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Clinical Microbiology Winter > Week 1: Specimens/Processing/Stains/ID Methods > Flashcards

Week 1: Specimens/Processing/Stains/ID Methods Flashcards

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1
Q

List types of wet mounts

A
  • Direct wet mount
  • Hanging-drop wet mount
  • KOH wet mount
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2
Q

Must use sterile slide and supplies but Darrell said…

A

We don’t actually use sterile slides :P

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3
Q

How do you smear prep swab specimens?

A
  • 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
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4
Q

How do you smear prep thick liquid or semi-solid specimens?

A
  • Immerse swab in specimen for several seconds and then roll onto slide
  • Feces
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5
Q

How do you smear prep thick, granular, or mucoid specimens?

A

Prepare same as peripheral blood smear because want thick and thin areas

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6
Q

How do you smear prep thin fluid specimens?

A
  • 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
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7
Q

How do you smear prep a bacterial colony from growth on media?

A
  • 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
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8
Q

Methods to fix slides?

A
  • Methanol
  • Heat fix at 56°C for several minutes
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9
Q

What is the procedure for the most common stain?

A

Gram stain

  1. Fix
  2. Crystal violet
  3. Iodine
  4. Decolorization
  5. Safranin counter-stain
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10
Q

What color is Gram variable bacteria?

A

Both pink and purple

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11
Q

Causes of gram variable appearance?

A

Antibiotic therapy or old age can compromise cell wall integrity such that it decolorizes easily

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12
Q

Which magnification used for Gram stain?

A

100x

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13
Q

List factors that affect gram reaction, morphology, and interpretation

A
  • 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
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14
Q

What are two acid-fast stain methods? Describe each

A

Ziehl-Neelsen” carbolfuchin stain requires heat to enter cell
Kinyoun: carbolfuchin stain enters cell due to high phenol concentration in stain

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15
Q

Counterstain for acid-fast stain? How to interpret results?

A

Methylene blue or malachite green

Positive = red
Negative = blue or green

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16
Q

Describe 3 fluorochrome stains (enhance bacterial detection when small number of microbes, much debris, many WBCs)

A
  • 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
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17
Q

How do you prepare specimens for culture setup?

A
  • Concentration: centrifuge or filter
  • Homogenization: grind using mortar and pestle/tissue grinder
  • Decontamination: Destroy normal flora with reagents in order to isolate mycobacteria
18
Q

List types of media

A
  • Nutrient (BHI, blood, chocolate, soy)
  • Differential (hemolysis on blood agar)
  • Selective (MAC, CNA)
  • Enrichment broth (thioglycolate, LIM, GN)
19
Q

T/F: Darrell thinks it’s okay to use cotton swabs to inoculate media

A

FALSE. He said that cotton inhibits some organisms so don’t use!

20
Q

What are the urine loop volume sizes and what dilution factors are they when quantitating colonies?

A
  • 0.01ml is 1:100
  • 0.001 ml is 1:1000
21
Q

What are capnophiles?

A

Require 5-10% CO2

22
Q

Incubator conditions for ambient air and CO2 incubators?

A
  • Ambient: 35°C, high humidity
  • CO2: 5-10% CO2 + 35°C room air
23
Q

Incubator conditions for microaerophiles?

A

85% N2, 10% CO2, and 5% O2

24
Q

Incubator conditions for anaerobes?

A

5% H2, 5-10% CO2, 85-90% N2, 35°C, high humidity

25
Q

What temperatures do the following grow at: psychrophiles, mesophiles, thermophiles

A

Psychrophiles: 4-20°C
Mesophiles: 30-45°C
Thermophiles: 50 to greater than 200°C

Most human pathogens grow at 35-37°C

26
Q

Length of incubation?

A
  • 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”
27
Q

Criteria for organism ID?

A
  • Macroscopic colony morphology
  • Environmental growth requirements
  • Microscopic morphology + staining traits
  • Nutritional requirements + metabolism
  • Antibiotic resistance/susceptibility
28
Q

Blood agar (BA/BAP) media

A

Supports growth of most organisms excluding fastidious ones. Enriched and non-selective

29
Q

Chocolate agar (CHOC)

A

Supports growth of most microbes including highly fastidious organisms (e.g., Neisseria and Haemophilus). Enriched and non-selective

30
Q

MacConkey (MAC)

A

Supports growth of most Gram-negative bacilli/rods including enteric. Selective and differential. Differentiates between lactose- fermenting and non-lactose-fermenting Gram-neg

31
Q

Colistin nalidixic acid (CNA)

A

Supports growth of Gram-positive cocci. Selective

32
Q

Types of hemolysis?

A

Alpha = partial
Beta = complete
Gamma = no hemolysis

33
Q

List types of morphological traits

A
  • Size
  • Shape
  • Color
  • Hemolysis
  • Elevation
  • Margin (edges)
  • Density
  • Surface appearance (dull/shiny)
  • Consistency (creamy, dry, waxy, mucoid)
  • Odor (don’t intentionally sniff)
34
Q

Which organisms smells like fruit/grapes?

A

Pseudomonas aeruginosa

35
Q

Which organism smells like dirt?

A

Nocardia

36
Q

Which organism smells like bleach?

A

Eikenella

37
Q

Which is the first test in any ID scheme?

A

Gram stain

38
Q

List single enzyme rapid tests

A
  • Catalase
  • Oxidase
  • Spot Indole
  • Urease
  • PYR
  • Slide coagulase
39
Q

What is TSI slant?

A

Triple sugar iron agar test. It tests microbe’s ability to ferment sugars and produce hydrogen sulfide

40
Q

MALDI TOF

A

Matrix-assisted laser desorption ionization: time of flight

41
Q

List metabolic pathway enzyme test types

A
  • Carb oxidation and fermentation
  • Amino acid degradation
  • Single substrate use
  • Usually overnight
42
Q

Inhibitory profile tests

A
  • Test ability of microbe to grow in presence of one or more inhibitory substances
  • E.g., NaCl, Esculin hydrolysis, chemical susceptibility

Clinical Microbiology Winter (4 decks)

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