Week 1: Specimens/Processing/Stains/ID Methods Flashcards
List types of wet mounts
- Direct wet mount
- Hanging-drop wet mount
- KOH wet mount
Must use sterile slide and supplies but Darrell said…
We don’t actually use sterile slides :P
How do you smear prep swab specimens?
- Use separate swab to prep slide from swab used to inoculate plates…or if use one swab do smear last
- Do rolling technique to get all swab surfaces on slide
How do you smear prep thick liquid or semi-solid specimens?
- Immerse swab in specimen for several seconds and then roll onto slide
- Feces
How do you smear prep thick, granular, or mucoid specimens?
Prepare same as peripheral blood smear because want thick and thin areas
How do you smear prep thin fluid specimens?
- Cytocentrifuge sterile body fluids if available
- Drop on slide but DO NOT spread
- CSF, urine…etc
- Draw circle around specimen on reverse side of slide
How do you smear prep a bacterial colony from growth on media?
- Touch top portion of isolated colony with sterile loop and suspend in small saline/water drop on slide
- Best if young (less than 24 hrs)
- Broth smears improve morphology
Methods to fix slides?
- Methanol
- Heat fix at 56°C for several minutes
What is the procedure for the most common stain?
Gram stain
- Fix
- Crystal violet
- Iodine
- Decolorization
- Safranin counter-stain
What color is Gram variable bacteria?
Both pink and purple
Causes of gram variable appearance?
Antibiotic therapy or old age can compromise cell wall integrity such that it decolorizes easily
Which magnification used for Gram stain?
100x
List factors that affect gram reaction, morphology, and interpretation
- Smear too thick/thin
- Too much heat fixing
- Decolorization done wrong
- Antibiotics
- Liquid versus solid media
- Too young/old colony
- Microbes have autolytic enzyme system
- Precipitated stain
- Mucus and other protein material present
- Counterstained too long or not enough time
What are two acid-fast stain methods? Describe each
Ziehl-Neelsen” carbolfuchin stain requires heat to enter cell
Kinyoun: carbolfuchin stain enters cell due to high phenol concentration in stain
Counterstain for acid-fast stain? How to interpret results?
Methylene blue or malachite green
Positive = red
Negative = blue or green
Describe 3 fluorochrome stains (enhance bacterial detection when small number of microbes, much debris, many WBCs)
-
Acridine orange: binds nucleic acids. Bacteria/yeast = red/orange while tissue cells = yellowish-green or black
Auramine-Rhodamine: for acid fast bacilli/tuberculosis specimens
Calcofluor white: binds cell wall chitin of fungi
How do you prepare specimens for culture setup?
- Concentration: centrifuge or filter
- Homogenization: grind using mortar and pestle/tissue grinder
- Decontamination: Destroy normal flora with reagents in order to isolate mycobacteria
List types of media
- Nutrient (BHI, blood, chocolate, soy)
- Differential (hemolysis on blood agar)
- Selective (MAC, CNA)
- Enrichment broth (thioglycolate, LIM, GN)
T/F: Darrell thinks it’s okay to use cotton swabs to inoculate media
FALSE. He said that cotton inhibits some organisms so don’t use!
What are the urine loop volume sizes and what dilution factors are they when quantitating colonies?
- 0.01ml is 1:100
- 0.001 ml is 1:1000
What are capnophiles?
Require 5-10% CO2
Incubator conditions for ambient air and CO2 incubators?
- Ambient: 35°C, high humidity
- CO2: 5-10% CO2 + 35°C room air
Incubator conditions for microaerophiles?
85% N2, 10% CO2, and 5% O2
Incubator conditions for anaerobes?
5% H2, 5-10% CO2, 85-90% N2, 35°C, high humidity
What temperatures do the following grow at: psychrophiles, mesophiles, thermophiles
Psychrophiles: 4-20°C
Mesophiles: 30-45°C
Thermophiles: 50 to greater than 200°C
Most human pathogens grow at 35-37°C
Length of incubation?
- Routine cultures grown 48-72 hrs (2-3 days)
- Anaerobic broths held for 5-7 days
- Mtb held up to 8 wks before deemed “no growth”
Criteria for organism ID?
- Macroscopic colony morphology
- Environmental growth requirements
- Microscopic morphology + staining traits
- Nutritional requirements + metabolism
- Antibiotic resistance/susceptibility
Blood agar (BA/BAP) media
Supports growth of most organisms excluding fastidious ones. Enriched and non-selective
Chocolate agar (CHOC)
Supports growth of most microbes including highly fastidious organisms (e.g., Neisseria and Haemophilus). Enriched and non-selective
MacConkey (MAC)
Supports growth of most Gram-negative bacilli/rods including enteric. Selective and differential. Differentiates between lactose- fermenting and non-lactose-fermenting Gram-neg
Colistin nalidixic acid (CNA)
Supports growth of Gram-positive cocci. Selective
Types of hemolysis?
Alpha = partial
Beta = complete
Gamma = no hemolysis
List types of morphological traits
- Size
- Shape
- Color
- Hemolysis
- Elevation
- Margin (edges)
- Density
- Surface appearance (dull/shiny)
- Consistency (creamy, dry, waxy, mucoid)
- Odor (don’t intentionally sniff)
Which organisms smells like fruit/grapes?
Pseudomonas aeruginosa
Which organism smells like dirt?
Nocardia
Which organism smells like bleach?
Eikenella
Which is the first test in any ID scheme?
Gram stain
List single enzyme rapid tests
- Catalase
- Oxidase
- Spot Indole
- Urease
- PYR
- Slide coagulase
What is TSI slant?
Triple sugar iron agar test. It tests microbe’s ability to ferment sugars and produce hydrogen sulfide
MALDI TOF
Matrix-assisted laser desorption ionization: time of flight
List metabolic pathway enzyme test types
- Carb oxidation and fermentation
- Amino acid degradation
- Single substrate use
- Usually overnight
Inhibitory profile tests
- Test ability of microbe to grow in presence of one or more inhibitory substances
- E.g., NaCl, Esculin hydrolysis, chemical susceptibility
