Garlanda 1 Flashcards
How can labs id organisms
directly (microscopically, culture, genome)
indirectly (finding antibodies to that organism)
What are the 5 types of lab tests
microscopy culture immunologic tests nucleic acid based ID non nuclei acid based ID
The 5 I’s of sample processing
inoculation incubation isolation inspection identification
What word is most important in inoculation
medium
define culture
the visible growth of the microbe in or on the medium
what is the gold standard for ID
culture
in the isolation stage what are the microbes in
colonies on solid media, or turbidity (free floating cells) in broths. Note subculturing occurs here
How do non nucleic acid tests (NNAT) work
they use phenotypic (functional or morphologic) characteristics
tests are done in what order
sequentially, which the results of the last one helping guide the next one
for lesions where should you sample
the leading edge
fill in the collection method for the following locations skin, membrane blood CSF stomach urine lungs diseased tissue
sterile swab (NYLON) needle aspiration from vein needle aspiration from subarachnoid space intubation catheter with midstream urine sputum surgical removal biopsy
contamination of urine sample
all non surgical samples become contaminated with urogenital flora during collection. these bacteria witll replicate if the specimen is not quickly stored in a preservation tube at 4C
when should you collect samples for blood parasite infections
during a febrile episode or every 6 hours for a 24 hour period
Quantity of CSF culture needed
2 ml from tubes 2, 3, or 4
For anaerobic culture what are 3 considerations
- you should not obtain sample from areas where normal flora is present
- fluid specimens (abscess) are superior to swab specimens
- transport fluid in a syringe or an anaerobic transport vial
- are best collected with metal (needle aspiration or scapel)
T/F a large piece of tissue (5-10 mm) willl protect anareobes in the center
T
How to collect chlamydia/N. gonorrhea culture
chlamydia in universal transport medium, GC culture in amies and charcoal or tranport to lab for immediate plating
is culture an option for tissue sent in formalin
no
is bacterial culture possible in sent in viral transport media
no, most VTM contain antibiotics
when to test for microbiology
before giving antibiotics
Levels of biosafety and examples
BSL 1: non pathogenic for healthy ppl. Use soap/disinfectants E.g: non pathogenic E.coli
BSL 2: moderately hazardous. Use safety cabinets. E.g MRSA, heptatitis
BSL 3: use cabinets, rooms with HEPA filters, double doors. E.g. TB, B antracis, yellow fever, SARS/COV2
BSL 4: fatal dx E.g. ebola, smallpox, lassa fever viruses. Use separate building, airlocks, showers, vacuum. Filter air and water
HEPA (high efficiency particulate air) filters can be placed on
safety cabinets, operating rooms, rooms of infected or immunocompromised pts
When do you need microscopic examination of tissue in a culture
to distinguish invasive disease from surface colonisation
Visibility of fungi can be improved by
applying 10% KOH to dissolve surrounding skin tissue and non fungal organisms so that you only have the fungus left, and its easier to observe
what volume is needed for microscopic detection of microbe
1 x 10^4-5/mL (this is why most specimens are concentrated by centrifugation before examination
Three kinds of microscopy
- bright field
- dark field (high contrast, for colorless cells, and pale objects)
- phase microscopy: for living cells, is high contrast
this includes differential interference contrast microscope (aka nomarski): for 3D look
Dyes used as stains are usually
salts
chromophore is
the colored portion of the dyes
acidic dyes stain:
basic dyes stain:
which is more common
alkaline structures
acidic structures
basic dyes are more common since most cells are negatively charged
examples of simple stains
crystal violet, safranin, methylene blue
examples of differential stains
gram stain, acid fast stain, endospore stain, histological stain (gomori methenamine silver for fungi, and haematoxylins and eosin)
acid fast stain work on
mycobacteria and nocardia, cryptosporidium (waxy lipids in the cell wall)
THIS NEEDS HEAT ITS V IMPORTANT TO GET THE CARBOFULSHIN DYE INTO THE ACID FAST CELLS
then you use HCL to remove excess carbofulshin
and dye the remaining cells with methylene blue
india ink (colloidal carbon stain) is for
cryptococcus neofromans and encapsulated fungi, here the background is stained
silver stains are used for
spirochetes, H pyloris, microsporidia, bartonella henselae (Cat scratch disease)
wright stain and giemsa stain are for
parasites and intracellular organisms
trichrome stain (gomori wheatley stain) and iron hematoxylin stain are for
intestinal protozoa.
Gomori is for microsporidia (can miss helminth eggs and larvae, and not great for cryptosporidium)
Iron stains cell inclusions and nuclei. Helminth eggs may stain too dark to permit ID
endospore stain (schaeffer-fulton stain) is for
clostridum and bacillus (the spore wall will have the green dye (circles))
eg of special stains
- negative stain: for capsule, acidic dyes are replused by negative charges on the capsule
- flagellar stain: important to ID species
T/F fluorescent stains allow detection at lower concentration
true (<1 x 10 ^4 cells/ml)
E.g. of fluorescent stains
- Acridine orange (for bacteria or fungi): binds to nucleic acids. At low ph 3.5-4 they stain bright orange
- Auramine-rhodamine and Auramine 0 for mycobacteria
- Calcofluor white for fungi and dermatophytes
Define direct or indirect immunofluoresence
coupling a fluorescent dye to an antibody directed at a pathogen.
After the dye is covalently linked to an antibody, the dye antibody binds to the antibodies target making the target visible under the light
examples of general purpose media for culture
blood agar (for culture of fastidious organisms and to differentiate hemolytic organisms), chocolate agar
streak plates use a
sequential pattern of streaks
what is nutrient broth
beef extract and peptones in water
what is nutrient agar
agar 1.5%, complex polysaccharide from cell wall of red algae
defined or synthetic media are
Is medium where all chemicals used are known, no yeast, plant or animal tissue is present
For Autotrophs: Inorganic salts and a source of CO2
For Chemoheterotrophs: glucose,
amino acids, vitamins
(Fastidious organisms)
complex media is
Undefined composition. From partial digestion of different protein
sources. E.g. nutrient broth, Trypticase soy agar, MacConkey agar….
Addition of blood: enriched medium.
selective media is
Media that allows the growth of certain organisms while inhibiting the growth of others
Eosin, methylene blue, crystal violet dyes, bile salts, to inhibit Gram +.
- NaCl for salt tolerant: S. aureus.
- Sabouraud dextrose agar, low pH, for fungi (bacteria inhibited).
- Enrichment culture, or cold enrichment for V. cholerae.
differential media is
one component is used in a differential manner by diverse
microorganisms.
- BLOOD AGAR: Streptococcus pneumoniae partial digests blood: alpha-hemolysis;
Streptococcus pyogenes completely digests: beta-hemolysis; Enterococcus foecalis
does not digest: gamma-hemolysis.
- PH-SENSITIVE DYE (red to yellow when ph decreases, when specific carbohydrate is metabolised, and gas could be produced)
macConkey agar
Is for the culture and differentiation of enteric bacteria based on the ability to ferment lactose
(is for gram - bacteria that ferment lactose)
IT IS DIFFERENTIAL MEDIA
S aureus will not grow
E coli will grow (gram neg)
Salmonella enterica will grow yellow
anaerobic media is for
obligate anaerobes.
Reducing media: Sodium thyglicolate combines with free O2.
Use of anaerobic culture systems
and anaerobic glove box
how to do carbohydrate utilization test, and hydrogen sulfide test
slide 59
- H2S: tests for sulfur reducing compunds
- CO2: tests for ability to ferment a carb, and produce gas
chromogenic culture is
Culture medium used to isolate, identify, and differentiate specific
microorganism from a heterogeneous population. The medium contains
chromogenic substrate which is utilized by the microorganism to give colored
colonies that is specific for each microorganism. Depending on the color of the
result, the presence or absence of target organism is determined and also
accurately differentiated from others.
Classical culture media are based on the principle of change in color of the pH
indicator, whereas Chromogenic media are based on enzymatic utilization of
chromogenic substrates.
Principle of Chromogenic Media
Chromogenic media contains soluble colorless molecules called
chromogens. Chromogens are composed of two parts: a substrate ( which is the
target of specific enzymatic activity of the microorganism) and a chromophore.
Due to reduced solubility, chromophore forms a precipitate that imparts unique
color to the colony. Depending on the purpose chromogenic media may also
contain inhibitors
