Microbiology MBBS > Microscopy, sterilisation,... > Flashcards

Microscopy, sterilisation,... Flashcards

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1
Q

Different types of microscopes

A
  1. Light
  2. Compound (bright field, dark ground, phase contrast)
  3. Fluorescence (UV)
  4. Electron microscope
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2
Q

Maximum theoretical resolving power of oil immersion and electron microscope

A

Oil immersion: 0.2 micrometer
1000X

Electron microscope: 0.005 nm

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3
Q

Stains used for fluorescence microscopy

A

Acridine orange
Auramine O
Fluorescein

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4
Q

Uses of fluorescence microscopy

A
  1. Auramine phenol staining
  2. Direct fluorescent antibody staining
  3. Indirect “”
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5
Q

First electron microscope was designed by

A

Ernst Ruska in 1931

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6
Q

Dark field microscope or dark ground microscope

A

Used for living, unstained or thin bacteria like spirochaetes in a dark background
It has a special condenser

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7
Q

Applications of phase contrast microscope

A
  1. Studying microbial motility
  2. Determining shape of living cell
  3. Detecting bacterial components such as endospores and inclusions bodies which become clearly visible because they have refractive index markedly different from water
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8
Q

Applications of fluorescence microscope

A
  1. Epifluorescence microscope (simple)
    A) autofluorescence (cyclospora)
    B) microbes coated with a fluorescent dye
    Acridine orange dye for malaria parasite (quantitative buffy coat examination)
    Auramine phenol for tubercle bacteria
    C) immunofluorescence (direct or indirect)
  2. Confocal microscope
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9
Q

Steps for preparation of a thin specimen of bacteria for transmission electron microscope

A
  1. Fixation: via glutaraldehyde or OsO4 for stabilisation
  2. Dehydration: acetone or ethanol
  3. Embedding: in a plastic polymer then hardened
  4. Slicing: by a ultramicrotome knife
    Mounted on a copper slide
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10
Q

Measures to increase the contrast of electron microscope

A
  1. Staining: via lead citrate and uranyl acetate
  2. Negative staining: specimen is spread out on a thin film with heavy metals like phosphotungstinic acid or uranyl acetate
    Structure of viruses, bacterial gas vacuoles,…
  3. Shadowing: specimen is coated with a thin film of platinum or other heavy metal at 45•
    For virus morphology, bacterial flagella and plasmids,…
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11
Q

Sterilisation, disinfection, antisepsis

A

Sterilisation: destruction of all forms of life including spores
Disinfection: destruction of only pathogenic organisms
Antisepsis: disinfection of living tissues

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12
Q

Order of susceptibility of organisms

A 
Prions ➡️ bacterial spores 
➡️ cysts of protozoa ➡️ mycobacteria 
➡️ non enveloped viruses ➡️ fungi 
➡️ gram +ve bacteria ➡️ gram -ve bacteria 
➡️ enveloped viruses
From difficulty to easy
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13
Q

Heat sterilisation

A 
Most effective
Mechanism:
1. Oxidative damage
2. Denaturation of proteins
3. Increased electrolytes to toxic levels
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14
Q

Examples of dry heat sterilisation

A
  1. Flaming:
    Disinfection of mouth of test tubes, cover slips, slides
  2. Red heat:
    Sterilisation of inoculating loops, straight wires, tips of forceps
  3. Incineration
  4. Hot air oven
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15
Q

Incineration (dry heat)

A

Sterilisation and reduction in volume of infectious hospital water
Reduction volume decreases by 80-85%
1° chamber: 650-750°C
2° chamber: 1050-800°C

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16
Q

Hot air oven is used for

A

Sterilisation of:

  1. Metallic instruments
  2. Glass ware
  3. Oils, jellies, powder, waxes
  4. Cotton swabs
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17
Q

Hot air oven

Temperature and precautions

A 
160°C for 2 hours (M/C)
170°C for 1 hour
180°C for 30 min
Precautions:
1. No rubber objects
2. No over loading
3. Cool for 2 hours before opening
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18
Q

Efficacy of hot air oven

A

Physical: temperature chart recorder
Chemical: Browne’s tube No 3
Biological: spores of C. tetani out B. subtilis

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19
Q

Moist heat

A

Mechanism:
Denaturation and coagulation of proteins
Superior to dry heat

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20
Q

Examples of moist heat less than 100°C

A
  1. Pasteurisation
  2. Serum bath: MH at 56°C for 1 hour for 3 consecutive days
  3. Vaccine bath:
    MH at 60°C for 1 hour for 3 consecutive days
  4. Inspissation:
    MH at 80-85°C for 30 min for 3 consecutive days
    Sterilisation of heat sensitive medium
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21
Q

Examples of pasteurisation

A 
1. Holder method:
 MH at 63°C for 30 min
2. Flash method:
 MH at 72°C for 15-20 sec followed by rapid cooling to <13°C
3. Very High Temperature method:
 MH at 149°C for 0.5 sec
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22
Q

Pathogenic bacteria surviving Holder method

A

Coxiella burnetti

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23
Q

Efficacy testing of pasteurisation

A
  1. Coliform test: superior
    Pasteurised milk overnight on McConkey agar, no gas and acid
  2. Phosphatase test: M/C
    Pasteurised milk and substrate (disodium phenyl PO4) for 2 hours
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24
Q

Moist heat at 100°C

A
  1. Tydallisation/fractional sterilisation

2. Autoclave

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25
Tydallisation (fractional sterilisation)
Moist heat sterilisation done in Koch and Arnold Steam steriliser for 20 min for 3 consecutive days Sterilisation of media: TCBS, XLD, DLA, selenite F broth, sugar solutions, gelatin media
26
Autoclave
Principle: Use of saturated steam under pressure •M/C used is moist heat at 121°C for 15-20 min at 15 psi •Moist heat at 134°C for 3 min at 30 psi
27
Uses of autoclave
Sterilising: 1. Dressing, linen 2. Non sharp metallic instruments 3. Aqueous solutions 4. Microbiological media 5. Plastic pipettes, tubes
28
Efficacy testing of autoclave
Physical: temperature chart recorder Chemical: Browne’s tube No 1, Bowie Dick tapes Biological: spores of B. stearothermophilus
29
UV rays wrt sterilisation
Non ionising with λ of 220-280 nm Acts via DNA damage Poor penetrating power and non-sporicidal Disinfection of biosafety cabinets, hospital corridors
30
Ionising radiation wrt sterilisation
γ rays for cold sterilisation Mechanism: DNA damage High penetration and sporicidal Efficacy testing using spores of B. pumilis
31
Uses of cold sterilisation
``` γ rays Disposable gloves, petriplate, syringes IV sets, foley’s catheters Sutures, implants, prosthesis Glassware Fabrics, contraceptive devices, cotton swabs ```
32
Filtration
``` For heat sensitive liquids Pre side: 0.45 μ Efficacy testing: Serratia marcescens Brevundimonas diminuta ```
33
Filtration is used for
``` Heat sensitive liquids lie Sera Antibiotic solutions Vaccines Sugar/urea solution Gelatin media ```
34
Types of filtration
1. Earthen wave filters 2. Asbestos filters 3. Sintered glass filters 4. Membrane filters/ Millipores
35
Earthen wave filters
``` Made of diatomaceous or porcelain earth Shaped in form of candles Eg., Pasteur chamberland filters Berkefeld filters Mandler filters ```
36
Asbestos filters
Made up of magnesium trisilicate Shape in form of discs. Eg., Seitz filters Has carcinogenic potential
37
Sintered glass filters
Shaped in the form of discs | Disadvantage: expensive
38
Millipores/Membrane filters
Made up of cellulose ester, polyesters Available in form of discs Single use discs M/C used filters
39
FDA classification of chemical disinfectants
``` 1. High level: Kills everything 2. Intermediate level: Does not kill all spores 3. Low level: Does not kill spores and not all viruses and mycobacterium ```
40
Examples of each level of FDA classification of chemical disinfectants
``` 1. High level: ETO, plasma aldehydes 2. Intermediate level: Phenol, halogens 3. Low level: Alcohols, surface acting agents ```
41
Alcohols as disinfectants
Mechanism: Coagulation of proteins, dehydration of cells 60-80% concentration Ethyl alcohol- spirit Isopropyl alcohol- hand disinfectant Methyl alcohol- fungal spores in biosafety cabinets
42
Aldehydes as disinfectants
``` Mechanism: alkylation of hydroxyl, amino and carbonyl residue of proteins Examples: 1. Formalin 2. Glutaraldehyde 3. Ortho-phthaldehyde ```
43
Uses of formaldehyde 40%
1. Fumigation of OTs, wards 2. Duckering- B. anthracis spores in animal wool 3. Preservation of anatomical specimens
44
Glutaraldehyde/cidex 2% uses
``` Sterilisation and disinfection of: 1. Anaesthetic equipment 2. Cysto/broncho/endoscopes, corrugated rubber tubings, endotracheal tubes Sterilisation in 6-8 hours Disinfection in 25-30 min Dilution reused for (10-14 days) ```
45
Ortho-phthaldehyde OPA 0.55%
Same uses as cidex Active at wide pH (cidex-alkaline pH) Less irritant Better mycobacteriocidal activity
46
Chlorine compounds disinfectants
Mechanism: oxidation of sulf-hydryl residues of proteins 1. Cl2 tablets: disinfection of water supplies 2. Sodium hypochlorite: •Disinfection of infectious hospital spills •Hospital floor
47
Iodine compounds as disinfectants
Skin antiseptics Mechanism: oxidation of sulf-hydryl residues of proteins 1. Tincture of iodine: 2% iodine in 70% alcohol 2. Betadine: Iodine coated onto a neutral carrier poly vinyl pyrrolidone
48
Phenolics - coal tar derivatives as disinfectants
``` Mechanism: Precipitation of proteins and DNA damage Active in organic matter Examples: 1. 5% phenol 2. 1-4% cresol 3. 2-5% lysol Uses: 1. Surface disinfection of hospital floors 2. Discard jars ```
49
Modified phenolics
``` Chlorinated biphenolics Lost corrosiveness ➡️ antisepsis Examples: 1. Chlorhexidine- hand disinfectant 2. Hexachlorophene 3. Chlorxylenol 4. Triclosan Savlon - chlorhexidine+cetrimide Dettol - 10% chlorxylenol ```
50
Surface acting disinfecting agents
Mechanism: damaging membranes Cationic: Cetrimonium bromide (Savlon), Benzalkonium chloride Anionic: soap
51
Ethylene oxide as disinfectants
9% of it + 91% of CO2 or HCFC Used for sterilising heat labile and moisture sensitive objects like: 1. Bedding, blankets, rubber and plastic objects 2. Disposables, heart lung machines, respirators, dental equipment
52
Cycles of ETO or ethylene oxide and efficacy
1 cycle time: 18-24 hours (aeration required) 2 types of sterilisers: 1. Cold cycle- 37-44°C 2. Hot cycle- 55°C Efficacy: spores of Bacillus atropheus (pigmented variant of B. subtilis)
53
Disadvantages of ETO as disinfectant
``` 1. Toxic: exposure monitoring required because of cataracts and neural disturbances 2. Carcinogenic 3. Irritant 4. Inflammable ```
54
Plasma (VHP) sterilisation
``` VHP vaporised H2O2 (59%) Steps: 1. Creation of vacuum 2. Introduce gas 3. Electromagnetic radiation (radiowaves or microwave) ➡️ releases toxic O2 radicals ```
55
Plasma steriliser (STERRAD)
Temperature: 30-60°C Cycle time: 60-75 min Efficacy: spores of B. stearothermophillus Use: sterilisation of heat sensitive metal or non-metal hospital devices Plasma sterile should be dry
56
Spaulding classification
Classification of medical devices into categories based on the risk of infection involved with their use: Critical, semicritical and non-critical
57
Critical category of Spaulding classification
Enter sterile tissues and vasculature of body Sterilised before use Eg., surgical instruments, implants, prosthesis, catheters, laproscopes, Foley’s catheter
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Semicritical category of Spaulding classification
Contact with mucus membranes and breaks in skin Eg., GI endoscopes Bronchoscope Cystoscope Anaesthetic equipment (Above eg. should have high level of disinfection or sterilisation) Oral and ractal thermometers (intermediate level)
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Non critical category of Spaulding classification
Contact with intact skin Eg., stethoscope, ECG electrode, bed pans Low level of disinfection is enough
60
Phenol coefficient
Efficacy of disinfectant | More than 1 ➡️ efficacious than phenol and vice versa
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Phenol coefficient statins
1. Serial dilution of new disinfectants along with phenol 2. Each of these solutions as standard inoculum of S. typhi or S. aureus 3. After every 5 min subculture into solid medium 4. We will find out highest dilution of new disinfectant and phenol which kills organism in 10 min but not in 5. 5. Solutions will be calculated respectively 6. Determine ratio
62
Test for efficacy of disinfectants (phenol coefficient)
1. Rideal Walker test 2. Chick Martin test: Better, superior test since more realistic Along with S. aureus, S. typhi, we also add 3% sterile faeces in this test
Microbiology MBBS
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