Wound healing and sterilization for MBBS students

Wound
healing
Vaibhav Trivedi
MBBS 2012
AIIMS RISHIKESH

FUNCTIONS OF THE SKIN
• Regulates body temperature.
• Prevents loss of essential body fluids, and penetration of toxic substances.
• Protection of the body from harmful effects of the sun and radiation.
• Excretes toxic substances with sweat ( waste removal).
• Mechanical support.
• Immunological function mediated by Langerhans cells.
• Sensory organ for touch, heat, cold, socio-sexual and emotional sensations.
• Vitamin D synthesis from its precursors under the effect of sunlight and
introversion of steroids.
3

Wound-definitions
- A loss of continuity of the skin or mucous membrane which may
involve soft tissues, muscles, bone and other anatomical structure.
4
- Any disruption to layers of the skin and underlying tissues
due to multiple causes including trauma, surgery, or a specific
disease state.

Open
• Open wounds can be classified according to the object that
caused the wound. The types of open wound are:
• Incisions or incised wounds, caused by a clean, sharp-edged
object such as a knife a razor or a glass splinter.
• Lacerations, irregular tear-like wounds caused by some blunt
trauma. Lacerations and incisions may appear linear (regular) or
stellate (irregular).
• Abrasions (grazes), superficial wounds in which the topmost
layer of the skin (the epidermis) is scraped off. Abrasions are
often caused by a sliding fall onto a rough surface.

• Puncture wounds, caused by an object puncturing
the skin, such as a nail or needle.
• Penetration wounds, caused by an object such as a
knife entering and coming out from the skin .
• Gunshot wounds, caused by a bullet or similar
projectile driving into or through the body. There
may be two wounds, one at the site of entry and one
at the site of exit.

Closed
• The types of closed wounds are:
• Contusions, more commonly known as bruises, caused by
a blunt force trauma that damages tissue under the skin.
• Hematomas, also called a blood tumor, caused by damage
to a blood vessel that in turn causes blood to collect under
the skin.
• Crush injury, caused by a great or extreme amount of
force applied over a long period of time.

CLASSIFICATION OF WOUNDS
• SUPERFICIAL
Involve only the epidermis and dermis and heal
without formation of granulation tissue and scar.
Deep
Involve layres deep to the dermis and heal by
formation of granulation tissue and scar.

CLASSIFICATION OF WOUNDS
• CLEAN
An operation carried out through clean non infected
skin under sterile conditions where the GI tract,GU
tract or respiratory tract is not breached.e.g. hernia
repair,varicose vein surgery.
Risk of wound infection should be less than 2%.

• CLEAN CONTAMINATED
An operation carried out under sterile conditios with
breaching of a hollow viscus other than colon,where
contamination is minimal,e.g.cholecystectomy.
Risk of wound infection should be less than 8%

• CONTAMINATED
An operation carried out where contamination has
occurred.e.g. by opening the colon,open fracture or
animal or human bites.
Risk of wound infection is 12%.

• DIRTY
An operation carried out in the presence of pus or a
perforated viscus.e.g.perforated appendicitis,fecal
peritonitis.
Risk of wound infection is 25%.

Introduction:
• A surgeon’s role in wound management is to create an
environment in which the healing process can proceed in
an optimal fashion.
• As noted by John Hunter, “. . . the injury alone has in all
cases a tendency to produce the disposition and the means
of a cure.”

Introduction contd..
The repair of tissue damage broadly separated into two processes,
regeneration and healing .
Regeneration refers to growth of cells and tissues to replace lost
structures.
Wound healing is the effort of tissues to restore normal function
and structure after injury
-To reform barriers to fluid loss and infection,
-limit further entry of foreign organisms and material,
-re-establish normal blood and lymphatic flow patterns,
-restore the mechanical integrity of the injured system.

History
The earliest accounts of wound healing date back to about 2000
B.C
 Galen of Pergamum emphasized the importance of maintaining a
moist environment to ensure adequate healing.
Ambriose Paré found that simply dressed gunshot wounds heal
faster and are less painful than when treated with boiling oil, the
previously accepted method.
Ignaz Philipp Semmelweis advocated need for washing hands
Joseph Lister began soaking his instruments in phenol and spraying
the operating rooms, reducing the mortality rates from 50 to 15%.

Phases of wound healing
Normal wound healing follows a predictable pattern that can be
divided into overlapping phases defined by characteristic cellular
populations and biochemical activities
(a) Hemostasis and Inflammation
(b) Proliferation
(c) Maturation and Remodeling

Wounding
• Blood vessels are disrupted, resulting in bleeding.
Hemostasis is the first goal achieved in the
healing process.
• Cellular damage occurs, this initiates an
inflammatory response.
• The inflammatory response triggers events that
have implications for the entire healing process.
• Step one then is hemostasis, resulting in Fibrin.

Early wound healing events
• Hemostasis
• Platelet aggregation
• Intrinsic and extrinsic coagulation cascade
• Thrombin, fibrin
• Vasoconstriction

I. Inflammatory Phase
 Represents the tissue’s attempt to limit damage
 Closely related with healing process
 Healing impossible without inflammation
The events can be divided into:
1. Vascular events
2. Cellular events

VASCULAR EVENTS
• Immediately after injury, intense vasoconstriction
leads to blanching, a process mediated by
epinephrine, NE, and prostaglandins released by
injured cells.
• Vasoconstriction reversed after 10min, by
vasodilatation.
• Now redness and warmth.
• Vasodilatation mediated by histamine, linins,
prostaglandins.

Inflammation
• As microvenules dilate, gaps form between the
endothelial cells,resulting in vascular
permeability. Plasma leaks out into extravascular
space.
• Leukocytes now migrate into the wound by
diapedesis, adhere to endothelial cells, to
wounded tissues.
• Alteration in pH from breakdown products of
tissue and bacteria, along with swelling causes the
pain.

Inflammation
• Neutrophils, macrophages and lymphocytes
come into wound.
• Neutrophils first on scene, engulf and clean up.
Macrophages then eat them or they die releasing
O2 radicals and destructive enzymes into wound.
• Monocytes migrate into extravascular space and
turn into macrophages.
• Macrophages very important in normal wound
healing.

Inflammation
• Macrophages eat bacteria, dead tissue, secrete
matrix metalloproteinases that break down
damaged matrix.
• Macrophages source of cytokines that stimulate
fibroblast proliferation, collagen production.
• Lymphocytes produce factors like FGF, EGF,
IL-2.
• At 48-72 hrs, macrophages outnumber neuts.
• By days 5-7 few remain.

Proliferation
• Mesenchymal cell chemotaxis
• Mesenchymal cell proliferation
• Angiogenesis
• Epithelialization

Proliferation
• Fibroblasts are the major mesenchymal cells involved
in wound healing,, although smooth muscle cells are
also involved.
• Normally reside in dermis, damaged by wounding.
• Macrophage products are chemotactic for fibroblasts.
PDGF, EGF, TGF, IL-1, lymphocytes are as well.

Proliferation
• Angiogenesis reconstructs vasculature in areas
damaged by wounding, stimulated by high lactate
levels, acidic pH, decreased O2 tension in tissues.
• Cytokines directly stimulate the endothelial cell
migration and proliferation required for
angiogenesis. Many are produced by Macs.
• FGF-1 is most potent angiogenic stimulant
identified. Heparin important as cofactor, TGF-
alpha, beta, prostaglandins also stimulate.

Epithelialization
• The process of epithelial renewal after injury.
• Particularly important in partial thickness
injuries, but plays a role in all healing.
• Partial thickness wounds have epidermis and
dermis damaged, with some dermis preserved.
Epithelial cells involved in healing come from
wound edges and sweat glands, sebaceous glands
in the more central portion of wound.

Epithelialization
• In contrast in an incisional wound, cellular migration
occurs over a short distance.
• Incisional wounds are re-epithelialized in 24-48h.
• The sequence of events here are cellular detachment,
migration, proliferation, differentiation.

Epithelialization
• First 24h, basal cell layer thickens, then elongate,
detach from basement membrane and migrate to
wound as a monolayer across denuded area.
• Generation of a provisional BM which includes
fibronectin, collagens type 1 and 5.
• Basal cells at edge of wound divide 48-72 h after
injury.
• Epithelial cells proliferation contributes new cells
to the monolayer. Contact inhibition when edges
come together.

Collagen
• Synthesized by fibroblasts beginning 3-5 days
after injury.
• Rate increases rapidly, and continues at a rapid
rate for 2-4 weeks in most wounds.
• As more collagen is synthesized, it gradually
replaces fibrin as the primary matrix in the
wound.
• After 4 weeks, synthesis declines, balancing
destruction by collagenase.

Collagen
• Age, tension, pressure and stress affect rate of
collagen synthesis.
• TGF-b stimulates it, glucocorticoids inhibit it.
• 19 types identified. Type 1(80-90%) most
common, found in all tissue. The primary
collagen in a healed wound.
• Type 3(10-20%) seen in early phases of wound
healing. Type V smooth muscle, Types 2,11
cartilage, Type 4 in BM.

Collagen
• Three polypeptide chains, right handed helix.
• Most polypeptide chains used in collagen assembly
are alpha chains.

Collagen
• Every third AA residue is Glycine.
• Another critical component is hydroxylation of
lysine and proline within the chains.
Hydroxyproline is necessary for this. Requires Vit
C, ferrous iron, and alpha ketoglutarate as co-
enzymes. Steroids suppress much of this,
resulting in underhydroxylated collagen, which is
incapable of making strong cross-links leading to
easy breakdown.

Wound Contraction
• Begins approximately 4-5 days after wounding.
• Represents centripetal movement of the wound edge
towards the center of the wound.
• Maximal contraction occurs for 12-15 days, although
it will continue longer if wound remains open.

Wound Contraction
• The wound edges move toward each other at an
average rate of 0.6 to .75 mm/day.
• Wound contraction depends on laxity of tissues, so a
buttocks wound will contract faster than a wound on
the scalp or pretibial area.
• Wound shape also a factor, square is faster than
circular.

Wound Contraction
• Contraction of a wound across a joint can cause
contracture.
• Can be limited by skin grafts, full better than split
thickness.
• The earlier the graft the less contraction.
• Splints temporarily slow contraction.

Remodeling
• After 21 days, net accumulation of collagen becomes
stable. Bursting strength is only 15% of normal at
this point. Remodeling dramatically increases this.
• 3-6 weeks after wounding greatest rate of increase, so
at 6 weeks you are at 80% to 90% of eventual
strength and at 6mos 90% of skin breaking strength.

Remodeling
• The number of intra and intermolecular cross-
links between collagen fibers increases
dramatically.
• A major contributor to the increase in wound
breaking strength.
• Quantity of Type 3 collagen decreases replaced
by Type 1 collagen
• Remodeling continues for 12 mos, so scar
revision should not be done prematurely.

Factors Affecting Wound Healing
• Age
• Infections
• Nutrition
• Hypoxia
• Anaemia
• Hypoperfusion
• Metabolic disorders
• Steroids and chemotherapeutic drugs
• Ionising radiation

Factors Affecting Wound Healing contd…
AGE
Aging produces intrinsic physiologic changes that result in delayed or
impaired wound healing.
 With aging, collagen undergoes qualitative and quantitative changes.
The increased incidence of cardiovascular disease, metabolic diseases
(diabetes mellitus, malnutrition, and vitamin deficiencies), cancer all
contribute to the higher incidence of wound problems in the elderly

Infections
Probably the most common cause of healing delays
• If the bacterial count in the wound exceeds 105organisms per gram
of tissue, or if any β-hemolytic streptococci are present, the wound
will not heal by any means.
• Bacteria prolong the inflammatory phase and interfere with
epithelialization, contraction, and collagen deposition.
• Endotoxins stimulate phagocytosis and release of collagenase
• Bacteria may accelerate expression or increase concentrations of

Nutrition
Precise calorie requirements for optimal healing has not been determined.
• Malnourished patients have diminished hydroxyproline accumulation (an
index of collagen deposition) into subcutaneously implanted
polytetrafluoroethylene tubes when compared to normally nourished
patients.
• Malnutrition correlates clinically with enhanced rates of wound
complications and increased wound failure after diverse surgical procedures.

Nutrition - Arginine
Arginine deficiency results in decreased wound-breaking strength and
wound collagen
The main effect of arginine on wound healing is to enhance wound
collagen deposition.
As increases in breaking strength during the first weeks of healing are
directly related to new collagen synthesis
Arginine supplementation may result in an improvement in wound strength
as a consequence of enhanced collagen deposition

Nutrition - Vitamin A
• Deficiency impairs wound healing, whereas supplemental vitamin A benefits
wound healing in non deficient humans and animals.
• Vitamin A increases the inflammatory response in wound healing, probably by
increasing the lability of lysosomal membranes.
• There is an increased influx of macrophages, with an increase in their activation
and increased collagen synthesis.
• Directly increases collagen production and epidermal growth factor receptors when
it is added in vitro to cultured fibroblasts.
• Supplemental vitamin A can reverse the inhibitory effects of corticosteroids on
wound healing.

Nutrition - Scurvy, or vitamin C deficiency
 Leads to a defect in wound healing, particularly via a failure in collagen synthesis
and cross-linking.
 Vitamin C is required for the conversion of proline and lysine to hydroxyproline
and hydroxylysine, respectively.
 Vitamin C deficiency has also been associated with an increased incidence of
wound infection
Zinc
 In deficiency states there is decreased fibroblast proliferation, decreased collagen
synthesis, impaired overall wound strength, and delayed epithelialization.

Hypoxia, Anemia, and Hypoperfusion
Low oxygen tension has a profoundly deleterious effect on all aspects of wound
healing.
• Fibroplasia is significantly impaired by local hypoxia.
• Optimal collagen synthesis requires oxygen as a cofactor
• Factors affecting local oxygen delivery
-systemic reasons (low volume or cardiac failure)
-local causes (arterial insufficiency, local vasoconstriction, or excessive tension
on tissues).
• The level of vasoconstriction of the subcutaneous capillary bed is exquisitely
responsive to fluid status, temperature, and hyperactive sympathetic tone as is
often induced by postoperative pain.

Steroids and Chemotherapeutic Drugs
• Large doses or chronic usage of glucocorticoids reduce collagen
synthesis and wound strength.
• Major effect is to inhibit the inflammatory phase of wound healing and
the release of lysosomal enzymes
• Steroids also inhibit epithelialization and contraction and contribute to
increased rates of wound infection, regardless of the time of
administration
• All chemotherapeutic antimetabolite drugs adversely affect wound
healing by inhibiting early cell proliferation and wound DNA and
protein synthesis

Metabolic Disorders - Diabetes Mellitus contd..
• Obesity, insulin resistance, hyperglycemia, and diabetic renal failure
contribute significantly and independently to the impaired wound
healing observed in diabetics.
• Reduced expression of growth factors like VEGF, IGF 1 FGF 1 KGF
and PDGF
• Diabetic fibroblasts and keratinocytes have reduced proliferation rates
and collagen production.

Metabolic Disorders
2. Uremia
• Associated with disordered wound healing.
• Experimentally, uremic animals demonstrate decreased wound
collagen synthesis and breaking strength

Ionizing Radiation
• Causes endothelial cell injury with endarteritis resulting in atrophy,
fibrosis, and delayed tissue repair
• Angiogenesis is not initiated
• Rapidly dividing cell populations like keratinocytes and fibroblasts are
most sensitive to radiation.

Local Factors
• Infection versus contamination
• Infection is when number or virulence of bacteria
exceed the ability of local defenses to control them.
• 100000 organisms per gram of tissue.
• Foreign bodies, hematomas promote infection,
impaired circulation, radiation.
• Systemic: AIDS, diabetes, uremia, cancer.

Local Factors
• Smoking stimulates vasoconstriction.
• Increases platelet adhesiveness
• Limits O2 carrying capacity
• Endothelial changes
• Diminished amount of collagen deposition.

Type of wound Features
Clean 1. No hollow viscus entered
2. Primary wound closure
3. No inflammation
4. No breaks in septic technique
5. Elective procedure
Clean contaminated 1. Hollow viscus entered but controlled
2. No inflammation
3. Primary wound closure
4. Minor break in aseptic technique
5. Mechanical drain used
6. Bowel preparation preop
Contaminated 1. Uncontrolled spillage from viscus
2. Inflammation apparent
3. Major break in aseptic technique
Dirty 1. Untreated, uncontrolled spillage from viscus
2.Pus in operative wound
3.Open suppurative wound, severe inflammation
Classification of Surgical Wounds

Animal Bites
• Wild Animals
• Bears, bison, moose, cougars, alligators.
• Injuries include puncture wounds, bites, lacerations,
bruises, rupture of internal organs, and evisceration.

Animal Bites
• Domestic animals
• Most animal bites are inflicted by dogs and cats.
• Cat bites frequently become infected.

Animal Bites
• What to do
• If not bleeding heavily, irrigate with water for 5 to 10
minutes.
• Remove foreign material.
• Control bleeding with pressure.
• If rabies is possible, wash with soap and water,
benzalkonium chloride, or povidone-iodine.

Animal Bites
• What to do
• After attack by a large animal, examine the victim for
internal injuries.
• Cover wounds with a sterile dressing.
• Evacuate.

Animal Bites
• Rabies
• A fatal viral infection of the brain.
• Only affects warm-blooded animals.
• Animals most commonly infected include skunks,
raccoons, and bats.

Animal Bites
• Rabies
• Consider rabies in the following situations:
• In an area or country where rabies is endemic
• If a bite by a dog, cat, skunk, raccoon, or fox is unprovoked and the
skin is broken
• If the victim was bitten by a bat
• If the victim was bitten by a large carnivore
• If an already open wound is licked by a potentially rabid animal

Animal Bites
• Rabies
• What to do
• Wash the bite vigorously with soap and water or irrigate with
benzalkonium chloride.
• Brain of infected animal must be examined. It may be
necessary to capture or kill the animal.

Human Bites
• What to do
• Wash with soap and water for 5 to 10 minutes.
• Control bleeding with pressure.
• Cover the wound with a sterile dressing.
• Seek medical care and tetanus immunizations, if necessary.

Snake Bites
• Pit viper
• Coral snake
© AbleStock
Courtesy of Luther C. Goldman/U.S. Fish & Wildlife Service

Snake Bites
• Pit viper snake bites
• What to look for
• Severe burning pain at bite site
• Two small puncture wounds
• Swelling
• Discoloration and blood-filled blisters
• In severe cases: nausea, vomiting, sweating, weakness,
bleeding, coma, and death.

Snake Bites
• What to do
• Get the victim away from the snake.
• Do not attempt oral suction or incising the skin.
• Keep the victim quiet.
• Evacuate immediately.

Snake Bites
• What to do
• Antivenin should be given within 4 to 6 hours.
• Immobilize affected limb.
• If there is no immediate reaction, start to walk slowly with
the victim to the trailhead.

Snake Bites
• Coral snake bite
• Respiratory depression.
• Double vision.
• Difficulty swallowing.
• Several hours can pass before onset of symptoms.
Absence of immediate symptoms does not mean it is a
harmless bite.

Snake Bites (6 of 8)
• Coral snake bite
• What to do
• Keep victim calm.
• Clean the bite with soap and water.
• Wrap a bitten limb with an elastic bandage.
• Evacuate the victim to a hospital for antivenin.

Snake Bites
• Nonpoisonous snake bite
• Horseshoe-shaped tooth marks
• Some swelling and tenderness
• No evidence of significant envenomation

Snake Bites
• Nonpoisonous snake bite
• What to do
• Clean bite with soap and water.
• Care for the bite as a minor wound.
• A tetanus booster may be needed.

Hypertrophic Scars and Keloids
• Excessive healing results in a raised, thickened
scar, with both functional and cosmetic
complications.
• If it stays within margins of wound it is
hypertrophic. Keloids extend beyond the
confines of the original injury.
• Dark skinned, ages of 2-40. Wound in the
presternal or deltoid area, wounds that cross
langerhans lines.

Keloids and Hypertrophic Scars
• Keloids more familial
• Hypertrophic scars develop soon after injury, keloids
up to a year later.
• Hypertrophic scars may subside in time, keloids
rarely do.
• Hypertrophic scars more likely to cause contracture
over joint surface.

Keloids and Hypertrophic Scars
• Both from an overall increase in the quantity of
collagen synthesized.
• Recent evidence suggests that the fibroblasts within
keloids are different from those within normal
dermis in terms of their responsiveness.
• No modality of treatment is predictably effective for
these lesions.

STERILISATION
the complete removal or destruction of all
forms of
microbial life
bacteria,
viruses,
fungi
spores
 Probabilistic notion
 No absolute assurance that there is 0
microorganism

STERILISATION
 Sterility assurance level (SAL) used as measure of
sterility
SAL = probability of survival of a microorganism
after sterilization process
 Expressed as log10 (probability of survival)
 SAL of 6 = < 1 chance in a million (10-6 ) that a
particular item is contaminated
 SAL = 6 acceptable for critical item.

DISINFECTION
• Not all microbial forms
• Main difference with sterilization = the lack of
sporocidal activity
• Categorized into 3 levels:
• High,
• Intermediate
• Low

OTHER DEFINITIONS
• Cleaning : the removal of adherent visible soil
(blood, protein substance and debris), dust or
other foreign material by manual or chemical
process
• Sanitizing : process that reduces microbial
population on object to a safe level
• Decontamination : process that removes
pathogenic microorganisms from an object to
make it safe to handle

OTHER DEFINITIONS
• Antiseptics = chemicals to prevent growth or
destroy
– Used on living tissues
– Regulated by FDA
• Disinfectants = chemicals used for disinfection
– Used for objects
– Regulated by EPA
• AOAC (Association of Official Analytical
Chemists) tests are used on antiseptics and
disinfectants

Factors Affecting Effectiveness of
Disinfection
• Cleaning
– Residual particles harbor & shelter from disinfectant
– Organic load restrict disinfectants effectiveness of alcohol, phenols,
chlorine & iodines
• Nature of object: crevices, hinges, lumens more
difficult to disinfect.
• Concentration of disinfectant:
– Diluted during application
– Lose potency with time

Factors Affecting Effectiveness of
Disinfection
• Time of contact
• Physical and chemical environment:
temperature, water hardness, pH

PHYSICAL AGENTS
1. SUNLIGHT: bactericidal activity due to
ultraviolet rays
2. DRYING: 4/5th
of weight of the bacterial cell
is due to water, but this method is highly
unreliable
3. RADIATION:
Non ionizing- like UV rays & IR rays
Ionizing- like X rays, gamma rays & cosmic rays

Non ionizing radiations
• UV RAYS are used to sterilize internal Surfaces of
safety cabinets, entry ways, Operation theaters etc.
• IR RAYS are used for rapid mass sterilization of pre
packed items such as syringes, catheters etc.

Ultraviolet irradiation: mechanismUltraviolet irradiation: mechanism
 Physical processPhysical process
 Energy absorbed byEnergy absorbed by
DNADNA
 pyrimidine dimers,pyrimidine dimers,
strand breaks, otherstrand breaks, other
damagesdamages
 inhibits replicationinhibits replication
UV
A
C
GTAAC
TT A
G
G C
T
DNA

UV disinfection: other applications
• Disinfection of air
• Surface disinfectant
• Hospital/food production
• Industrial application
• Cooling tower (Legionella control)
• Pharmaceuticals (disinfection of blood components and
derivatives)

Ionizing radiations
• Gamma radiation emitted from a radioactive element,
usually Co 60,provides a reliable means of sterilizing
plastic and other materials that are heat sensitive.
• Used commercially for sterilization of packaged
disposable articles such as syringes

APPLICATION OF HEAT
• Thermal death time (TDT) is the length of time
required to kill all bacteria in a liquid culture at a
given temperature
• Thermal death point (TDP) is the lowest temperature
at which all bacteria in a liquid culture will be killed in
10 minutes
• Decimal reduction time (DRT) is the length of time
in which 90% of a bacterial population will be killed
at a given temperature (especially useful in canning
industry

HEAT
• DRY HEAT
• Mechanism of action
1. protein denaturation
2. Oxidative damage
3. Toxic effects of
increased levels of
electrolytes
• MOIST HEAT
• Mechanism of action
1. Protein denaturation &
coagulation
2. Latent heat liberated
when steam condenses
on cooler surface
3. Hydrolysis &
breakdown of bacterial
proteins

DRY HEAT
• Red heat : for inoculating wires, loops, points of
Forceps etc
• Flaming : for scalpels, needles, culture tubes etc
• Hot air oven : for sterilizing
glassware,forceps,scissors,scalpels,glass
syringes,swabs,pharmaceutical products like liquid
paraffin, fats & grease.

HOT AIR OVEN
• Electrically heated, and provided with a fan or a
blower to ensure rapid & uniform heating
• Time the HOLDING PERIOD of 1hr at 160^C
when the thermometer first shows that the oven has
reached 160^C.
• Then the oven is allowed to cool gradually for about
2hrs before the door is opened.

Sterilization control for hot air oven
• Spores of non toxigenic strains of clostridium tetani
• Paper strips impregnated with 106spores are sterilized and
inoculated in thioglycollate or cooked meat media and
incubated for sterility test under strict anaerobic conditions
for 5 days at 37^C.
• Browne’s tube which shows a green color after
sterilization
• Thermocouples

MOIST HEAT
Sterilization
By
Moist heat
Moist heat at
Below 100^C
Moist heat
At 100^C
Moist heat
At above 100^C
e.g.. pasteurization
•Boiling
•Tyndallization
Autoclave

Moist heat at below 1000
C
• PASTEURISATION
• HOLDER METHOD : milk heated at 630
C for 30 min
• FLASH METHOD : milk heated at 720
C for 15 sec
• Both followed by cooling rapidly to 130
C or lower
• VACCINE BATH
• Vaccines prepared from cultures of non sporing bacteria
sterilized by heating in vaccine bath at a low temperature
of 600
C for 1 hr.

Moist heat at 1000
C
• BOILING
• Most vegetative forms almost immediately killed at 90-
1000
C but sporing bacteria require prolonged periods of
boiling
• Hard water should not be used
• 2% sodium bicarbonate may be added to promote
sterilization
• Koch or Arnold steam sterilizer is used

TYNDALLIZATION
• Means intermittent exposure at 1000
C
• Principle : that one exposure kills vegetative
organisms, between heatings the spores being in a
favorable nutrient medium become vegetative forms
which get killed during subsequent heating.
• Used for gelatin media, media containing sugars
• May fail to kill thermophilic,anaerobic and other
bacteria whose spores do not germinate in a
particular medium

Moist heat at above 1000
C
AUTOCLAVES

AUTOCLAVES
• Saturated steam under pressure.
• Cheap & nontoxic
• Penetrates fabric
• Method of choice for all items except those which
are moisture or heat sensitive.
• 4 parameters of importance
• Pressure
• Temperature
• Time
• Steam
• Air must be removed and steam must reach the
item for required time at required temperature

PRINCIPLE
• Water boils when its
vapour pressure equals
that of surrounding
atmosphere.
PV=nRT
• When pressure inside the
closed vessel increases,
the temp at which water
boils also increases

MECHANISM
• When steam comes in contact with a cooler surface it
condenses to water and gives up its latent heat to that
surface (1600 ml steam at 100^C at atmospheric
pressure condenses into 1 ml of water at 100^C and
releases 518cal of heat) and moisture which together
denature microbial proteins

Sterilization cycle
• Sterilization cycle includes
1-warming of the chamber
2-vacuum extraction
3-pre-steam penetration time
4-steam penetration time
5-holding time
6-cooling time

STERILIZATION TIMES
• AUTOCLAVES
• 1150
C, 10 lb/in2
for 45 min
• 1210
C, 15 lb/in2
for 15-20 min
• 1340
C, 30 lb/in2
for 3 min
• HOT AIR OVEN
• 1600
C for 45 min
• 1700
C for 18 min
• 1800
C for 7.5 min

Types of autoclaves
• According to structure
• Simple non jacketed autoclave
• Steam jacketed autoclave with automatic air and condensate discharge
• High pre-vacuum sterilizers
• According to function
• Simple lab autoclave
• Transportable bench top autoclave
• Large simple autoclave
• Downward displacement lab autoclaves
• Media preparators
• Multi purpose lab autoclave

USES
• Culture media
• Aqueous solutions
• Empty bottles and
impervious containers
• Surgical instruments
• Wrapped dry goods and
dressings

What should not be autoclaved?
• Items containing solvents, volatiles or corrosive
chemicals
• Radioactive material

Safety Procedures
• Follow manufacturers’ guidelines
• Do not open when chamber is pressurized
• Avoid standing directly in front of autoclave door
when opening
• Place autoclave on preventive maintenance schedule
• Cool to below 80^Cbefore opening
• Bottles must not be overfilled

STERILIZATION
INDICATORS
• AUTOMATIC PROCESS CONTROL
• Carries through the sterilization cycle according to a pre
selected scheme for duration,temperature,and pressure of each
stage
• RECORDING THERMOMETER
• Graphic record of temperature changes in chamber discharge
channel avoiding errors in timing the holding period
• THERMOCOUPLE
• When kept inside the test article and attached to
potentiometer, it indicates the temperature inside the test
article during autoclaving

STERILIZATION
INDICATORS
• CHEMICAL INDICATORS
• BROWNE’S STERILIZER has red solution which turns
green when heated at 115^C for 25min(type 1),or
15min(type 2), or at 160^C for 60min(type 3)
• Stored at 20^C to avoid pre mature color change
• ADHESIVE TAPES
• Bowie-Dick autoclave tape test for steam penetration

Biological indicators
• Organism : Bacillus stearothermophilus,a thermophile that
requires to be cultivated 55-60^C.Its spores are killed at
121^C in 12min
• Preparation: culture grown aerobically on nutrient
agar for 5days is suspended in sterile water to a
concentration of one million spores per ml.small
strips of filter paper soaked in the suspension are
dried at room temp and packed in envelopes

Biological indicators
• Procedure
• placed in the centre of the largest or most densely packed
items and some in the coolest part
• After autoclaving, envelope is cut with a sterile scissors and
strip transferred to a recovery medium,eg.thioglycolate broth
with strict precautions against contamination
• Tube incubated for 7 days at 55^C and examined for growth.
An unautoclaved spore strip is used as positive control and
uninoculated tube of medium as negative control
• Results in terms of degree of heat resistance of preparation
used

TECHNIQUE
• Forced passage of liquids through a filter of porosity
small enough to retain any microorganism contained
in them.
• A positive or negative pressure is necessary to draw
the fluid through the filter into a sterile container e.g..
A filtering flask
• Used for sterilizing sera, solutions of sugars,
vaccines, antibiotics used for culture media

TYPES OF FILTERS
• Earthenware candles
• Berkefeld,Mandler
• Chamberland
• Asbestos and asbestos paper discs,e.g.Seitz
• Sintered glass filters
• Cellulose membrane filters
• Gradocol and modern membrane filters
• Syringe,pressure,vacuum membrane filters
• High efficiency particle arresters(HEPA)

ALCOHOLS
• Ethyl alcohol and isopropyl alcohol are most frequently used
• Act by denaturing bacterial proteins
• Concentration required : 60-90% in water
• Protein slows its action,1%mineral acid or alkali enhances it
• Effective against both gram+ve and –ve bacteria, not sporicidal
• Isopropyl alcohol is preferred as it is better fat solvent, more
bactericidal and less volatile
• Flammable
• Methyl alcohol is effective against fungal spores and used for treating
cabinets and incubators affected by them
• Can be removed from disinfected articles by flaming

ALDEHYDES
• FORMALDEHYDE
• Active against the amino group of protein molecule
• Markedly bactericidal,sporicidal and virucidal
• Commercial formalin is 40% (w/v) solution of formaldehyde
in water with 10%methanol to prevent polymerization
• Borax formaldehyde solution with 0.5%sodium tetraborate
and 4%formaldehyde in water is used to disinfect clean metal
instruments
• For gaseous disinfection,eg. For fumigating wards, sick rooms
etc the atmosphere should have high relative humidity, over
60% and temp of at least 18^C

ALDEHYDE
• GLUTARALDEHYDE
• Markedly bactericidal,virucidal including HIV and
HBV,mycobacteria and to a lesser extent spores
• Concentration required 2%
• Ability to penetrate organic material is poor
• Less corrosive
• Can be used to disinfect
cystoscopes,endoscopes,corrugated rubber tubes, face
masks, metal instruments,polythene tubes

PHENOLS
• Obtained by distillation of coal tar between temp 170-270^C
• active against gram+ve and –ve bacteria,mycobacteria,but little
activity against spores and viruses
• Act by causing cell membrane damage, releasing cell contents
and causing lysis
• Resistant to inactivation by organic matter
• Phenolic products like Lysol and cresol are good general
disinfectants but are toxic to humans
• Related products chlorophenols and chloroxyphenols are less
toxic, less irritant but inactive against pseudomonas

- LYSOL is used to disinfect linen
- 3%lysol,0.5% phenol or 0.1% p-chloro-m-cresol is used for
preserving sera and vaccines
- hexachlorophene is toxic,chlorhexidine is a nontoxic skin
antiseptic most active against gram+ve and fairly against gram-ve
ones

ETHYLENE OXIDE
• Colourless liquid with a boiling point of 10.7^C
• At normal temp and pressure, it is a highly penetrating gas with
ethereal smell
• Acts by alkylating the amino,carboxyl,hydroxyl and sulphydryl
groups in protein molecule and also by damaging DNA and RNA
• Highly inflammable, and highly explosive at a concentration of
>3% in air, carcinogenic and mutagenic
• Explosive tendency eliminated by mixing with inert gases like CO2
and N2 to a concentration of 10%
• Active against most organisms including spores and viruses

ETO GAS
• TEST ORGANISM : Bacillus subtilis var.globigii
• USES : heat and moisture sensitive articles like plastic and
rubber articles,blankets,pharmaceutical
products,sutures,heart lung machines. Not for fumigating
rooms since explosive
• Objects to be sterilized are put in a cabinet from which air
has been removed using vacuum, and a non explosive
mixture containing ETO gas is introduced to a pressure
of 5-30 lb/in2
above atm pressure,temp to be maintained
at 45-55^C and relative humidity 20-40%

HALOGENS• IODINE
• Aqueous and alcoholic solutions used as skin disinfectants
• Actively bactericidal,virucidal,and fairly active against spores
• Iodine compounds with non ionic wetting or surface active agents called
IODOPHORES are more active and efficient
• It inhibits protein synthesis and oxidizes –SH groups of amino acids

CHLORINE
• Reactions for free chlorine formation:
Cl2 (g) + H2O <=> HOCl + Cl-
+ H+
HOCl <=> OCl-
+ H+
(at pH >7.6)
• Three different methods of application
• Cl2 (gas)
• NaOCl (liquid)
• Ca(OCl)2 (solid)
• Chloramine formation
• HOCl + NH3 <=> NH2Cl (monochloramine) + H2O
• NH2Cl + HOCl <=> NHCl2 (dichloramine) + H2O
• NHCl2 + HOCl <=> NCl3 (trichloramine) + H2O

CHLORINEChlorine is used in free, hypochlorite as well as chloramine form
• Kills vegetative bacteria
• Kills viruses like HIV,HBV
• It is very effective against HBV and HIV viruses, therefore its use is
recommended at 1:10 dilution for disinfection of blood spills. It is also
recommended for disinfection of counter tops and work surfaces,
hydrotherapy tanks, laundry. used for disinfection of water supplies and
swimming pools
• not much useful against spores

DYES
• ANILINE DYES
• Brilliant green, malachite green and crystal violet
• React with acid groups in cell
• More active against gram+ve than gram-ve bacteria, no activity against
tubercle bacilli
• Non irritant,non toxic
• Inhibited by organic material
• ACRIDINE DYES
• Proflavine,acriflavine,euflavine,aminacrine
• Impair DNA complexes of organisms and destroy reproductive capacity
of the cell
• More active against gram+ve bacteria than gram-ve

OTHER CHEMICAL AGENTS
• METALLIC SALTS
• Salts of Ag,Cu and Hg,eg.merthiolate,a proprietary name for
sodium ethylmercuriosalicylate is used for preservation of
antitoxins and sera in 1:10000 dilution
• Protein coagulants and combine with free –SH groups of cell
enzymes
• SURFACE ACTIVE AGENTS
• Alter energy relationship at interfaces producing a reduction
of interfacial tension
• 4 groups : cationic,anionic,nonionic and amphoteric(Tego
compounds)

Other chemical agents
• LIQUID PERACETIC ACID (STERIS ® )
• Uses a solution of peracetic acid with H2O2
• Peracetic acid disrupts and denatures proteins,sporicidal
• Extra oxygen rapidly inactivates many cell systems
• corrosive and irritant
• Sterilant for endoscopes
• HYDROGEN PEROXIDE PLASMA STERILIZATION
(STERRAD ®)
• Radio frequency emissions applied to the H2O2 producing active
radicals and reactive chemical species
• Plasma reverts to water and CO2 on removing energy
• Sporicidal,bactericidal,virucidal,fungicidal,mycobactercidal
• Not effective below 42^Cor in lipid contamination

ype of Disinfectant: H = High level; I = Intermediate level; L = Low lev

SPAULDING
CLASSIFICATION
Eagle H. Spaulding believed that how an object
will be disinfected
depended on the object’s intended use

Processing “Critical” Patient Care
Objects
• Classification: Critical objects enter normally sterile
tissue or vascular system, or through which blood
flows.
• Object: Sterility.
• Level germicidal action: Kill all microorganisms,
including bacterial spores.
• Examples: Surgical instruments and devices; cardiac
catheters; implants; etc.
• Method: Steam, ETO, hydrogen peroxide plasma,
ozone or chemical sterilization

Chemical Sterilization of “Critical
Objects”
• Glutaraldehyde (> 2.0%)
• Hydrogen peroxide-HP (7.5%)
• Peracetic acid-PA (0.2%)
• HP (1.0%) and PA (0.08%)
• HP (7.5%) and PA (0.23%)
• Glut (1.12%) and Phenol/phenate (1.93%)
Exposure time per manufacturers’ recommendations

Processing “Semicritical”
Patient Care Objects
• Classification: Semicritical objects come in contact
with mucous membranes or skin that is not intact.
• Object: Free of all microorganisms except high
numbers of bacterial spores.
• Level germicidal action: Kills all microorganisms
except high numbers of bacterial spores.
• Examples: Respiratory therapy and anesthesia
equipment, GI endoscopes, thermometer, etc.
• Method: High-level disinfection

High Level Disinfection of
“Semicritical Objects”
Exposure Time > 12 m-30m, 20oC
Germicide Concentration
• Glutaraldehyde > 2.0%
• Ortho-phthalaldehyde (12 m) 0.55%
• Hydrogen peroxide* 7.5%
• Hydrogen peroxide and peracetic acid* 1.0%/0.08%
• Hydrogen peroxide and peracetic acid* 7.5%/0.23%
• Hypochlorite (free chlorine)* 650-675 ppm
• Glut and phenol/phenate** 1.21%/1.93%
*May cause cosmetic and functional damage; **efficacy not verified

Processing “Noncritical”
Patient Care Objects
• Classification: Noncritical objects will not come in
contact with mucous membranes or skin that is not
intact.
• Object: Can be expected to be contaminated with
some microorganisms.
• Level germicidal action: Kill vegetative bacteria, fungi
and lipid viruses.
• Examples: Bedpans; crutches; bed rails; EKG leads;
bedside tables; walls, floors and furniture.
• Method: Low-level disinfection

Low level disinfection
for
“non critical objects”
• Ethyl alcohol ≤ 10mn
• Isopropyl alcohol ≤10mn
• Chlorine 100 ppm ≤10mn
• Phenolic germicidal solution ≤10mn
• Iodophor germicidal solution
• Quaternary germicidal ≤10mn

Decreasing Order of Resistance of Microorganisms
to
Disinfectants/Sterilants
• Prions
• Spores
• Mycobacteria
• Non-Enveloped Viruses
• Fungi
• Bacteria
• Enveloped Viruses

PRION STERILIZATION
• Critical/Semicritical-devices contaminated with
high-risk tissue from high risk patients requires
special prion reprocessing
• NaOH and steam sterilization (e.g., 1N NaOH 1h, 1210
C
for 30 m)
• 1340
C for 18m (prevacuum)
• 1320
C for 60m (gravity)
• No low temperature sterilization technology
effective

EVALUATING DISINFECTANTS
• PHENOL COEFFICIENT
• RIDEAL WALKER TEST : suspensions with equal numbers of typhoid
bacilli are submitted to action of varying concentrations of phenol and
of disinfectant to be tested
• Dilution of disinfectant sterilizing the suspension = phenol coefficient
corresponding dilution of phenol
• CHICK MARTIN TEST : the disinfectant acts in presence of organic
matter
Both these fall short of simulating natural conditions
• DILUTION TEST
• Drying bacteria to surface followed by exposure to disinfectant and
subsequent washing and inoculation in sterile broth

FILTER PAPER/DISC DIFFUSION METHOD
placement of disinfectant impregnated filter paper on well inoculated agar
Hypochlorous acid
Phenol
Lysol
Nisin
E.coli

CONTACT ME FOR MORE
SLIDES
VAIBHAV TRIVEDI
vaibhavclinic@gmail.com
9557311796

Wound healing and sterilization for MBBS students

Recommended

Recommended

More Related Content

What's hot

What's hot (20)

Viewers also liked

Viewers also liked (17)

Similar to Wound healing and sterilization for MBBS students

Similar to Wound healing and sterilization for MBBS students (20)

Recently uploaded

Recently uploaded (20)

Wound healing and sterilization for MBBS students

Editor's Notes