HOST PATHOGENS INTERACTION…….

Origen of microbial flora

The fetus is in a sterile environment during first few days of life

New born……. Many micro organisms

Once established onto or into a particular body site……..

Symbiosis, commensalism, parasitism.

Characteristics of indigenous flora

• Indigenous flora• Body sites …same or different flora• Resident microbial flora• Transient flora

• Carriers• Carrier state• Carrier state may be acute or chronic • Colonizing….host resistance….microbial flora

provide protection

Factors that determine the composition of the usual microbial flora

• Presence of organisms …. Influenced by nutritional and environmental fectors.

Composition of the microbial flora at different body sites

• Human host…colonized by….app. 100 microbs• Effectiveness of host…..low incidence of

infection• Clinical microbiologist must be able to

recognize and identify the types of microbs …at diff body sites

Usual flora of the skin

• Skin has numbr of mechanisms...prevent infections

• Skin…..vide veriety of microbs• Reduce but not eliminated by scrubbing and

washing

• Composition of flora….depends…• Concentrate….. Moist areas• Aerobic diptheroids…moist areas• staphylococcus …hair follicles• Inhibit pathogenic bacteria• P.acnes colonize deep sebaceous glands

Usual flora of the mouth

• Contain larg number of bacteria• Many bind… buccal mucosa & tooth surface• Bacterial plaques..1011

streptococci• Plaque…..low oxidation potential at tooth

surface…growth of anarobes

Usual flora of the Respiratory tract

• Upper….lower tracts• Upper….mouth,nasopharynx,oropharynx,larynx• Lower….tracea,bronchi,lungs…protected by cilia• Usual flora of the mouth….streptocococci• Upper….mouth,nasopharynx,oropharynx…

microbs show some diff• Staphylococcus….30% of normal individuals

colonize anterior nares

• Individuals ….hospitalized…upper r.t…..by gram negative bacteria.

• Orophyarynx….streptococci• A number of species…...s.mutans, s.milleri,

s.sanguis,• Hospitalizwd patients…..gram negative rods

Usual flora of the gastrointestinal tract

• Comprisis….esophagus,stomach,s.intestine,colon

• Equiped…defenses & antimicrobial fectors• Ingesting organisms• Don’t multiply ….esophagus, stomach• In ingested food…as transient flora.• Most mirobs susceptible...acid ph…destroy….

Exception sper forming becteria,cysts

• Stomach acidty….reduce• Smal intestine….few microbs• In the colon….108 -1011

• Anarobs…90% large intestine• Gram positive cocci, yeasts, pseudomonas• Population….antibiotics• In some cases …microbs supressed….othr able

to proliferate…enterocolititis

Role of the microbial flora in the pathogenesis of infectious disease

• Microbial flora….provide some benefits… symbiotic relationship

• Some opportunists….when damage..disturbed or change…or immune system

• Trauma….accidentalor surgical….not part of microbial flora…

• Host immune response….reduces…due to ….drugs…

• Lymphoma, leukemia….reduce immune response

• Microbial flora…initiat an infection….in patients with chronic illnesses

Role of the microbial flora in the host defense against infectious disease

• Beneficial effects• Immunological competence• Immune system developed or undeveloped• Newborn….not developed

• Micobial flora…block colonization by extragenious pathogens

• Indigenous flora altered• gastroenteritis• Pathogenic species • C. albcans

Microbial flora…

• Important role in health and diseases• Eradication….negative effects• Knowledge in…clinical samples

Sterile Techniques

Sterile Techniques

STERILISATION: any process that effectively kills or

eliminates transmissible agents (such as fungi, bacteria, viruses, spore forms, etc.) from a surface, equipment, article of food or medication, or biological culture medium. Sterilization can be achieved through application of heat, chemicals, irradiation, high pressure or filtration.

STERILISATION Types

PHYSICAL METHODSA. Heat :

1. Dry heat2 Moist heat

B. Radiations1. Ultraviolet radiations2. Ionizing radiations

C. FiltrationII. CHEMICAL METHODS

Heat sterilization

Dry Heat sterilization1. Dry heat, as the name indicates, utilizes hot air that is either free from

water vapour, or has very little of it, and where this moisture plays a minimal or no role in the process of sterilization 1. Hot air ovens :

Hot air ovens are electrical devices used in sterilization. The oven uses dry heat to sterilize articles. Generally, they can be operated from 50 to 300 °C. There is a thermostat controlling the temperature. These are digitally controlled to maintain the temperature. Their double walled insulation keeps the heat in and conserves energy, the inner layer being a poor conductor and outer layer being metallic. There is also an air filled space in between to aid insulation. An air circulating fan helps in uniform distribution of the heat. These are fitted with the adjustable wire mesh plated trays or aluminium trays and may have an on/off rocker switch, as well as indicators and controls for temperature and holding time. The capacities of these ovens vary.

Mechanism of killing by dry heat

1. Dry heat kills the organisms by destructive oxidation of essential cell constituents

2. Killing of the most resistant spores by dry heat requires a temperature of about 160 °C for 60 minutes

3. Dry heat is employed for glassware; syringes, metal instruments and paper wrapped goods, which are not spoiled by high temperatures.

4. It is also used for anhydrous fats, oils and powders that are impermeable to moisture.

METHODS OF STERILIZATION BY DRY HEAT

• RED HEAT• FLAMING• HOT AIR OVEN• INFRARED RADIATIONS

1.RED HEAT

• Inoculating wires, points of forceps and searing spatulas are sterilized by holding them in the flame of Bunsen burner until they are seen to be red-hot.

2. FLAMING

• This method is used for sterilizing scalpel, mouth of culture tubes, glass slides etc. It involves passing of an article through Bunsen flame without allowing it to become red-hot.

3. HOT AIR OVEN

• This is the main means of sterilization by dry heat. Exposure at a temperature of 160 °C for 1 hour is generally employed.

4. INFRARED RADIATIONS

• Source employed is an electrically heated element, the infra red rays are directed on to the object to be sterilized and temperature of 180 °C can be obtained.

Steam sterilization

A widely-used method for heat sterilization is the autoclave, sometimes called a converter. Autoclaves commonly use steam heated to 121 °C or 134 °C. To achieve sterility, a holding time of at least 15 minutes at 121 °C or 3 minutes at 134 °C is required. Proper autoclave treatment will inactivate all fungi, bacteria, viruses and also bacterial spores, which can be quite resistant. It will not necessarily eliminate all prions. For prion elimination, various recommendations state 121–132 °C (270 °F) for 60 minutes or 134 °C (273 °F) for at least 18 minutes. The prion that causes the disease scrapie (strain 263K) is inactivated relatively quickly by such sterilization procedures

Moist Heat sterilization

METHODS OF STERILIZATION BY MOIST HEAT

Moist heat can be employed at1. Temperature below 100 °C2. Temperature of 100 °C3. Temperature above 100 °C

MOIST HEAT BELOW 100 °C

EXAMPLE: Pasteurization of milk In Pasteurization of milk the temperature

employed is either 63 °C for 30 minutes or 72 °C for 20 seconds. All nonspore forming pathogens in milk like Salmonellae, M.tuberculosis are killed.

MOIST HEAT ABOVE 100°CSterilization in an autoclave1. Autoclaving is the most reliable method. It is the method

most widely used fo sterilization of culture media and surgical supplies

2. When water is boiled within a closed vessel at an increased pressure, the temperature at which it boils and the steam it forms will rise above 100 °C

3. This principle is used in the autoclave 4. Normally autoclaving is done at 15 lbs. (pounds per sq. inch

pressure) and 115 °C for 15 minutes

Mechanism of killing by moist heat

1. Moist heat kills the organisms by coagulating and denaturing their enzymes and structural protein.

2. Sterilization by moist heat of the most resistant spores generally requires 121 °C for 15-30 minutes.

3. Moist heat is used for the sterilization of culture media, and all other materials through which steam can penetrate

4. Moist heat is more effective than dry heat5. Sterilization can be done at lower temperatures in a given

time at a shorter duration at the same temperature.

FACTORS INFLUENZING STERILIZATION BY HEAT

1. The temperature and time: they are inversely related, shorter time is sufficient at high temperatures.Number of microorganisms and spores:

2. The number of survivors diminished exponentially with the duration of heating

3. Depends on the species, strains and spore forming ability of the microbes.

4. Thermal death point is the lowest temperature to give complete killing in aqueous suspension within 10 minutes

5. Depends on the nature of material: a high content of organic substances generally tends to protect spores and vegetative organisms against heat.

6. Presence of organic or inorganic disinfectants facilitates killing by heat

7. pH also plays an important role in the killing of microorganisms

MERITS AND DEMERITS OFHEAT STERILIZATION

Advantages of heat sterilization1. Sterilization is very effective2. Instruments are standardized to deliver the

required effective heat3. Heat deliver system can be monitored

effectively with various controls like pressure gauge, temperature meters etc

4. Established quality control methods available

Disadvantages of heat sterilization

1. Steam impermeable materials like fats, oils and powders can not be sterilized by autoclaving.

2. Heat sensitive materials can not be sterilized by heatExamples:1. Serum can not be sterilized2. Antibiotics3. Plastic materials4. Vaccines5. Rubbers3. Presence of organic matters interfere with effective

sterilization4. Dangers of explosion when high pressure is used

STERILIZATION BY FILTRATION

STERILIZATION BY FILTRATION

1. When fluids are passed through bacteria stopping filters, they are made free from bacteria.

2. It is useful for making preparations of soluble products of bacterial growth such as toxins

3. Liquids that would be damaged by heat such as serum and antibiotic solutions can be sterilized by filtration

4. Efficient filters should be able to retain Serratia marcescens

Chemical sterilization

Chemical sterilization

• sterilization includes both liquid and gas chemicals

• Chemical Sterilizing With Alcoholal• In many experimental procedures, the most effective

way to sterilize objects is with ethanol. Either 95% or 70% will work. The latter is actually more effective, but the former is often more convenient.

Vapors and Gases

• formaldehyde, beta-propiolactone and ethylene oxide are not routinely used in laboratory sterilization practices. These sterilants are used in hospitals and commercial facilities where closed systems controlling temperature, humidity, and concentration are required to achieve sterilization using these agents.