
List and describe the different types of culture media (agar)
- Non-selective media: No inhibitory substance and supports growth.
- Selective media: Contains inhibitory substances or antimicrobials which suppresses some organisms and allows others to grow.
- Differential media: Contains substances which allow detection of organism characteristics. (Used for detection…not to be confused with "selective" media, which actually suppresses)
- Broth media: Used to propagate very small numbers to organisms or hard to grow pathogens. (High risk of growing contaminants)
Identification of gram-neg rods on MacConkey agar is used in what?
"Old school" culture identification
Describe the "current school" culture identification.
- STEP 3 ALTERNATIVE PROCESS: automated processor (Vitek, Microscan, Pheonix)
- Bacteria is added to specific automated "cards" (for gram-neg and gram-pos) with different antibiotics.
- Identification and sensitivity results in 4 hours (Just for step 3. All 3 steps still takes about 3 days, so first best guess is important).
What are the 3 types of susceptibility testing methods?
- Kirby-Bauer Disk Diffusion
- Microbroth Dilution
- (Not necessary to do all 3, just choose one)
Describe the steps of the Kirby-Bauer disk diffusion and how to interpret the results.
- 1. Cover agar with isolated organism
- 2. Drop antibiotic disks on to agar
- 3. Incubation and growth (24-48 hours)
- 4. Measure zone of inhibition (diameter of clear area around disk in mm)
- 5. Interpretation of results: Sensitive (larger zone), immediate, resistant (smaller zone)
- Don't use zone to compare different antibiotics ON PLATE. Can't say one antibiotic is better than the other. (Need BREAKPOINTS or PD to interpret results)
Describe the Microbroth Dilution and how to interpret the results.
- Organisms are tested against 2-fold serial dilutions to determine MIC.
- Several wells are filled with the SAME CONCENTRATION of organism, then increasing concentration of antibiotics are added.
- Well incubated for 18-24 hours.
- The lowest concentrating with no visible growth is the MIC.
Describe the Epsilometer (E) test and how to analyze the results.
- 1. Agar is inoculated
- 2. Antibiotic impregnated strip is placed on agar.
- 3. Incubation and growth.
- 4. Analyze the zone of inhibition vs. number (MIC) on E test.
- DO NOT measure the zone diameter, that's only for the Kirby-Bauer.
- For the E-Test you look for the MIC (it's the lowest concentration on the strip that lies within the zone of inhibition).
What are some limitations for all susceptibility testing.
- Test do not account for patient-specific factors. (immune system, inoculum size, or site of infection)
- Not all resistant mechanisms can be detected.
- No test is 100% accurate.
T/F: An MIC represents the drug's potency.
- It only represents the RELATIONSHIP between ONE BUG and ONE DRUG!
- MIC alone can't tell you drug potency. (Need to compare MIC with PD for Potency)
- MIC alone can't tell you pathogen's virulence/sensitivity. (Need to compare it with BREAKPOINTS for that)
What would you use to interpret the following breakpoints, and what do they mean: ≥ 21, 16-20, ≤ 15 mm.
- Use Kirby-Bauer because it's in millimeters, not concentration (which is MIC, used in E-test and Microbroth dilution)
- Equal to 21 mm or above would be susceptible (bigger zone = more bacteria killed)
- 16-20 mm is intermediate
- Equal to or less than 15 mm would be resistant.
Who determines breakpoints?
- FDA: Susceptibility break points (Just S, not I or R) must be determined prior to FDA approval). Not as complete as CSLI.
- CLSI(Clinical Laboratory Standards Institute): Non-gov agency that is more "clinical" or forward thinking than FDA.
How are breakpoints determined?
- Clinical, pharmacological, microbiological, and pharmacodynamic consideration are involved.
- AUC and Cmax are used as guidelines.
The relationship between a pathogen's MIC and an antibiotic is called what?
- Pharmacodynamics (PD)
- Just remember: MIC REPRESENTS the relationship one bug and one drug (given in mcg/mL).
- PD DESCRIBES the relationship between that bug and the drug (That's how you determine POTENCY, which leads to BREAKPOINTS)
What describes the relationship between the drug and the body?
Define synergy in terms of "logs."
- Combo kills increased > 2 logs above the MORE ACTIVE SINGLE agent.
- Or > 2 log decrease in cfu/mL with combo, compared to MORE ACTIVE SINGLE agent.
- (Look at graph…drops more than 2 logs)
Define synergy in terms of "MIC."
Combo results in ≥ 4 dilutions lower than EITHER single agents.
Explain Cidal vs Static.
- The difference is the speed at which it kills.
- Cidal: > 3 log kill in 24 hours (or 72 depending on study methodology)
- Static: < 3 log kill in 24 hours (or 72 depending on study methodology)
T/F: You can determine "cidal" if > 3 log kill in 6 hours.
- Because that's within 24 hours.
T/F: You can determine "static" if < 3 log kill in 6 hours.
- Because it still might reach > 3 before 24 hours.
T/F: You can determine if something is "cidal" or "static" by it's MOA.
- But generally speaking, CW inhibitors "TEND" to be "cidal" and all others "TEND" to be "static."
- Exceptions: Amino-glycosides are "cidal" against gram-neg bacteria.
- Exceptions: All current antibiotics are "static" against Enterococcus sp. (can make "cidal" regimen with combo therapy of B-lactam + AG or Vanco + AG)
T/F: Cidal antibiotics are associated with better outcomes than static antibiotics.