Microbial Control

Your Cell Phone Is 10 Times Dirtier Than a Toilet

Seat
http://time.com/4908654/cell-phone-bacteria/

Abigail Abrams August 23, 2017

• Cellphones carry 10 times more bacteria than most toilet seats, so it
shouldn't be surprising that a man in Uganda reportedly contracted
Ebola after stealing one.

• He stole the phone from a quarantined ward of a hospital, near the

site of a recent Ebola outbreak.

• While toilets tend to get cleaned frequently, because people

associate the bathroom with germs, cellphones and other commonly
handled objects — like remote controls— are often left out of the
cleaning routine.

• Cellphones pick up germs all the time; some people talk on their
phone on toilets.
• However, the amount of germs on a phone isn't a problem — it’s the
sharing of phones between people. Without sharing, each phone
carries just one set of germs, and won't get its owner sick.

• The problem with phones is that we're in constant contact with

them, and they spend a lot of time in close proximity to our faces
and mouths.

• And, because it's an electronic device, most people are hesitant

about cleaning them.
 5

• This is also this case with remote controls, which, are also often used
by people when they're sick.

• Remotes are more frequently shared, too, so they're usually even

worse than phones for spreading germs.

• Other common culprits that are hotspots of unseen disease include

office phones, shopping carts and the first-floor buttons of elevators.
SLOs
• Define sterilization, disinfection, antisepsis, sanitization, biocide, germicide,
bacteriostasis, and asepsis.
• Describe the microbial death curve.
• Describe the effects of microbial control agents on cellular structures.
• Compare effectiveness of moist heat (autoclaving, pasteurization) vs .dry heat.
• Describe how filtration, low temperature, high pressure, desiccation, and osmotic
pressure suppress microbial growth.
• Explain how radiation kills cells.
• List the factors related to effective disinfection.
• Interpret results of use-dilution tests and the disk-diffusion method.
• Identify some methods of action and preferred uses of chemical disinfectant
• Differentiate between halogens used as antiseptics and as disinfectants.
• Identify the appropriate uses for surface-active agents.
• List the advantages of glutaraldehyde over other chemical disinfectants.
• Identify the method of sterilizing plastic labware.
• Explain how microbial control is affected by the type of microbe.
 Controlling Microorganisms
• It is essential to be able to kill the undesirable microorganisms or
inhibit their growth to minimize their destructive effects.

• Physical, chemical, and mechanical methods to destroy or reduce

undesirable microbes in a given area.

• Primary targets are microorganisms capable of causing infection or

spoilage:
▫ vegetative bacterial cells and endospores
▫ fungal hyphae and spores, yeast
▫ protozoan trophozoites and cysts
▫ worms
▫ viruses
 8

Basic Principles of Microbial Control

Conditions Influencing Antimicrobial
Activity

• Under most circumstances, a microbial population is not killed

instantly by an agent but instead over a period of time

• The death of the population over time is exponential, similar to the

growth during log phase
Conditions Influencing Antimicrobial
Activity
• Several critical factors play key roles in determining the effectiveness
of an antimicrobial agent, including:
▫ Population size
▫ Types of organisms
▫ Concentration of the antimicrobial agent
▫ Duration of exposure
▫ Temperature
▫ pH
▫ Organic matter
▫ Biofilm formation

https://www.cdc.gov/infectioncontrol/guidelines/disinfection/
efficacy.html
 11

The Selection of Microbial Control Methods

• Factors Affecting the Efficacy of Antimicrobial

Methods

▫ Site to be treated
● Harsh chemicals and extreme heat cannot be used on humans,
animals, and fragile objects
● Method of microbial control based on site of medical procedure
 12

Relative susceptibilities of microbes to antimicrobial agents

Figure 9.2
Resistance of Microorganisms
Spores Sterilization
bacterial, fungal
Bacillus stearothermophilus
Bacillus subtilis
Clostridium sporogenes

High Level Disinfection

Mycobacteria, TB bacilli

Intermediate Disinfection
Hydrophilic viruses
Polio, Coxsackie, Rhino

Vegetative fungi &

Low Disinfection
bacteria
Lipophilic viruses Trichophyton, Cryptococcus,Candida
Pseudomonas, Staphylococcus,Salmonella
HSV, CMV, RSV, HBV, HIV
The Selection of Microbial Control Methods

• Factors Affecting the Efficacy of

Antimicrobial Methods

▫ Relative susceptibility of microorganisms

● Germicides classified as high, intermediate, or low effectiveness
● High-level kill all pathogens, including endospores
● Intermediate-level kill fungal spores, protozoan cysts, viruses,
and pathogenic bacteria
● Low-level kill vegetative bacteria, fungi, protozoa, and some
viruses
Rate of Microbial Death
Bacterial populations subjected to heat or antimicrobial chemicals
die at a constant rate.

Microbial Death
Curve, plotted
logarithmically, shows
this constant death rate
as a straight line.

Rate: 90% / min

Figure 7.1a
• A solution containing a million bacteria would take longer to sterilize
than one containing a half-million bacteria.
Methods of Sterilisation
 CLASSIFICATION

• Sterilization process can be basically separated as Terminal and

Non Terminal process based on stage at which the preparation is
subjected to the process of sterilization.
Terminal Sterilization: the process whereby product is sterilized
within its sterile barrier system. Different types of terminal
sterilization techniques employed are physical sterilization and
chemical sterilization.
Non Terminal Sterilization (Aseptic processing): process whereby
product is not sterilized in a sterile barrier system and hence not
protected from environmental and handling contamination after
the sterilization cycle. It is a processing technique ensures aseptic
guarantee level by sterilization filtration or aseptic operation under
sterile conditions.
 Method of Control

• physical or chemical?
▫ physical control includes heat, irradiation, filtration and
mechanical removal
▫ chemical control involves the use of antimicrobial chemicals
▫ depends on the situation
▫ degree of control required

antimicrobial
air filters chemicals
Physical Methods of Microbial Control

Heat is very commonly used, heat may be moist or dry.

• Heat

▫ Thermal death point (TDP): Lowest temperature at which all cells in

a culture are killed a given time.

▫ Thermal death time (TDT): Time to kill all cells in a culture at a given
temperature
 24

Physical Methods of Microbial Control

• Heat-Related Methods
▫ Effects of high temperatures

● Denature proteins
● Interfere with integrity of cytoplasmic membrane and cell wall
● Disrupt structure and function of nucleic acids
▫ Thermal death point
● Lowest temperature that kills all cells in broth in 10 min
▫ Thermal death time
● Time to sterilize volume of liquid at set temperature
 25

Physical Methods of Microbial Control

• Heat-Related Methods
▫ Moist heat

● Used to disinfect (kill organisms and remove spores), sanitize

(kill organisms but not necessarily their spores), and sterilize
(kill all organisms and spores)
● Denatures proteins and destroys cytoplasmic membranes
● More effective than dry heat
● Methods of microbial control using moist heat
● Boiling
● Autoclaving
● Pasteurization
● Ultrahigh-temperature sterilization
 26

Physical Methods of Microbial Control

• Heat-Related Methods
▫ Moist heat

● Boiling
● Kills vegetative cells of bacteria and fungi, protozoan
trophozoites, and most viruses
● Boiling time is critical
▫ Different elevations require different boiling times
● Endospores, protozoan cysts, and some viruses can survive
boiling
Moist Heat

• Moist heat denatures

proteins
• Autoclave: Steam under
pressure
• Pressure applied to boiling
water prevents steam from
escaping
• Boiling temperature
increases as pressure
increases
• Autoclave conditions –
121ºC, 15 psi, 15 min
 28

The relationship between temperature and pressure

Figure 9.5
 29

Physical Methods of Microbial Control

• Heat-Related Methods
▫ Moist heat

● Pasteurization
● Used for milk, ice cream, yogurt, and fruit juices
● Not sterilization
▫ Heat-tolerant microbes survive
● Pasteurization of milk
▫ Batch method
▫ Flash pasteurization (High temp, short time)
▫ Ultrahigh-temperature pasteurization (very high temp,
very short time)
Physical Methods of Microbial Control

• Pasteurization reduces spoilage

organisms and pathogens
• Equivalent treatments
▫ 63°C for 30 min
▫ High-temperature short-time 72°C for
15 sec
▫ Ultra-high-temperature: 140°C for <1
sec
▫ Thermoduric organisms survive
Physical Methods of Microbial Control
• Heat-Related Methods
▫ Moist heat

● Ultrahigh-temperature sterilization
● 140ºC for 1 sec, then rapid cooling
● Treated liquids can be stored at room temperature
Physical Methods of Microbial Control

• Heat-Related Methods
▫ Dry heat

● Used for materials that cannot be sterilized with moist heat

● Denatures proteins and oxidizes metabolic and structural
chemicals
● Requires higher temperatures for longer time than moist heat
● Incineration is ultimate means of sterilization
Heat Sterilization

Dry Heat:
▪ Examples of dry heat sterilization are
▪ 1) Incineration
▪ 2) Red heat
▪ 3) flamming

Direct Flaming: Used to sterilize inoculating loops and needles.

Heat metal until it has a red glow.
Incineration: Effective way to sterilize disposable items (paper
cups, dressings) and biological waste.
Hot Air Sterilization: Place objects in an oven. Require 2 hours
at 170oC for sterilization. Dry heat is transfers heat less
effectively to a cool body, than moist heat.
• Hot air oven
There are various temperatures and periods of treatment for dry
heat depending on the pharmacopeia.
The U.S Pharmacopeia states that the dry heat sterilization
process for containers for sterile pharmaceutical products should be
at a temperature of 160-170 0C for a period of 2-4 hr.
The British Pharmacopeia states that items sterilized by dry
heat should be kept at a temperature not less than 160 0C for at least
1 hr.
The benefit of dry heat includes good penetrability and non
corrosive nature which makes it applicable for sterilizing glass ware
and metal surgical instruments also non aqueous thermo stable
liquids and thermo stable powders.
• Hot Air Oven :-
Dry heat sterilization is usually carried
out in a hot air oven , which consists of
the following..
1) An insulated chamber surrounded by
an outer case containing electric heater.
2) A fan.
3) Thermocouples.
4) Temperature Sensor.
5) Door locking controls.
▪ Operation :
Articles to be sterilized are first wrapped
or enclosed in a container of card board
paper or aluminium. Hot Air Oven

Then the material are arranged to

ensure uninterrupted air flow.
Precautions:

•Glass wares should be dry.

•Oven should not be over loaded.
•Articles are to be arranged in a manner to allow free circular of air.
•Door of the Oven should be opened after it cools down (2 hours).

Temperature (C) Holding time

(minutes)
160 45

170 18

180 7.5

190 1.5

37
Physical Methods of Microbial Control

• Refrigeration and Freezing

Low Temperature: Effect depends on microbe and treatment applied.

● Refrigeration: Temperatures from 0 to 7oC. Bacteriostatic effect.
Reduces metabolic rate of most microbes so they cannot reproduce or
produce toxins.
● Freezing: Temperatures below 0oC.

▫Decrease microbial metabolism, growth, and reproduction

● Chemical reactions occur slower at low temperatures
● Liquid water not available
▫Psychrophilic microbes can multiply in refrigerated foods
▫Refrigeration halts growth of most pathogens
▫Slow freezing more effective than quick freezing
▫Organisms vary in susceptibility to freezing
Physical Methods of Microbial Control

• Dessication and Lyophilization

▫ Dessication is drying (98% of the water is removed) inhibits

growth due to removal of water
▫ Lyophilization (freeze-drying)
● Substance is rapidly frozen and sealed in a vacuum
● Substance may also be turned into a powder
▫ Used for long-term preservation of microbial cultures
● Prevents formation of damaging ice crystals
Physical Methods of Microbial Control

Desiccation: In the absence of water, microbes cannot grow or reproduce,

but some may remain viable for years. After water becomes available, they
start growing again.

Susceptibility to desiccation varies widely:

Neisseria gonnorrhea: Only survives about one hour.
Mycobacterium tuberculosis: May survive several months.
Viruses are fairly resistant to desiccation.
Clostridium spp. and Bacillus spp.: May survive decades.
Physical Methods of Microbial Control

Filtration: Removal of microbes by passage of a liquid or gas through

a screen like material with small pores. Used to sterilize heat
sensitive materials like vaccines, enzymes, antibiotics, and some
culture media.

Membrane Filters: Uniform pore size. Used in industry and

research. Different sizes:
0.22 and 0.45um Pores: Used to filter most bacteria. Don’t
retain spirochetes, mycoplasmas and viruses.
0.01 um Pores: Retain all viruses and some large proteins.

High Efficiency Particulate Air Filters (HEPA): Used in

operating rooms to remove bacteria from air.
Physical Methods of Microbial Control

Filtration
• used for heat sensitive fluids
• air
 44

Filtration equipment used for microbial control

45
 46

The role of HEPA filters in biological safety

cabinets

High-Efficiency Particulate
Arresting (HEPA) air
filters are used in medical
facilities, automobiles,
aircraft, and homes. The
filter must remove 99.97%
of all particles greater than
0.3 micrometer from the
air that passes through.

Figure 9.10
Physical Methods of Microbial Control
• Osmotic Pressure

Osmotic Pressure: The use of high concentrations of salts and sugars in

foods is used to increase the osmotic pressure and create a hypertonic
environment 🡪 inhibit growth
Plasmolysis: As water leaves the cell, plasma membrane shrinks away
from cell wall.
● Yeasts and molds: More resistant to high osmotic pressures.
● Staphylococci spp. that live on skin are fairly resistant to high osmotic
pressure.

▫ Cells in hypertonic solution of salt or sugar lose water

▫ Fungi have greater ability than bacteria to survive hypertonic
environments
Physical Methods of Microbial Control

Electromagnetic spectrum, shorter wavelength

means great energy and can penetrate further.
• Radiation damages DNA
• Two types used:
▫ Ionizing radiation (X rays, gamma rays,
electron beams)
▫ Non-ionizing radiation (UV)
▫ (Microwaves kill by heat; not especially
antimicrobial)
Electromagnetic Spectrum

Figure 7.5
 50

Physical Methods of Microbial Control

• Radiation
▫ Ionizing radiation

● Wavelengths shorter than 1 nm

● Electron beams, gamma rays
● Ejects electrons from atoms to create ions
● Ions disrupt hydrogen bonding, cause oxidation, and create
hydroxide ions
● Hydroxide ions denature other molecules (DNA)
● Electron beams – effective at killing but do not penetrate well
● Gamma rays – penetrate well but require hours to kill microbes
 51

Increased shelf life of food achieved by ionizing

radiation

radura
Physical Methods of Microbial Control
• Radiation
▫ Nonionizing radiation (UV)
● Wavelengths greater than 1 nm
● Excites electrons, causing them to make new covalent bonds
● Affects 3-D structure of proteins and nucleic acids
● UV light causes pyrimidine dimers in DNA which cause mutations.
● UV light does not penetrate well
● Suitable for disinfecting air, transparent fluids, and surfaces of objects
● Used to disinfect operating rooms, nurseries, cafeterias.
Disadvantages: Damages skin, eyes. Doesn’t penetrate paper, glass, and
cloth.
 53

Physical Methods of Microbial Control

• Biosafety Levels
▫ Four levels of safety in labs dealing with pathogens

● Biosafety Level l (BSL-1)

● Handling pathogens that do not cause disease in healthy
humans
● Biosafety Level 2 (BSL-2)
● Handling of moderately hazardous agents
● Biosafety Level 3 (BSL-3)
● Handling of microbes in safety cabinets
● Biosafety Level 4 (BSL-4)
● Handling of microbes that cause severe or fatal disease
A BSL-4 worker carries
Ebola virus cultures
 Agent
Moist Heat, boiling
Moist Heat, Autoclaving
Moist Heat, Pasteurization
Dry Heat, Flaming
Dry Heat, Hot air oven
Filtration
Cold, Lyophilization (also
desiccation)
Cold, Refrigeration
Osmotic Pressure, Addition of
salt or sugar
Radiation, UV
Radiation, X-rays
Strong vis. Light
Mechanisms of Action
Denatures proteins
Denatures proteins
Denatures proteins
Incineration of contaminants
Oxidation & Denatures proteins
Separation of bacteria from
liquid (HEPA or ULPA: from air)
Desiccation and low
temperature
Decreased chemical reaction
rate
Plasmolysis of contaminants
DNA damage (thymine dimers)
DNA damage
Comments
Kills vegetative bacterial cells and viruses
Endospores survive
121°C at 15 p.s.i. for 30 min kills
everything
Kills pathogens in food products
Used for inoculating loop
170°C for 2 hours; Used for glassware &
instrument sterilization
Used for heat sensitive liquids
Used for food & drug preservation; Does
not necessarily kill so used for Long-term
storage of bacterial cultures
Bacteriostatic
Used in food preservation (less effective
against fungi)
Limited penetration
Used for sterilizing medical supplies
Line-drying laundry
 Control of M.O's by chemical agents

• Also called as BIOCIDES

• Factors influencing on microbial chemical agent :

The kinds of microorganisms potentially present

The concentration and nature of the disinfectant to be used
The disinfectant should be stable upon storage , odorless or with
odor, soluble in water and lipids for permeation into
microorganisms, and a low surface tension so that it can enter
cracks in surfaces
The main factor is : chemical must be toxic for infectious agents,
it should not be toxic to people or corrosive for common
materials in practice, this balance between effectiveness and low
toxicity for animals is hard to achieve.
Chemical Methods of Microbial Control
• Affect microbes’ cell walls, cytoplasmic membranes, proteins, or
DNA
• Effect varies with differing environmental conditions
• Often more effective against enveloped viruses and vegetative cells
of bacteria, fungi, and protozoa
 Chemical Methods of Microbial Control
• Principles of effective disinfection
▫ Concentration of disinfectant
▫ Organic matter
▫ pH
▫ Time

Often chemical methods are compared to Phenol. If more effective given a

phenol coefficient of >1. If less effective give a phenol coefficient of
<1
Chemical Methods of Microbial Control

• Phenol and Phenolics

▫ Intermediate- to low-level disinfectants

▫ Denature proteins and disrupt cell membranes
▫ Effective in presence of organic matter
▫ Remain active for prolonged time
▫ Commonly used in health care settings, labs, and homes
▫ Have disagreeable odor and possible side effects
• Example :- phenyl ethanol for eye drops and contact lens solution,
benzyl alcohol in injections.

PHENYL BENZYL
ETHANOL ALOHOL
 61

Phenol Coefficient
• The number obtained by dividing the degree of dilution of test
disinfectant by the degree of dilution of phenol in a certain span
of time.
• Comparison of the efficacy of an antimicrobial agent compared to phenol.
• The phenol coefficient of less than 1 indicates that the antimicrobial agent is
less effective than phenol.
• Method: the highest dilution level of antimicrobial compound that kills
microbes in 10 minutes but does not kill in 5 minutes divided by the highest
dilution level of phenol that kills microbes in 10 minutes but does not kill in
5 minutes
• Phenol Coefficient =
• (highest dilution antiseptic, where bacteria do not grow at 10' but grow at 5’)
divided by
• (highest dilution of phenol, where bacteria do not grow at 10', but grow at
5')
 Dilution ratio Time (minute)
5 10
Fenol 1: 50 + -
1:100 + -
1:150 - -
Wipol 1:25 - +
1:50 - -
1:75 + -

Phenol coefficient data:

*(+) bacterial colonies grow
(-) no growth of bacterial colonies

Calculation:
Phenol coefficient= 75/100 = 0,75
 63

https://www.biologyonline.com/dictionary/phenol-coefficient
Chemical Methods of Microbial Control

• Alcohols

▫ Intermediate-level disinfectants
▫ Denature proteins and disrupt cytoplasmic membranes
▫ More effective than soap in removing bacteria from hands
▫ Swabbing of skin with 70% ethanol prior to injection
▫ Kill bacteria, fungi, but not endospores or naked viruses.
▫ Not good for open wounds, because cause proteins to coagulate.
● Ethanol: Drinking alcohol. Optimum concentration is 70%.
● Isopropanol: Rubbing alcohol. Better disinfectant than ethanol. Also
cheaper and less volatile.
Chemical Methods of Microbial Control

• Halogens
• Intermediate-level antimicrobial chemicals, effective alone or in compounds
• Believed to damage enzymes via oxidation or by denaturation
● A. Iodine: Iodine tincture (alcohol solution) was one of first antiseptics
used.
● B. Chlorine: When mixed in water forms hypochlorous acid:
Cl2 + H2O ------> H+ + Cl- + HOCl
Hypochlorous acid
Used to disinfect drinking water, pools, and sewage.

• The halogens Iodine and Chlorine are important antimicrobial agents.

• Widely used in numerous applications
Iodine
• Used as a skin antiseptic and kills by oxidizing cell constituents and
iodinating cell proteins.
• At high conc it may kill some spores .
• Iodine often has been applied as tincture of iodine, 2% or more iodine in
water – ethanol solution of potassium iodide (lugol’s iodine)
• Although it is an effective antiseptic, the skin may be damaged, a stain is
left, and iodine allergies can result.
• The staining and irritant properties of iodine have resulted in the
development of IODOPHORES, mixtures of iodine with surface active
agents which hold the iodine in micellar combination from which it
released slowly.
• Some popular brands are Wescodyne for skin and Betadine (polyvinyl
pyrrolidone iodine formulated as 10% povidone iodine) used as a non
staining, non irritant antiseptic for wounds.
Chlorine

• Chlorine is usual disinfectant for municipal water supplies.

• One potential problem is that chorine reacts with organic compounds to
form carcinogenic trihalomethanes which must be monitored in drinking
water.
• Ozone some times has been used successfully as a alternative to
chlorination in Europe and Canada.
• Traditional chlorine containing pharmaceutical formulations are Eusol-
chlorinated lime and boric acid solution and Dakin’s solution (surgical
chlorinated soda solution) both of which are designed to provide slow
release of chlorine.
• Sometimes alternatively Chloramine T used for prolonged release of
chlorine.
• Halazone slowly released chloride when added to water and disinfects.
Chemical Methods of Microbial Control

• Oxidizing Agents

▫ Peroxides, ozone, and peracetic acid

▫ Kill by oxidation of microbial enzymes, disrupt membranes and proteins
▫ High-level disinfectants and antiseptics
▫ Example: Hydrogen peroxide (H2O2)< Ozone, Peraetic Acid
▫ Peracetic acid is an effective sporocide used to sterilize equipment
Chemical Methods of Microbial Control
Types of Disinfectants

Oxidizing Agents:

A. Ozone:
● Used along with chlorine to disinfect water.
● Helps neutralize unpleasant tastes and odors.
● More effective killing agent than chlorine, but less stable and more
expensive.
● Highly reactive form of oxygen.
● Made by exposing oxygen to electricity or UV light

B. Hydrogen Peroxide (H2O2):

● Not good for open wounds because quickly broken down by catalase
present in human cells.
● Effective in disinfection of inanimate objects
Chemical Methods of Microbial Control
• Surfactants

▫ “Surface active” chemicals

● Reduce surface tension of solvents
▫ Soaps and detergents
● Soaps have hydrophilic and hydrophobic ends
● Good degerming agents but not antimicrobial
● Detergents are positively charged organic surfactants
▫ Quats (Quaternary ammonium cations)
● Low-level disinfectants; disrupts cell membranes
● Ideal for many medical and industrial applications
● Good against fungi, amoeba, and enveloped viruses, but not
endospores, Mycobacterium tuberculosis and non-enveloped
viruses.
Chemical Methods of Microbial Control

• Heavy Metals

▫ Heavy-metal ions denature proteins

▫ Include copper, selenium, mercury, silver, and zinc.
▫ Very tiny amounts are effective.
▫ Low-level bacteriostatic and fungistatic agents
▫ 1% silver nitrate to prevent blindness on infants against gonorrheal eye
infections caused by N. gonorrhoeae (now has ben replaced by
erythromycin)
▫ Organic mercury compounds like merthiolate and mercurochrome are
used to disinfect skin wounds. Thimerosal used to preserve vaccines
▫ Copper inhibits algal growth in pools and fish tanks
Chemical Methods of Microbial Control

• Aldehydes

▫ Compounds containing terminal –CHO groups

▫ Cross-link functional groups to denature proteins and inactivate
nucleic acids
▫ Glutaraldehyde disinfects and sterilizes
▫ Formalin used in embalming and disinfection of rooms and
instruments
Chemical Methods of Microbial Control
Types of Disinfectants

Aldehydes:

A. Formaldehyde:
● Excellent disinfectant, 2% aqueous solution.
● Commonly used as formalin, a 37% aqueous solution.
● Formalin was used extensively to preserve biological specimens and
inactivate viruses and bacteria in vaccines.
● Irritates mucous membranes, strong odor.

B. Glutaraldehyde:
● Less irritating and more effective than formaldehyde.
● Commonly used to disinfect hospital instruments.
Chemical Methods of Microbial Control

• Gaseous Agents

▫ Microbicidal and sporicidal gases used in closed chambers to

sterilize items
▫ Denature proteins and DNA by cross-linking functional groups
▫ Used in hospitals and dental offices
▫ Disadvantages
● Can be hazardous to people
● Often highly explosive
● Extremely poisonous
● Potentially carcinogenic
 GASEOUS STERILIZATION

• The chemically reactive gases such as formaldehyde and ethylene oxide

(CH2)2O possess biocidal activity.
• Ethylene oxide is a liquid at temperature below 10.8 0C above this
temperature it vaporizes rapidly.
• Ethylene oxide is a colorless, odorless, & flammable gas.
• So this flammable nature overcome by preparing mixtures of ethylene
oxide in carbon dioxide which is now available commercially.
• The CO2 – Ethylene oxide are non flammable and there is NO alteration
of the microbial activity of the ethylene oxide.
• The mechanism of Anti microbial action of these gases assumed to be
ALKYLATION of sulfhydryl , amino, hydroxyl and carboxylic groups on
proteins and amino groups of nucleic acids.
• Both of these gases being Alkylating agents are potentially Mutagenic &
Carcinogenic.
• They also produce acute toxicity including irradiation of the skin ,
conjunctiva & nasal mucosa.
• It is used for sterilizing heat or moisture sensitive materials in hospitals,
industry & laboratories has become Universal.
Chemical Methods of Microbial Control
• Enzymes

▫ Antimicrobial enzymes act against microorganisms

▫ Human tears contain lysozyme
● Digests peptidoglycan cell wall of bacteria
▫ Enzymes to control microbes in the environment
● Lysozyme used to reduce the number of bacteria in cheese
● Prionzyme can remove prions on medical instruments
 Agent Mechanisms of Action Comments
Surfactants Membrane Disruption; Soaps; detergents
increased penetration
Quats (cationic Denature proteins; Disrupts Antiseptic - benzalconium chloride,
detergent) lipids Cepacol; Disinfectant
Organic acids and High/low pH Mold and Fungi inhibitors; e.g., benzoate
bases of soda
Heavy Metals Denature protein Antiseptic & Disinfectant; Silver Nitrate

Halogens Oxidizing agent Antiseptic - Iodine (Betadine)

Disrupts cell membrane Disinfectant - Chlorine (Chlorox)
Alcohols Denatures proteins; Disrupts Antiseptic & Disinfectant
lipids Ethanol and isopropyl
Phenolics Disrupts cell membrane Disinfectant
Irritating odor
Aldehydes Denature proteins Gluteraldehyde - disinfectant (Cidex);
Formaldehyde - disinfectant

Ethylene Oxide Denaturing proteins Used in a closed chamber to sterilize

Oxidizing agents Denature proteins Hydrogen peroxide – antiseptic;

Hydrogen peroxide – disinfectan; Benzoyl
peroxide – antiseptic
 STERILIZATION INDICATORS

Mechanical Monitors:
❑ Devices that record time, temperature &
pressure.

Biological Indicators:
❑ They are standardized preparation of spores.
❑ A positive biological indicator is indicative of
possible sterilization process failure.
❑ They should be used atleast once a week but
❑ Time needed for incubation is long.
E.g. spores of Bacillus sterothermophilus.
Efficient Autoclaving

• All instruments must be double

wrapped in linen or special paper
or placed in a special metal box
equipped with a filter before
sterilization.
• The white stripes on the tape
change to black when the
appropriate conditions
(temperature) have been met.
• Expiration dates should be printed
on all equipment packs.
• There should be a uniform
development of bars throughout
the length of the strips.
• Ready made plastic bags with
strips printed with a sensitive ink.
Control of autoclave efficiency

• Bowie-Dick test: Used for high-vacuum

autoclaves, Done every day:
▫ In the middle of a test pack of towel, a
paper on to which a strip of a specific
tape is put, for testing.
▫ Uniform development of dark color
indicates that the steam has passed
freely and rapidly to the center of the
load.
• Biological sterilization indicators:
Spores of a non-pathogenic organism:
Done weekly
▫ They are killed at 121°C after 15 minutes.
▫ Attempts to culture them is
subsequently made

Autoclaves should be tested periodically

with Biological Indicators like of
Bacillus sterothermophilus to ensure
proper function.
Biological indicators

• Preparations of selected microorganisms with high resistance towards

specific sterilization process.

• 3 types:
▫ Spores that are added to a carrier (strips or disk of filter paper, glass,
plastic, or other materials).
▫ Spore suspension that is inoculated on or into representative units of
the products to be sterilized.
▫ A shelf-contained indicator 🡪 contain growth medium with/without
spore suspension, dye
 STERILIZATION INDICATORS
Chemical Indicators:
❑These are more practical means & detect problems immediately.
❑The CDC & all major U.S organizations standards & guidelines advocate that a
chemical indicator be attached to every package that goes through a
sterilization cycle & within each package to be sterilized in what is expected
to be the most difficult-to-sterilize location.

These are divided into 6 classes, higher the class, more sensitive the indicator
Class 1-
These are Internal & External Process Indicator
These inform that item has been exposed to sterilization process.
E.g. External Process Indicator – Autoclave Tape.
STERILIZATION INDICATORS

Class 2
❑E.g. Bowie-Dick test for vacuum steam sterilizer.
❑They only access Vacuum Pump efficiency & detect the
presence of air leaks &/or gases in steam.

Class 3
❑E.g. Temperature Tube.
❑Contains chemicals that melts & sometimes changes color
when the appropriate temp is attained.

Class 4
❑Respond to one or more sterilization parameters.
❑Contains Ink that changes color when exposed to correct
combination of sterilization parameters.
 STERILIZATION INDICATORS

Class 5
❑Known as Integrating Indicators or Integrators
❑Respond to all parameters of sterilization over a specified range
of temperatures.

Class 6
❑These are emulating indicators.
❑These are designed to react to all critical parameters over a
specified range of sterilization cycles for which the stated values
are based on the settings of the selected sterilization cycles
Biological indicators:

• These indicators use heat-resistant bacterial endospores to demonstrate

whether or not sterilization has been achieved.
• If the bacterial endospores have been killed after sterilization, you can
assume that all microorganisms have been killed as well.
• After the sterilization process the strips are placed in a broth that
supports aerobic growth and incubated for 7 days.
• The advantage of this method is that it directly measures the effectiveness
of sterilization.
• The disadvantage is that this indicator is not immediate, as are mechanical
and chemical indicators.
• Bacterial culture results are needed before sterilization effectiveness
can be determined.
 88

Biological indicators:

• Geobacillus stearothermophilus (formerly Bacillus stearothermophilus) is

a thermophilic, aerobic bacterium, which produces heat-resistant spores.
• It forms biofilms attaching to stainless steel surfaces in dairy plants.
• Although the bacterium has no public health significance, its presence
in canned foods, milk powders, and ingredients is of interest. It is the
typical species responsible for the thermophilic flat sour spoilage of
low-acid canned foods.
• The maximum growth temperature of G. stearothermophillus is between
65–75°C, with a minimum growth temperature of 40°C, and a limited
tolerance to acid. The bacterium does not grow at 37°C; its optimum
growth is at 55°C.
• Minimum pH for growth of B. stearothermophilus is 5.2; the minimum
water activity (aw) for growth at optimum temperature is 0.93.
 89

Sterility indicator
STORAGE

• After sterilization the sterilized items are kept in different racks as per
labeling.

• Supplied as per the demand of different area.

• To ensure continuous availability of sterile supply five times of daily

requirement should be available in storage.
 REMEMBER

• What went inside the machines is the

STERILISED material
• Personnel are only DISINFECTED
 CONCLUSION

• Sterilization is a special process because it’s efficacy cannot be verified by

simple inspection and testing on the final product. For this reason ,
sterilization process have to be validated before use, the performance of
the process monitored routinely and equipment regularly maintained.
• Risk assessment: Factors affecting sterilization effectiveness during
sterilization process is important.
• Biological indicators required to assess sterilization process.
Chemical Methods of Microbial Control

• Antimicrobials

▫ Antibiotics, semi-synthetic, and synthetic chemicals

▫ Typically used for treatment of disease
▫ Some used for antimicrobial control outside the body
Chemical Methods of Microbial Control

• Development of Resistant Microbes

▫ Little evidence that products containing antiseptic and

disinfecting chemicals is beneficial to human or animal health
▫ Use of such products promotes development of resistant microbes
Mechanisms of Antimicrobial Action

• Inhibition of bacterial wall synthesis

• Disruption of existing cytoplasmic membranes
• Inhibition of Protein Synthesis
• Inhibition of Nucleic Acid Synthesis
• Inhibition of Metabolic Pathways
Basic Principles of Microbial Control

• Action of Antimicrobial Agents

▫ Alteration of cell walls and membranes

● Cell wall maintains integrity of cell

● Cells burst due to osmotic effects when damaged
● Cytoplasmic membrane controls passage of chemicals into and
out of cell
● Cellular contents leak out when damaged
● Non-enveloped viruses have greater tolerance of harsh
conditions
Mechanisms of Antimicrobial Action

• Inhibition of Cell Wall Synthesis

▫ Inhibition of bacterial wall synthesis

● Most common agents prevent cross-linkage of NAM-NAG

subunits
● Beta-lactams are most prominent in this group
● Functional groups are beta-lactam rings
● Beta-lactams bind to enzymes that cross-link NAM-NAG
subunits
● Bacteria have weakened cell walls and eventually lyse
Mechanisms of Antimicrobial Action

• Inhibition of Cell Wall Synthesis

▫ Inhibition of synthesis of bacterial walls

● Semi-synthetic derivatives of beta-lactams

● More stable in acidic environments
● More readily absorbed
● Less susceptible to deactivation
● More active against more types of bacteria
● Simplest beta-lactams – effective only against aerobic
Gram-negatives
Mechanisms of Antimicrobial Action

• Inhibition of Cell Wall Synthesis

▫ Inhibition of synthesis of bacterial walls

● Prevent bacteria from increasing amount of peptidoglycan

● Have no effect on existing peptidoglycan layer
● Effective only for growing cells
Mechanisms of Antimicrobial Action

• Disruption of Cytoplasmic Membranes

▫ Some drugs form channels through cytoplasmic membrane and

damage its integrity
▫ Amphotericin B attaches to ergosterol in fungal membranes
● Humans somewhat susceptible because cholesterol similar to
ergosterol
● Bacteria lack sterols; not susceptible
Mechanisms of Antimicrobial Action

• Disruption of Cytoplasmic Membranes

▫ Azoles and allyamines inhibit ergosterol synthesis

▫ Polymyxin disrupts cytoplasmic membranes of Gram-negatives
● Oral form is toxic to human kidneys, so only used topically
▫ Some parasitic drugs act against cytoplasmic membranes
Basic Principles of Microbial Control

• Action of Antimicrobial Agents

▫ Damage to proteins and nucleic acids

● Protein function depends on 3-D shape

● Extreme heat or certain chemicals denature proteins
● Chemicals, radiation, and heat can alter or destroy nucleic acids
● Can produce fatal mutants
● Can halt protein synthesis through action on RNA
Mechanisms of Antimicrobial Action

• Inhibition of Protein Synthesis

▫ Prokaryotic ribosomes are 70S (30S and 50S)

▫ Eukaryotic ribosomes are 80S (40S and 60S)
▫ Drugs can selectively target translation
▫ Mitochondria of animals and humans contain 70S ribosomes
● Can be harmful
Mechanisms of Antimicrobial Action
• Inhibition of Metabolic Pathways

▫ Antimetabolic agents can be effective when pathogen and host

metabolic processes differ
▫ Quinolones interfere with the metabolism of malaria parasites
▫ Heavy metals inactivate enzymes
▫ Some agents disrupt glucose uptake by many protozoa and
parasitic worms
▫ Some drugs block activation of viruses
Diffusion Susceptibility Test:
Zone of inhibition
Minimum inhibitory concentration test
A minimum bactericidal concentration test
The Selection of Microbial Control Methods

• Methods for Evaluating Disinfectants and

Antiseptics

▫ Phenol coefficient

● Evaluates efficacy of disinfectants and antiseptics by comparing

an agent’s ability to control microbes to phenol

● Greater than 1.0 indicates agent is more effective than phenol

● Has been replaced by newer methods
The Selection of Microbial Control Methods

• Methods for Evaluating Disinfectants and

Antiseptics

– Use-dilution test

• Metal cylinders dipped into broth cultures of bacteria

• Contaminated cylinder immersed into dilution of disinfectant
• Cylinders removed and placed into tube of medium to see how
much bacteria survived
• Most effective agents entirely prevent growth at highest dilution
• Current standard test in the U.S.
• New standard procedure being developed
The Selection of Microbial Control Methods
• Methods for Evaluating Disinfectants and
Antiseptics
▫ Kelsey-Sykes capacity test

● Alternative assessment approved by the European Union

● Bacterial suspensions added to the chemical being tested
● Samples removed at predetermined times and incubated
● Lack of bacterial reproduction reveals minimum time required
for the disinfectant to be effective
The Selection of Microbial Control Methods

• Methods for Evaluating Disinfectants and

Antiseptics

▫ In-use test

● Swabs taken from objects before and after application of

disinfectant or antiseptic
● Swabs inoculated into growth medium and incubated
● Medium monitored for growth
● Accurate determination of proper strength and application
procedure for each specific situation
https://www.publichealthontario.ca/en/eRepository/Case_
Study_%20Pathogens_Spices_2016.pdf