Sterilization, Morphology and Microscope PDF

Sterilization is killing or removing all types of microorganisms from a certain material.  Killing the Physical Heat microorganisms methods sterilization Chemical Radiation Sterilization...

Sterilization is killing or removing all types of microorganisms from a certain material.  Killing the Physical Heat microorganisms methods sterilization Chemical Radiation Sterilization methods Removing the microorganisms Filtration  Dry heat Direct flaming Hot air (Bunsen flame) (oven) Moist heat With steam under the With steam under pressure atmospheric pressure higher than the atmospheric (Tyndallization) pressure (Autoclaving) (Arnold apparatus) (Autoclave) All materials and tools must be sterilized without any change in their chemical and physical features  Physical Sterilization Methods I. Heat Sterilization Viable Microbial Cells - Denaturation of the cell proteins and enzymes - Disruption of the nucleic acids Loss of cell function and cell ability to reproduce Dead Microbial Cells  The time required for moist heat sterilization is shorter than that for dry heat sterilization (why) ❑ Water is a good heat conductor whereas the air is a bad heat conductor, so the moist heat penetrates the microbial cells faster than the dry heat. ❑ The presence of water molecules in the moist heat helps to disrupt the hydrogen bonds and other interactions that hold proteins in their shapes. This causes denaturation of the cell proteins.  A. Dry heat sterilization - Direct Flaming Bunsen flame is used for this purpose. The temperature of Bunsen flame is 1560° c. The instruments sterilized by direct flaming must be heated to red heat without any change in their chemical and physical features.  outer red cone the upward flow of air reduces the chance of microorganisms presence in the working area Inner blue cone Air – hole is completely opened The barb is connected to a gas nozzle on the laboratory bench with rubber tubing.  Right flame Wrong flame ❖ The air around Bunsen flame in a circle with a radius of 15 cm is sterilized. ❖ The upward flow of air reduces the chance of microorganisms presence in the working area. ❖ No air around Bunsen flame helps to maintain the sterile area around it. Sterilization by direct flaming is usual for: Inoculating wire and loop. Forceps, scissors and scalpels after dipping in ethanol (why). These tools are manufactured from aluminum which is heated slowly, so they must be dipped in ethanol to heat rapidly without any change in their features.  A. Dry heat sterilization - Hot Air The hot air oven : ❑ Is double wall equipment. ❑ Provided with perforated shelves. ❑ Operated by electric power. ❑ Controlled thermostatically. ❑ The tools must be heated to 160° c /3 h or 180° c / 2 h. Hot Air Sterilizer Sterilization in the hot air oven is usual for: Glass test-tubes, flasks and bottles after plugging their mouth with cotton plug. Glass Petri dishes after their packing in stainless steel containers. Glass pipettes after plugging their mouth with cotton plug and packing them in stainless steel containers.  The instruments must be heated to 160°c / 3 hours or 180°c / 2 hours. The time of sterilization is calculated after reaching the temperature to the required level. Glassware must be dried before placing in the oven. Loading the oven should be taken place when the oven is cold. Spaces, between the tools in the oven, should be left (why) for circulation of the hot air through the load. The oven should be allowed to cool to the room temperature before the door is opened (why) to prevent the recontamination and cracking the glasswares.  B. Moist heat sterilization - With steam under pressure (Autoclaving) The most effective sterilization system is steam under pressure. The temperature used in this system is above the boiling temperature of water, so the sterilization adopting this system must be completed in a pressurized container.  The autoclave is a closable double wall construction pressure vessel that can be filled with steam under pressure greater than atmospheric pressure. The autoclave is equipped with: ❑ Temperature gauge to control the temperature.. ❑ Pressure gauge (manometer) to control the pressure. ❑ Timer to control the time of sterilization. ❑ Safety valve which is closed when the inner space is entirely filled with steam. Autoclave The actual temperature inside the autoclave depends on the steam pressure. Pressure Temperature Time 0.5 atmospheric pressure 111°c 30 min. 1.0 atmospheric pressure 121°c 15 – 20 min. 1.5 atmospheric pressure 128°c 5 min. The pressure is directly proportional with the temperature. The time is inversely proportional with the temperature. It is very important to ensure that air is completely removed from the autoclave before sterilization begins (why) Because The air is a bad heat conductor so a mixture of air and steam results in a lower temperature for any given pressure.  Pressure % of steam and air Temperature 1.0 atmospheric 100 % steam pressure 121°c (√) 1.0 atmospheric 75 % steam + 25 % air 115°c pressure (X) 1.0 atmospheric 50 % steam + 50 % air pressure (X) 112°c The autoclaving is effective for the sterilization of: Culture media containing heat stable ingredients (capable of withstanding the high temperature, over 100°c, without decomposition). So, the culture media containing milk, gelatin and sugars are not sterilized by autoclaving. Rubbery tools. Lab coats. 1.The water must be added into the autoclave before sterilization begins. 2. Loading should be taken place when the autoclave is cold. 3. The time of sterilization is calculated after reaching the temperature and pressure to the required level. 4. The inner space of the autoclave must be entirely filled with the steam (why). 5. After sterilization, the autoclave is not opened until the pressure reduces to the atmospheric pressure. 6. The neutral media must be autoclaved for a longer time than the acidic or alkaline media (why). because the microbial load in the neutral media is more than that in the acidic or alkaline media. 5. The slimy solutions must be autoclaved for a longer time than the true liquids (why). because the slimy solution coats the microbial cells with a slimy layer which causing the slowdown of heat transfer to the cell.  B. Moist heat sterilization - With steam (Tyndallization) Intermittent sterilization It is a sterilization method in steam at atmospheric pressure. The tyndallization is performed by heating at 100°c / 30 minutes on each of 3 successive days interspersed with incubation at 30 – 37°c. The apparatus used in this system is Arnold apparatus which is a closable vessel.  Culture medium 1st day Culture medium (spores) ( vegetative cells + 100°c/30 min spores) 30°c/24 h. Culture medium Sterile culture ( vegetative cells + medium spores) 2nd day 100°c/30 min 3rd day 100°c/30 min Culture medium 30°c/24 h. Culture medium ( vegetative cells) (spores) ❑ The tyndallization is effective for the sterilization of culture media containing autoclaving sensitive ingredients (ingredients that are labile at the high temperature, over 100°c) as milk, gelatin and sugars. 1. The water must be added into Arnold before sterilization begins to generate the steam. 2. Loading should be taken place when Arnold is cold. 3. The time of sterilization is calculated after reaching the temperature 100°c.  Physical Sterilization Methods II. Sterilization by Radiation The radiation as a sterilization method has several effects to kill the microbial cells: ❑ Alterations in the nucleic acids which prevent the DNA replication. ❑ Destruction of the genetic material. ❑ Denaturation of the cellular proteins and enzymes. The energy of radiation is inversely proportional with the wave length. (The shorter wave length, the higher energy). The types of radiation used as sterilizing agent are: Electromagnetic rays. Heating radiations. Ultraviolet light. Ionizing radiations (α, β, ϒ and x rays). Sterilization by radiation is used for: ❑ Heat-sensitive solid materials as plastic pipettes, Petri-dishes and plastic syringes. ❑ The air in surgery rooms. ❑ In some cases, the radiation is used for foods.  Chemical Sterilization The effect of chemicals as a sterilization method includes: ❑ Denaturation of the cell proteins and genetic material. ❑ Permeabilization of the cell membrane (loss of the selective permeability), that means the transport into and out of the cell is impaired.  Chemical Sterilization The chemicals used as sterilizing agents are classified into 2 groups: A. Disinfectants B. Antiseptics A chemical agent used to A chemical agent used to destroy the microorganisms. inhibit the microbial growth.  A.Disinfectants B. Antiseptics Ex: Phenol solution 5% Ex: Preservatives as Cresol solution 5% benzoic acid, lactic acid Formaldehyde solution sorbic acid, acetic acid 0.25% and their salts. Chloroform solution 0.25% Mercuric chloride Alcohol The culture media are not sterilized by chemicals (why) because the residues of chemicals will prevent or inhibit the growth of the desired microorganisms. The blood is sterilized chemically as follows: Addition of formaldehyde solution 0.25% to the blood for 30 minutes. Heating the mixture in a water bath at 57°c to remove formaldehyde.  Sterilization by filtration Sterilization by filtration is passage of the heat-sensitive liquids through a filter with small pore size to retain the microbial cells. When the pore diameter is: ❑ 0.45 µm, the filter will retain the bacterial cells. ❑ 0.22 µm, the filter will retain the bacterial cells and viral particles.  Filtration is used to sterilize the heat-sensitive liquids as: Carbohydrates (as pentoses) Enzymes Hormones Vitamins Toxins Blood serum Amino acids Urea Sodium bicarbonate (converted to sodium carbonate with heating)  Types of filters 1. Membrane filter made of cellulose acetate or nitrate. 2. Sintered-glass filter made of glass fibers. 3. Seitz filter made from asbestos.  Filtration Unit Funnel Filter holder Rubber disk Rubber tubing Suction flask Vacuum flask Büchner flask The filtrate without microbial cells (sterile liquid) will be Vacuum pump collected in the vacuum flask 1. The filter and suction flask must be sterilized by autoclaving before starting the filtration process. 2. After filtration, the filter must be autoclaved for the safe disposal.  Syringe filter (Disposable filter) A syringe filter (sometimes called a wheel filter if it has a wheel-like shape) is a single-use filter cartridge. It is attached to the end of a syringe for use. A syringe filter generally consists of a plastic housing with a membrane that uses as a filter. These syringe filter units are used for filtration of small volume of liquids. How to use the syringe filter? Sterilized syringe filter Sterilized syringe 1. Loading the sample into the syringe. 2. Attaching the filter with a twisting movement. 3. Holding the assembled syringe and filter vertically on sterilized bottle or tube. 4. Pressing the syringe plunger to push sample through the filter. 5. After filtration: Closing the container containing the sterilized filtrate. Autoclaving the filter and syringe after placing them in autoclavable bag. This step is very important for safe disposing of the filter and its syringe.  Sterility test It is a method to establish the presence or absence of viable microorganisms in all culture media and filtrates which are required to be sterile. The culture medium is The filtrates are tested for the tested for the sterility sterility by as follows: its incubation at 30 - Inoculation of the sterile 37°c / 24h. The efficient culture medium, which is sterilization is indicated tested previously for the by absence of the sterility, with a small microbial growth. volume of the filtrate. Incubation of the inoculated medium at 30- 37°c / 24h. Testing the result: The efficient sterilization is indicated by absence of the microbial growth.  How to use the autoclave as Arnold? By opening the safety valve to prevent generation of pressure higher than the atmospheric pressure. ❖ The distilled water is not sterilized by tyndallization (why) because it does not contain nutrients which permit the germination of spores to vegetative cells to kill at 100°c. ❖ The boiling is not considered as a sterilization method (why) because it is sufficient only to kill the non-spore forming microorganisms. Many bacterial spores are resistant to boiling that means they will survive.  You have a culture medium containing gelatin in a glass bottle. The bottle is plugged with rubber plug. How to sterilize it? 1. Replacement of the rubber plug with cotton plug. 2. Wrapping the rubber plug with the aluminum foil. 3. Sterilization of the medium by tyndallization. 4. Sterilization of the rubber plug by autoclaving. 5. In the working area of the Bunsen flame: Unwrapping the rubber plug. Closing the bottle containing sterilized medium with rubber plug instead of the cotton one.  You have a culture medium in a glass bottle. The bottle is plugged with rubber plug. How to sterilize it? 1. Replacement of the rubber plug with cotton plug. 2. Wrapping the rubber plug with the aluminum foil. 3. Sterilization of the medium and rubber plug by autoclaving. 4. In the working area of the Bunsen flame: Unwrapping the rubber plug. Closing the bottle containing sterilized medium with rubber plug instead of the cotton one. 1. Spherical Cells (cocci): They are found in different arrangements: 2. Short Rods: They are found as single cells.  Non-sporulated long  Sporulated long rods rods, found in chains. 4. Spiral Shape (Spirillum) 5. Vibrio Shape 6. Actinomycetes: Long, thin, branched and aseptate hyphae, reproduce by conidia. 1. Molds: Large, thick, 2. Yeasts: Large oval cells, branched and septate found as single cells or in hyphae, reproduce by non-uniform groups, conidia. reproduce by budding.  To know the morphology of the microbial cells, you must do 2 things: 1. Preparation of the stained smear. 2. Microscopic examination of the stained smear. Smear is a spread thin film of the microbial cells on the slide for the microscopic examination  Microscope Mechanical Optical Parts Parts 1. Base. 2. Stage (to hold the slide). 3. Arm (to carry the microscope). 4. Focusing Knobs: a. Coarse adjustment knob (used to help the sample on the slide to be into view). b. Fine adjustment knob (used to focus the sample sharply). 1. Mirror: to direct the light to the slide through the condenser. 2. Condenser consists of several lenses and iris diaphragm to control and concentrate the light on the slide. 3. Ocular lens. 4. Objective lenses which are attached to the nosepiece. ✓ Low-power objective lens (8 – 10x). ✓ High-power objective lens (40 – 45x). ✓ Oil-immersion lens (90 – 100x). Condenser could be Iris diaphragm could moved up (ranking up) to be opened to obtain obtain light with high light with high intensity intensity or or moved down (ranking closed to obtain light down) to obtain light with low intensity. with low intensity.  What is the relation between the microscopic magnification and lighting intensity? magnification of the microscope lighting intensity  The optical parts controlling the lighting intensity  are 1.1. Condenser ( Racking up or Racking down). 2.2. Iris Diaphragm (Opening or closing). 3. Mirror (in the microscopes with external light source). Objective Lens Lighting Condenser Iris Lens Magnification intensity Diaphragm Low- Racking power (8 – 10x) Weak down to the Closed lens lowest point High- In the middle Partially power (40 – 45x) Medium (slightly opened lens raised) Racking up Oil- (90 – 100x) Strong to the Completely highest opened immersion point lens (completely raised  Objective lens (low or high power) Air Glass slide Air Lighting  Objective lens (oil-immersion) cedar-wood oil Glass slide Air Lighting Using cedar-wood oil in the microscopic examination of stained smears (why). Because Light is refracted when it passes between glass and air because the refractive index of glass and air is different. The refractive index of glass and cedar oil is the same. So, when immersion oil (cedar-wood oil) is placed between the slide and the oil- immersion lens, the light continues without refraction. Steps of the Microscopic Examination of Stained Smears Using the Oil-Immersion Lens: 1. 1. Racking up the condenser. 2. 2. Opening the iris diaphragm. 3. 3. Placing 1 drop of cedar oil on the smear. 4. 4. Placing the slide on the stage. 5. Lowering the oil-immersion lens, using the coarse adjustment knob, until the oil is flattened. 6. Using the coarse adjustment knob with very very very slow movement to come the cells into view. 7. Using the fine adjustment knob to focus the cells sharply. Next week, you must have the following tools: 1. Lab coat. 2. Slides. 3. Slide cover. 4. Wooden or plastic slide box to maintain the stained slides to be submitted at the end of semester. 5. Matches. 6. Small towel. 7. Wooden clip (clothes peg).