Download Sterilization & Disinfection

Sterilization
&
Disinfection
By
Prof. Dr. Zainalabideen A Abdulla,
DTM&H., MRCPI, Ph.D., FRCPath. (U.K.)
LEARNING OBJECTIVES
1. Differentiate between sterilization,
disinfection, and sanitization
2. Differentiate between bactericidal and
bacteriostatic agents
3. Explain the process of pasteurization and
lyophilization
4. List several methods used to inhibit the
growth of microorganisms
5. Identify several factors that can influence
the effectiveness of disinfectants
Sterilization
Destruction or elimination of all microbes
including cells, spores, and viruses
- Sterile = Devoid of microbial life
- Physical or chemical methods in healthcare
• Dry heat
• Autoclaving (steam under pressure)
• Ethylene oxide gas
• Formaldehyde
• Radiation (UV, gamma rays)
Disinfection
The elimination of most or all pathogens (except
bacterial spores) from nonliving objects; using:
1. Liquid chemicals (disinfectants)
2. Wet pasteurization
Antiseptics: Disinfectants used on living
tissues as the skin
Sanitization: Reduction of the microbial
population to safe levels, e.g. in resturants
Microbicidal agents
- Suffix: cide or cidal = “killing”
Examples:
• Bactericidal = Kill bacteria
• Sporicidal = Kill bacterial spores
• Viricidal = Kill viruses
• Fungicidal = Kill fungi
• Pseudomonicidal= Kill Pseudomonas
• Tuberculocidal = Kill M. tuberculosis
Pasteurization
- A method to disinfect liquids (e.g. milk)
- Is not sterilization
- Not all microbes are killed
- 63 Celsius 30 minutes
72 Celsius 15 seconds
140 Celsius 4 seconds
Microbistatic agents
- Drugs or chemicals that inhibit reproduction
and metabolism of microorganisms (NOT
necessarily kill them)
- Freeze drying & rapid freezing: Bacteriostatic
- Freeze drying = Lyophilization; preserve m.o.
Sepsis: Presence of pathogens in blood or
tissues
Asepsis: Absence of pathogens
- Medical or Surgical aseptic techniques, e.g.,
hand hygiene, sterile gloves, masks, and gowns
Antisepsis: Prevention of infection
Antiseptic technique: Use of antiseptics
Physical Methods
Heat
- The most common type of sterilization for
inanimate objects able to with stand high
temperatures
- Effect depends on:
1. Temperature
2. Time
- Pathogenic > susceptible than nonpathogenic
Thermal Death Point (TDP)
Lowest temp. that kill all organism in a standardized
pure culture within specified period
Thermal Death Time (TDT)
The length of time to sterilize a pure culture at a
specified temperature
Dry heat
- 160 – 165 Celsius 2 hours or
- 170 – 180 Celsius 1 hour
- In oven
- Items: Metals, glassware, oils, waxes
Incineration: Burning; contaminated disposable
materials; must NOT overloaded with protein
and moist materials as feces, vomitus or pus
Flaming: Bunsen burner flame or electrical heat
device for wires, loops or forceps
Moist heat
1. Boiling for 30 minutes, e.g. metal (needles),
glass. Not effective against spores, viruses and
Mycobacterium.
2. Autoclave (Moist heat + Pressure):
- 15 psi + 121.5 Celsius for 20 minutes
- Kills spores, viruses, and vegetative form
- Rubber can be autoclaved
- Indicators for completion: • Autoclave tape
or • Solutions of bacterial spores
- Keep cans open, bottles covered loosely
Cold
- Refrigeration: Slows metabolism & growth rate
- Slow freezing: Form crystals & rupture cells
- Rapid freezing (liquid nitrogen): Keep/Preserve
• Freeze- Thawing of food: Unsafe (spores
vegetative forms
Desiccation
- Dried clinical specimens and dust may contain
viable microorganisms.
Radiation
- UV: • Sterilize vaccines, antisera, toxins
• May cause skin cancer, eye damage
- X-ray, gamma and beta ray:
• Gamma ray sterilizes food as meat
from Salmonella & Campylobacter
Ultrasonic Waves
- For cleaning delicate equipment
- Tank of water + short US waves then sterilize
- In dental or medical clinics, e.g. glassware
Filtration
- Different pore sizes; separate organisms
(bacteria, viruses, others) from liquid or gases
- Micro-pore filters
- High-Efficiency Particulate Air (HEPA) used
in operating rooms, patient room to filter air
Gaseous Atmosphere
- Aerobs/ Microaerophiles: Remove O2
- Anaerobic: Provide O2
• Hyperbaric Oxygen Chamber
(increased pressure) to kill m.o.
Disinfectants (Chemical Agents)
- Temporal or permanent
- Factors affecting disinfectants:
• Prior cleaning/proteinaceous materials
• Organic matter load
• Bio-burden (microbial)
• Concentration
• Time
• Physical nature
• Temperature/ pH
cont./… Disinfectants
- Susceptible: Vegetative state, fungi, protozoa,
most viruses
- Resistant: Mycobacteria, bacterial endospore,
Pseudomonas spp., fungal spores,
hepatitis viruses
- Degree of resistance (see Table)
cont./… Disinfectants
- Never use disinfectant if physical sterilization
is possible
- Do not destroy all bacteria & bacterial spores
- Spore/ Mycobacteria/Viruses destruction:
• Formaldehyde / fumigation
• Ethylene oxide
Disinfectant characteristics
• Broad
• Fast-acting
• Not affected by organic materials
• Non-Toxic; Evaporation-Concentration
• Leave residual anti-microbial film
• Soluble in water
• Inexpensive
• Stable
• Odorless
Antiseptics
- Chemicals used safely on human tissues
such as skin
- Reduce numbers of organisms on surfaces
- Used at surgical incisions, at pores of folds
of the skin
- Antiseptic soup + Brushing
Factors affecting microbial growth
1. Availability of nutrients: 92 natural elements
2. Moisture: Cells composed of 70-95% water
3. Temperature: Optimum
Minimum
Maximum
cont./…
cont./… temperature/ factors
- Thermophiles (love heat)
e.g. Cyanobacteria, Algae
- Mesophiles (moderate temperature)
e.g. most bacteria (37 Celsius)
- Psychrophiles (love cold)
e.g. Psychrotrophs (4 C; bread molds)
Psychroduric (warm to very cold)
4. pH
- Neutral – slightly alkaline (7.0-7.4); most
- Acidophiles (2.0-5.0); human stomach m.o.
- Alkaliphiles (> 8.0); Vibrio cholerae
5. Osmotic pressure
- Solutes: Dissolved substances
- Hypertonic: Higher concentration than in a cell
- Osmosis: Movement of solvent (e.g. water)
• Crenation (shrink): RBC loss water
• Plasmolysis: Bacterial cell membrane
and cytoplasm shrink from cell wall
• Opposite: Swell (RBC)
Plasmoptysis (Bacteria)
Halophilic: Salt-loving organisms
e.g. Vibrio parahemolyticus
Haloduric: Do not prefer salty environment but
can tolerate it
e.g. Staphylococcus aureus
6. Barometric pressure
- 14.7 pounds per square inch (psi)- Normal
e.g. most bacteria
- Piezophiles: Tolerate very high pressure
e.g. Archaea
7. Gaseous atmosphere
- Discussed previously
Bacterial growth
- Increase in number of organisms (not size)
- Proliferation/multiplication
- Binary fission (two identical daughter cells)
- Form colonies (piles)
Generation time - Rapid and Slow growers:
• 20 min. E. coli, Staph., Strept., V. cholera
• 10 min. Pseudomonas, Clostridium
• 18-24 hour: Mycobacterium
Culture media
- Artificial media/ synthetic media
- Chemically defined: Known ingredients
- Complex medium: Exact contents unknown
- Solid + AGAR (polysaccharides from red
marine alga)
- liquid (broth)
Enriched medium
Extra rich supply (nutrients) added:
• Nutrient agar; PLUS:
• Blood agar: 5% sheep RBC
• Chocolate agar (lysed RBC- Hb available)
- For Fastidious microorganism
(complex nutritional requirements)
e.g. Neisseria gonorrheae,
Haemophilus influenzae
Selective medium
Inhibitors added to discourage unwanted microbes
- MacConkey’s : • Selective for gram • Inhibit gram +
- Mannitol salt agar: • For haloduric
Differential medium
Differentiate between microorganisms:
• MacConkey’s agar: Lactose sugar
- Lactose fermenter (LF)
- Non-Lactose fermenter (NLF)
• MSA
• Blood agar: Hemolysis (alpha, beta, gamma)
• Thioglycollate broth (THIO): O2 gradient
Inoculation of culture medium
- Streaking
- Aseptic technique- Prevent contamination:
• Professionals
• Environment
• Specimens/ cultures/subcultures
Incubation
- Incubators:
• CO2 (5% to 10%), Capnophiles
• Non-CO2 (20% to 21% O2)
• Anaerobic (Devoid of O2)
- Pure culture: One bacterial species
- 35 to 37 C
Bacterial population count
- Contamination of fluid/food
- Total number: Spectrophotometry- turbidity
- Viable count: . serial dilutions
. Aliquots & subcultures
No. of colonies X Dilution = Count per ONE ml
Q. If 0.1 ml inoculated?-
X 10
- UTI (viable count important)
Bacterial population growth curve
- Logarithm of numbers of bacteria (viable) vs
Time
- Four phases
• Lag phase
• Logarithmic (exponential) phase
• Stationary phase
• Death (decline) phase
Lag phase
- Prepare for cell division
- Do not increase in numbers
Logarithmic (Log) growth phase
- Multiply rapidly; growth rate is the greatest
- Usually short
Stationary phase
- Division slows (nutrients
, wastes
- Numbers of dividing = dying bacteria
- Density of culture: Greatest
)
Death (Decline) phase
- Death, or spores formed
- Change morphology, e.g. filamentous form
protoplast/ spheroblast (L-form- no cell wall)
Virus cultures
- Embryonated chicken eggs, Lab animals,
cell culture (monkey, rabbit, human, etc)
- Cytopathic effect (CPE); examination
Fungal culture
- Brain-Heart infusion
- Sabouraud Dextrose Agar (SDA)
- Anti-microbial agents added, pH 5.6
Protozoa culture: Example Amoeba