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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
Q

Tydallisation (fractional sterilisation)

A

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
Q

Autoclave

A

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
Q

Uses of autoclave

A

Sterilising:

  1. Dressing, linen
  2. Non sharp metallic instruments
  3. Aqueous solutions
  4. Microbiological media
  5. Plastic pipettes, tubes
28
Q

Efficacy testing of autoclave

A

Physical: temperature chart recorder
Chemical: Browne’s tube No 1, Bowie Dick tapes
Biological: spores of B. stearothermophilus

29
Q

UV rays wrt sterilisation

A

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
Q

Ionising radiation wrt sterilisation

A

γ rays for cold sterilisation
Mechanism: DNA damage
High penetration and sporicidal
Efficacy testing using spores of B. pumilis

31
Q

Uses of cold sterilisation

A 
γ rays
Disposable gloves, petriplate, syringes
IV sets, foley’s catheters
Sutures, implants, prosthesis
Glassware 
Fabrics, contraceptive devices, cotton swabs
32
Q

Filtration

A 
For heat sensitive liquids 
Pre side: 0.45 μ
Efficacy testing:
 Serratia marcescens 
 Brevundimonas diminuta
33
Q

Filtration is used for

A 
Heat sensitive liquids lie
 Sera
 Antibiotic solutions
 Vaccines
 Sugar/urea solution
 Gelatin media
34
Q

Types of filtration

A
  1. Earthen wave filters
  2. Asbestos filters
  3. Sintered glass filters
  4. Membrane filters/ Millipores
35
Q

Earthen wave filters

A 
Made of diatomaceous or porcelain earth
Shaped in form of candles
Eg., Pasteur chamberland filters
 Berkefeld filters
 Mandler filters
36
Q

Asbestos filters

A

Made up of magnesium trisilicate
Shape in form of discs. Eg., Seitz filters
Has carcinogenic potential

37
Q

Sintered glass filters

A

Shaped in the form of discs

Disadvantage: expensive

38
Q

Millipores/Membrane filters

A

Made up of cellulose ester, polyesters
Available in form of discs
Single use discs
M/C used filters

39
Q

FDA classification of chemical disinfectants

A 
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
Q

Examples of each level of FDA classification of chemical disinfectants

A 
1. High level:
 ETO, plasma aldehydes
2. Intermediate level:
 Phenol, halogens
3. Low level:
 Alcohols, surface acting agents
41
Q

Alcohols as disinfectants

A

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
Q

Aldehydes as disinfectants

A 
Mechanism: alkylation of hydroxyl, amino and carbonyl residue of proteins
Examples:
1. Formalin 
2. Glutaraldehyde
3. Ortho-phthaldehyde
43
Q

Uses of formaldehyde 40%

A
  1. Fumigation of OTs, wards
  2. Duckering- B. anthracis spores in animal wool
  3. Preservation of anatomical specimens
44
Q

Glutaraldehyde/cidex 2% uses

A 
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
Q

Ortho-phthaldehyde OPA 0.55%

A

Same uses as cidex
Active at wide pH (cidex-alkaline pH)
Less irritant
Better mycobacteriocidal activity

46
Q

Chlorine compounds disinfectants

A

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
Q

Iodine compounds as disinfectants

A

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
Q

Phenolics - coal tar derivatives as disinfectants

A 
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
Q

Modified phenolics

A 
Chlorinated biphenolics
Lost corrosiveness ➡️ antisepsis
Examples:
1. Chlorhexidine- hand disinfectant
2. Hexachlorophene
3. Chlorxylenol
4. Triclosan
Savlon - chlorhexidine+cetrimide
Dettol - 10% chlorxylenol
50
Q

Surface acting disinfecting agents

A

Mechanism: damaging membranes
Cationic:
Cetrimonium bromide (Savlon), Benzalkonium chloride
Anionic: soap

51
Q

Ethylene oxide as disinfectants

A

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
Q

Cycles of ETO or ethylene oxide and efficacy

A

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
Q

Disadvantages of ETO as disinfectant

A 
1. Toxic:
 exposure monitoring required because of cataracts and neural disturbances
2. Carcinogenic
3. Irritant
4. Inflammable
54
Q

Plasma (VHP) sterilisation

A 
VHP vaporised H2O2 (59%)
Steps:
1. Creation of vacuum
2. Introduce gas
3. Electromagnetic radiation (radiowaves or microwave) ➡️ releases toxic O2 radicals
55
Q

Plasma steriliser (STERRAD)

A

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
Q

Spaulding classification

A

Classification of medical devices into categories based on the risk of infection involved with their use:
Critical, semicritical and non-critical

57
Q

Critical category of Spaulding classification

A

Enter sterile tissues and vasculature of body
Sterilised before use
Eg., surgical instruments, implants, prosthesis, catheters, laproscopes, Foley’s catheter

58
Q

Semicritical category of Spaulding classification

A

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)

59
Q

Non critical category of Spaulding classification

A

Contact with intact skin
Eg., stethoscope, ECG electrode, bed pans
Low level of disinfection is enough

60
Q

Phenol coefficient

A

Efficacy of disinfectant

More than 1 ➡️ efficacious than phenol and vice versa

61
Q

Phenol coefficient statins

A
  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
Q

Test for efficacy of disinfectants (phenol coefficient)

A
  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 (16 decks)

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