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
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)
Use the right tube. (Different tubes uses different techniques for specific organisms)
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
(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)