Microbio 1 - Bacteriology > Bacterial Diagnostics (III) > Flashcards

Bacterial Diagnostics (III) Flashcards

(54 cards)

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1
Q

Why do we burn the slide before microscopy?

A

To clean the slide from any fats on the surface by waving it over the bunsen burner a few times (5)

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2
Q

Steps of fixing bacterial smear

A

1) Clean slide using flame from fats
2) Apply sample (liquid drop or solid mixed into water drop)
3) Fix sample/dry on slide with bunsen flame (~3 times)

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3
Q

Native Preparations

A

Unstained preparation examined by bright field, dark field, or phase-contrast microscopy
- Live microbes
- Morphology
- Motility

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4
Q

Types of Native prep

A
  • Wet mount
  • Hanging drop
  • Vital staining
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5
Q

Wet mount prep

A

1) Drop of bacterial suspension into center of slide
2) Cover with coverslip
3) Examine slide under low power

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6
Q

Hanging drop prep

A

1) Drop of bacterial suspension on coverslip
2) Vaseline dotted on slide
3) Place slide over coverslip where dot is in center
4) Flip and now drop suspended
= used to see motility of bacteria

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7
Q

Vital Staining method

A

1) Make a wet mount prep
2) One drop of Diluted Methylene Blue or Lugol’s iodine to edge of coverslip
3) Wait 10 mins
4) Examine under microscope

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8
Q

Gram stain prep

A

1) Smear specimen & fix (heat)
2) Crystal violet stain & wait 1-2 mins
3) Rinse with water
4) Iodine (lugol’s) & wait 1 min
5) Rinse with water
6) Decolorize with 96% alcohol
7) Safranin for 30s
8) Dry with filter paper & examine under microscope

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9
Q

Neisser Stain use

A

Detects volutin granules in Corynebacterium staining them dark blue/purple

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10
Q

Neisser Stain Prep

A

1) Stain fixed smear with 2:1 mix of Neisser I & II for 10 minutes
2) Rinse slide with water
3) Stain with Crysoidine dye for 2 mins
4) Repour dye in bottle
5) Dry slide & examine

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11
Q

Acid-Fast stain facts

A
  • Stains Mycobacteria
  • Carbol-Fuchsin & Phenol
  • aka Ziehl-Neelsen stain
  • Stains lipid rich non-gram staining bacteria
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12
Q

Steps of Acid-Fast stain

A

1) Fix smear
2) Primary stain with Carbol-Fuchsin & apply heat to penetrate mycolic acid
3) Wash with water
4) Decolorization with Acidic alcohol (96% ethyl alc)
5) Counter stain using Loffler’s Methylene blue or Malachite-green staining background to show the acid-fast bacilli

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13
Q

Capsule Stain methods

A
  • Negative stain
  • Direct Fluorescent AB stain
  • Welch method
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14
Q

Negative Capsule stain

A

Staining darkens background
1) Drop of specimen on slide with India Ink
2) Allow to dry & fix (no heat, could distort capsule)
3) Stain Fuchsin for 1 min
4) Rinse
5) Dry & view with immersion oil

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15
Q

Welch Capsule stain method

A

Special capsule stain
1) Hot crystal violet solution
2) Wash with Copper-Sulfate solution
3) Cell & background stain but capsule very light

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16
Q

Detection of Spores

A

Stained using simple dyes like Methylene-blue, Malachite green or carbol-fuchsin
Malachite green:
1) Primary stain malachite green
2) Rinse
3) Counterstain Safranin
= Spores Green, Cells red/pink

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17
Q

Detection of Flagella

A
  • Flagella stains: Colloidal susp. of Tannic Acid Salts
  • Motility test medium
  • Directly by Wet mount
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18
Q

Microscopes used in Mirobiology

A
  • Light (bright field) microscope: resolves 250nm, max 1500x
  • Electron microscope: resolves 0.1nm, max 500,000x
  • Dark field microscope
  • Fluorescent microscope
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19
Q

Relative sizes of Organisms (V, B, Protozoa, Parasites)

A
  • Bacteria = 0.3-30 µm
  • Viruses = 10-300 nm
  • Protozoa, Fungi = 5-100 µm
  • Parasites = 200+ µm
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20
Q

Composition of culture media

A
  • Nutrition
  • Carbon, Nitrogen, Sulphur, Phosphate
  • Minerals (Mg)
  • Water
  • pH 7.2-7.4 (general)
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21
Q

Bacteria Temp classification

A
  • Psychrophiles: 15-20
  • Thermophiles: 50-60
  • Mesophiles: 30-37
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22
Q

Pure culture

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A

Population of bacteria belonging to 1 species isolated at a certain time from a certain origin

23
Q

Bacterial Isolate

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A

After mixed culture 1 colony can be grown on its own medium to have the same type of species (e.g. stool isolate of salmonella)

24
Q

Bacterial strain

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A

Population of bacteria originated from 1 colony maintained for a long time in lab conditions

25
Aerobic culture specs
- 35-37˚C - Normal pressure (1atm) - High humidity - Normal air - Higher CO2
26
Normal vs Microaerophil gas %
- Normal: 21% O2 - Micro: 5-10% O2, 8-10% CO2
27
Methods of Anaerobic Culture
- Anaerostat (pump out) - Gas pack Jar (H2 = H2O) - Liquid media + Wax - Chemical method (addition of O2 reducer like thioglycolate) - Fortner-technique (aerobe + anaerobe, use up O2)
28
Obligate I.C Bacteria
- Chlamydia Trachomatis - Rickettsiae - Myobacterium Leprae - Treponema Pallidum - Coxiella Burnetti
29
Uses of Lab animals
- Symptoms of infections - Maintenance of Bacterial colonies (testes) - Demonstration of Bacteria Exotoxin (römer test diphteria) - Determination of Virulence - Biological activity test (LAL) - Monoclonal antibody production, or for passive immunity
30
MIC
Minimal Inh Conc - Lowest conc of a drug which inhibits growth of bacteria (not killed) - Lower MIC means higher bacterial susceptibility - mg/l or µg/l
31
MBC
Min bactericidal conc - Lowest conc of drug that kills 99.9% of certain quantity of bacteria - Important for life threatening conditions
32
Determination of MIC/MBC
- Tube dilution test (MIC) - Micro-dulution test (96) - Agar Plate Dilution - Disc diffusion Test (lawn culture) - Punching Test (liquid diff) - E-test: Plastic strip with conc. of AB changing to read the MIC number
33
Russel Medium
- Glucose, Lactose, Sucrose - Andrade indicator (red in acid) - Fermentation of lactose+glucose = all turns red - Glucose only the bottom yellow & slant red
34
TSI (tripple sugar Iron agar)
- Glucose, Lactose, Sucrose - Iron + pH indicator - H2S production = Black precipitate - Turns yellow in acid
35
ONPG test
- Detects slow lactose fermenters - Detects B-galactosidase - Yellow = color change
36
Ureum-Indole test
- Urease Test, Ammonia, high pH, Phenol red turns Pink - Tryptophanase enzyme breaks tryptophan to Indole = Kovacs reagent positive is Red
37
Types of Serology Agglutination
- Slide agglutination (test antigen) - Tube agglutination (test antibodies, titer) - Passive agglutination (either ab/ag on latex beads)
38
Types of Serology Percipitation
- Liquid phase (ring) - Semi-solid phase (agar) - ELEK's Test (all in equivalence zone)
39
Complement Fixation Test
It’s a blood test used to check if a person has antibodies against a specific antigen Complement is added that later lyses RBCs if not bound
40
Complement Fixation Steps
1) Serum from patient (w or wo Abs) 2) Heated to eliminate natural complement proteins 3) Standard complement proteins added 4) Known antigen added 5) RBC + Antibody for sheep RBC added 6) If serum contained antibodies against our antigen the complement is bound and can not lyse RBCs
41
Immunofluorescent method
Uses fluorescently-labeled antibodies to detect specific antigens on or inside cells/tissues, viewed under a fluorescence microscope. - Antibody is tagged with a fluorescent dye like FITC (fluorescein isothiocyanate). = Direct & Indirect methods
42
Direct Immunofluorescence (DIF)
- One-step method - The primary antibody is directly labeled with the fluorescent dye. - Used to detect antigen in tissues or cells. = antigen detection
43
Indirect Immunofluorescence (IIF)
- Two-step method 1) First, an unlabeled primary antibody binds the antigen. 2) Then, a fluorescently labeled secondary antibody (e.g. FITC-labeled goat anti-rabbit IgG) binds to the primary antibody. = stronger signal as more FITC can bind 1 primary Ab
44
ELISA
- Direct: Detects Antibodies - Indirect / Sandwich: Detects soluble antigens
45
Direct ELISA
1) Antibodies in serum bind antigen on surface 2) Wash off unbound Abs 3) Add enzyme-conjugated anti-human Abs 4) Wash off unbound 5) Add enzyme substrate to form chromophore 6) Spectrophotometer Analysis
46
Sandwich ELISA
1) Specific Antibody fixed on plate 2) Add patient serum & wash off 3) Add second antigen specific antibodies & wash 4) Add enzyme-conjugated anti-human Abs & wash 5) Add enzyme substrate forming chromophore 6) Specrophotometer analysis
47
Western Blot
Confirmation test after ELISA 1) Antigens separated using SDS-page 2) Transferred to NC paper 3) Paper incubated with Serum (has antigen-spec Abs) 4) Add enzyme-conjugated antihuman Abs 5) Add substrate and observe
48
Bacterial Typing Methods
- Serotyping (slide agglutination) - Phage typing - Pulsed-field gel electrophoresis - Multi-locus sequence typing - Whole genome sequencing - Matrix Assisted laser desorption ionization time of flight
49
Diagnostic Skin test Examples
- Dick test - Schick test - Tuberculin/Mantoux test - Allergy skin test
50
Dick Test
For scarlet fever susceptibility 1) Inject 0.1ml of scarlet fever toxin intradermally 2) If red area >10mm forms, positive result (susceptible)
51
Schick Test
For Diphtheria susceptibility 1) Inject diphtheria toxin in one arm and heat-inactivated/control in other - Positive: Wheal (5-10mm) in test arm only = susceptible - Pseudo-positive: Mild reaction in both arms, fades in 4 days = Immune but hypersensitive - Negative: No reaction = Immune - Combined: Erythema fades in control arm only = susceptible & hypersensitive
52
Tuberculin / Mantoux Test
Detect TB infection or exposure 1) Inject 0.1ml of PPD intradermally & leave for 48-72h - Positive: >10mm lesion - Negative no lesion - False positive: BCG vaccine, or other Mycobacteria - False negative: AIDS, Malnutrition, steroid use
53
Measurement of Toxicity
LD50 - Lethal dose - Dose of substance required to kill 50% of a test population
54
Types of Serotyping methods
- Latex agglutination - Enzyme immunoassay (urine) - Lance-field classification - Quellung reaction (S.pneum swell) - Enterobacteriaceae serotyping (K, O, H Antigens)
Microbio 1 - Bacteriology
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