What are concentration techniques?
-parasite diagnosis from feces, vomit, sputum
1. Flotation
2. Sedimentation
3. Baermann
Describe fecal floats.
-parasites inhabiting GI tract, liver, bile duct
-diff in SG between parasites eggs, larvae, cysts, oocysts
-McMaster egg counting slide = determine egg type & level of infection from eggs per gram of feces
Describe the Baermann technique.
-extract nematode larvae
-warm water stimulates larvae to move out & gravity pulls them to bottom (+6 hrs)
Describe H&E.
-common tissue stain
-viral, bacteria, fungal, parasitic infection
-hematoxylin (basic) = stains acidic or neg charged [ig. Nuclei, chromatin) PURPLE
-eosin (acidic) = stains basic or pos charged (ig. Cytoplasmic & granules) & extracellular components (ig. Muscle, RBCs) RED/PINK
Describe viral inclusion bodies.
-H&E
-nuclear or cytoplasmic aggregates made of proteins = site of viral replication
-virus cause change in cell structure
Describe the romanowsky stain.
-histological staining
-rapid
-cytoplasmic detail
-modified wright-giemsa stain = differential used to study adherence of pathogenic bacteria to cells (ex. Blood cells, chromosomes)
STEPS:
Fixative (green in methanol) -> eosin G -> methylene blue ->
Describe gram-stain.
-differentiate between gram pos & gram neg bacteria
-based on peptidoglycan (not mycoplasma bc doesn’t have one)
-gram pos = purple
-gram neg = red/pink
STEPS:
Crystal violet -> iodine -> 95% ethyl alcohol -> safranin
Describe the different stains for bacterial structures.
- Acid fast = impenetrable cell wall (ex. Mycobacterium, cryptospordium)
- Capsule stain = neg staining, contrast translucent dark background using nigrosin or Congo red with stained cells but unstained capsule (ex. Bacillus anthracis, klebsiella pneumonia, clostridium spp)
- Endoscope = heating malachite green
- Flagella = thickened with mordant
Describe fungal & parasite stains.
- Lactophenol cotton blue = stains chitin in cell wall of fungi
- Gomori metheanime silver = dark brown stain of fungal cell wall (surrounding tissue is green)
- Periodic acid schiff = mucin stain
- Wheatleys trichome stain = detect Protozoa
Describe culture methods.
-bacteria & fungi
-viruses use cell cultures
Describe the pros & cons of culturing.
PROS
- ID
- AMR
- ability of microbe to cause disease
- microbial characteristics & genetics
CONS
- time consuming
- expensive
- unculturable
Describe fastidious bacteria.
-require specific nutrients & culture conditions
Ex. Temp (20-42c), pH, atmosphere, C & N, phos, sulfate, K, Mg, Ca, Fe, trace elements, vitamins, purine/pyramidine
What are the basic nutrient media?
- TSA
- LB
- MH
What are the enriched nutrient media?
- BA
- BH
- Chocolate
- Lysed-blood
Describe selective nutrient media types.
- Gram pos = PEA
- Fungi = SDA
- Gram neg = EMB
Describe differential media types.
- Blood agar = hemolysis
- MAC = gram neg, lactose ferm (pink)
- MSA = gram pos, mannitol ferm (yellow)
- CLED (urinary bacteria) = proteus, klebsiella lactose ferm
What are the different biochemical tests for bacteria ID?
- Enzyme production
-catalase = breaks down H2O2
-coagulase = cause fibrin in blood to clot
-urease = hydrolyses urea
-tryptophanase = ability to convert tryptophane to indole - Carbon source
- Carbohydrate ferm
Describe UTI culture paddles.
-semi quantitative colony count
-presumptive ID of common uropathogens
-37c
-18 to 24 hrs
-2 sides: EMB (gram neg) & non selective CLED
RESULTS
-colony density = degree of infection
-growth on EMB = gram neg
-color on CLED = lactose ferm (yellow)
Describe flexicult vet urine test.
-semi quantitative
-presumptive ID
-antibiotic susceptibility
-30 min of urine collection & 37c for 18-24hrs
-10^3 pathogen/ml = UTI infection
-no growth = bacteria susceptible to antibiotic
Describe onsite analysis at your practice.
-gross
-microscopic
-point of care tests
>urine paddles (UTI infections)
>direct parasite ID
>snap test
>agglutination test
Describe diagnostic lab tests.
-specimen culture
-antimicrobial susceptibility testing
-advanced immunochemical testing (ex. Immunofluorescence)
-molecular diagnostics
How to choose a good diagnostic lab?
-guidance for optimal specimen management
-immunochemical & molecular methods
-antimicrobial susceptibility testing
-quality assurance
-skills
-accredited
-standardization
Lactophenol cotton blue
Gomori methanamine silver
