wound healing and sterilization for mbbs students

Wound healing

Vaibhav Trivedi

MBBS 2012

AIIMS RISHIKESH

THE SKIN

2

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.

Classification

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%.

Wound Healing

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

Stages of Wound Healing

Phases of wound healing

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.

MATURATION AND REMODELLING

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…

AGEAging 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 MMPs, growth factors, and cytokines in chronic-type wounds.


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 - ArginineArginine 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.

Factors Affecting Wound Healing contd…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.

Factors Affecting Wound Healing contd…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 closure3. No inflammation4. No breaks in septic technique5. Elective procedure

Clean contaminated 1. Hollow viscus entered but controlled2. No inflammation3. Primary wound closure4. Minor break in aseptic technique5. Mechanical drain used6. Bowel preparation preop

Contaminated 1. Uncontrolled spillage from viscus2. Inflammation apparent3. Major break in aseptic technique

Dirty 1. Untreated, uncontrolled spillage from viscus 2.Pus in operative wound3.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

• Pit viper 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

• Pit viper 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• What to look for

• 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

• What to look for

• 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&

DISINFECTION

STERILISATION

the complete removal or destruction of all forms of

microbial lifebacteria,viruses,fungispores

Probabilistic notion No absolute assurance that there is 0

microorganism

STERILISATION

Sterility assurance level (SAL) used as measure ofsterilitySAL = probability of survival of a microorganismafter sterilization process Expressed as log10 (probability of survival) SAL of 6 = < 1 chance in a million (10-6 ) that aparticular 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 ofDisinfection

• 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 ofDisinfection

• 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 by Energy absorbed by

DNADNA pyrimidine dimers, pyrimidine dimers,

strand breaks, other strand breaks, other damagesdamages

inhibits replicationinhibits replication

UV

AC

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 106 spores 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 HEATSterilization

ByMoist heat

Moist heat atBelow 100^C

Moist heatAt 100^C

Moist heatAt above 100^C

e.g.. pasteurization•Boiling

•Tyndallization

Autoclave

Moist heat at below 1000C

• PASTEURISATION

• HOLDER METHOD : milk heated at 630C for 30 min

• FLASH METHOD : milk heated at 720C for 15 sec

• Both followed by cooling rapidly to 130C or lower

• VACCINE BATH

• Vaccines prepared from cultures of non sporing bacteria sterilized by heating in vaccine bath at a low temperature of 600C for 1 hr.

Moist heat at 1000C

• BOILING

• Most vegetative forms almost immediately killed at 90-1000C 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 1000C

• 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 1000C

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• 1150C, 10 lb/in2 for 45 min

• 1210C, 15 lb/in2 for 15-20 min

• 1340C, 30 lb/in2 for 3 min

• HOT AIR OVEN• 1600C for 45 min

• 1700C for 18 min

• 1800C 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

FILTRATION

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)

CHEMICAL AGENTS

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

http://www.encolombia.com/salud/primerosauxilios2.htm

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

* Type of Disinfectant: H = High level; I = Intermediate level; L = Low level

http://www.safety.queensu.ca/biocom/disin.htm

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)

• 1340C for 18m (prevacuum)

• 1320C 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 METHODplacement of disinfectant impregnated filter paper on well inoculated agar

Hypochlorous acid

Phenol

Lysol

NisinE.coli

http://www.srga.org/RAFMETOD/rafmet.htm

CONTACT ME FOR MORE SLIDES

VAIBHAV [email protected]

9557311796

mailto:[email protected]

wound healing and sterilization for mbbs students

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