Microbiology Ola Manual PDF

Laboratory Safety Procedure : 1. Microbiological procedures, including : a. Reporting all broken glassware to the instructor and receiving instructions for cleanup b. Methods for aseptic transfer c. Washing hands prior to and following laboratories and at any time contamin...

Laboratory Safety Procedure : 1. Microbiological procedures, including : a. Reporting all broken glassware to the instructor and receiving instructions for cleanup b. Methods for aseptic transfer c. Washing hands prior to and following laboratories and at any time contamination is suspected d. Never eating or drinking in the laboratory e. Disinfecting lab benches prior to and at the conclusion of each lab session f. Identification and proper disposal of different types of waste ,never applying cosmetics, including contact lenses, or placing objects (fingers, pencils) in the mouth or touching the face g. Reading and signing a laboratory safety agreement indicating that the student has read and understands the safety rules of the laboratory h. Good lab practice, including returning materials to proper locations. i. Appropriate labels and signs must be placed on all specimens or instruments j. Train faculty and staff in the use of MSDS 2. Protective procedures, including : a. Tying long hair back, wearing personal protective equipment (eye protection, coats, closed shoes b. always using appropriate pipetting devices and understanding that mouth pipetting is forbidden 3. Emergency procedures, including a. locating and properly using emergency equipment (eye-wash stations, first-aid kits, fire extinguishers, chemical safety showers, and emergency numbers) b. reporting all injuries immediately to the instructor and following proper steps in the event of an emergency Safety Data Sheet : Safety Data Sheet (SDS) , also referred to as Material safety Data Sheet (MSDS) , is a form containing information regarding the properties of a particular substance. The SDS includes physical data such as melting point , boiling point , information on the substance’s toxicity , reactivity , health effects , storage , and disposal Personal protection Equipment (PPE): PPE from an immediate barrier between the personnel and the hazardous agent , and they include item for personal protective such as : 1. Lab coat and Gowns 2. Gloves 3. Goggles 4. Face mask 5. Foot protection (Shoe cover) Lab Safety Equipment: 1. Safety shower , Eye wash , Fire blanket ,fire distinguisher 2. Sharp containers: for the disposable of Sharp objects, including tips, microscope slides, broken glass, glass tubes or bottles, scalpels, and needles. 3. Biologic Safety Cabinet: A BSC is a device that encloses a workspace in such a way as to protect workers from aerosol exposure to infectious disease agents. Air that contains the infectious material is sterilized, either by heat, ultraviolet light, or, most commonly, by passage through a HEPA filter that removes most particles larger than 0.3 mm in diameter. Most clinical laboratories use Type II A BSCs for processing of specimens and for work with isolates, especially those that present an aerosol hazard. Type I hoods are rarely used today Type III hoods are not commonly employed in diagnostic laboratories. In the class II and III cabinets, air is directed across the work surface from the top of the cabinet in a laminar fashion, minimizing contamination of the samples from aerosolized agents. These devices are sometimes referred to as laminar flow safety cabinets. The downward laminar flow also protects the work from unfiltered air that is drawn in through the front portal in the class II cabinets; the front intake air is directed by the laminar flow into the sump below the work surface where it is filtered before eventually joining the downward laminar flow. Biosafety Levels : 1. Biosafety Level 1 (BSL-1) : BSL-1 is the basic level of protection common to most research and clinical laboratories , and is appropriate for agents that are not known to cause disease in normal , health humans. BSL-1 agents include Bacillus subtilis and Naegleria gruberi 2. Biosafety Level 2 (BSL-2) : BSL-2 is appropriate for moderate – risk agents known to cause human disease of varying severity by ingestion or through percutaneous or mucous membrane exposure. Most cell culture labs should be at least BSL-2 , but the exact requirements depend upon the cell line used and the type of work conducted. Examples include: Salmonella, Staphylococcus aureus, Clostridium dificile,  3. Biosafety level 3 (BSL -3) BSL-3 is appropriate for indigenous or exotic agents with a known potential for aerosol transmission , and for agent that may cause serious and potentially lethal infections. Mycobacterium tuberculosis, and West Nile virus 4. Biosafety Level 4 (BSL -4 ) BSL-4 is appropriate for exotic agents that pose a high individual risk of life -threatening disease by infectious aerosols and for which no treatment is available. these agents are restricted to high containment laboratories. , such as Ebola virus Microbiology Laboratory Waste Disposal: All materials contaminated with potentially infectious agents must be decontaminated before disposal. These include unused portions of patient specimens, patient cultures, stock cultures of microorganisms, and disposable sharp instruments, such as glass microscope slides, glass tubes, scalpels, and syringes with needles. Infectious waste may be decontaminated by use of an autoclave, incinerator, or any one of several alternative waste-treatment methods. Some state or local municipalities permit blood, serum, urine, feces, and other body fluids to be carefully poured into a sanitary sewer. Infectious waste from microbiology laboratories is usually autoclaved on site or sent for incineration. Infectious waste (agar plates, plastic tubes, and reagent bottles) should be placed into two leak-proof, plastic bags for sturdiness; this is known as double bagging. Instrument and tools Used In Microbiology Labs : Bunsen Burner Swab Hot plate Wire Loop :To transfer MO Incubator Petri Dish Autoclave Burner Needle : For Stabbing in deep media Anaerobic jar : generate CO2  Observing Microorganism Through a Microscope : Microscopy is defined “magnify” (i.e., visually enlarge) objects too small to be visualized with the naked eye so that their characteristics are readily observable. Because most infectious agents cannot be detected with the Naked eye. In Microbiology we used the microscope to : a. Detect the microorganisms b. To see the characteristics for this MO (shape , color after stain , Morphology …… ) c. several staining procedures to be seen clearly. Units of Measurement: When measuring microorganisms, we use the metric system. Microorganisms are measured in even smaller units, such as Bacteria and Protozoa in micrometers and Viruses in nanometers. The basic 4 types of microscopy: o Bright-field microscopy (also known as light microscopy) o Fluorescence microscopy have the widest use and application within the clinical microbiology laboratory. o Dark-field and electron microscopes are not typically found within a clinical laboratory and are predominantly used in reference or research settings 1. Bright-field microscopy (also known as light microscopy) Refers to the use of any kind of microscope that uses visible light to observe specimens Compound Light Microscopy (LM) is a types of light microscopy , has a series of lenses and uses visible light as its source of illumination, With a compound light microscope, we can examine very small specimens as well as some of their fine detail.  Parts of compound microscope and functions , Focusing Knobs: The coarse adjustment and fine adjustment knobs on the side of the microscope are used for bringing objects into focus when studying an object on a slide. The magnification is achieved when light rays from an illuminator (the light source) pass through a condenser, which has lenses that direct the light rays through the specimen. From here, light rays pass into the objective lenses, the lenses closest to the specimen. The image of the specimen is magnified again by the ocular lens, or eyepiece. How we Calculate The Total Magnification ?  Oil immersion - 100x Oil Immersion Techniques : The oil immersion lens derives its name from the fact that a special mineral oil is placed between the lens and the microscope slide. The oil is used because it has the same refractive index as glass, which prevents the loss of light due to the bending of light rays as they pass through air. The use of oil in this way enhances the resolving power of the microscope Note: we use the oil immersion with 100× objective lens only. 2. Darkfield Microscopy : is used to examine live microorganisms that either are invisible in the ordinary light microscope, cannot be stained by standard methods. A darkfield microscope uses a darkfield condenser that contains an opaque disk. The disk Make the Light reflected off the specimen enters the objective lens. The specimen appears light against a black background. This technique is frequently used to examine very thin spirochetes, such as Treponema pallidum. 3.Fluorescence Microscopy : is commonly used for the rapid detection and identification of bacterial antigens in tissue smears, sections, and fluids. For example, a sputum specimen can be quickly screened for M. tuberculosis by staining it with a fluorescent dye that binds specifically to M. tuberculosis. Only the stained bacterium of interest will be visible when the specimen is viewed under the fluorescence Microscope  Uses UV light.  Fluorescent absorb UV light and emit visible light.   Cells may be stained with fluorescent dyes (Fluorochrome) , Which have special attractions.For example, the fluorochrome O, which glows yellow when exposed to ultraviolet light, is strongly absorbed by Mycobacterium tuberculosis 3.Electron Microscopy : Objects smaller than about 0.2 μm, such as viruses or the internal structures of cells, must be examined with an electron microscope. - Uses Electrons instead of Light - The shorter wavelength of electrons gives greater resolution - Images produced by electron microscopes are always black and white. Instead of using glass lenses, an electron microscope uses electromagnetic lenses to focus a beam of electrons onto a specimen. There are two types: the transmission electron microscope and the scanning electron microscope. Direct and Indirect Smears😊 Staining methods are either used directly with patient specimens or are applied to preparations made from microorganisms grown in culture. - A direct smear is a preparation of the primary clinical sample received in the laboratory for processing. Its provides a mechanism to identify the number and type of cells present in a specimen, including white blood cells, epithelial cells, and predominant organism type. - Preparation of an indirect smear indicates that the primary sample has been processed in culture and the smear contains organisms obtained after purification or growth on artificial media. Indirect smears may include preparation from solid or semisolid media or broth. Care should be taken to ensure the smear is not too thick when preparing the slide from solid media. In addition, smear from a liquid broth should not be diluted Preparation and Staining 😊 There are two principal ways of preparing a microbial specimen for observation with light microscope : 1. Wet Preparation: used to look at living microbes ,two types ( Wet mount and Hanging drop) 2. Stained smear: used to observe the morphology, staining property and arrangement of microbes. 1) Wet preparation types: there are two types of wet preparation used to observe living microbes, with the both types we use a single drop of liquid containing living microbes. a) Wet mount: is commonly used to see: The size and shape of individual organisms and the characteristics arrangement or grouping of bacterial cells. Generally, specimen samples are placed on the slide using a swab or a direct smear that contains patient material or by using a pipette into which liquid specimen has been aspirated, covered with cover glass and observed under 10x or 40x objective lens. b) Hanging drop: used to differentiate between motile and nonmotile microorganisms. - True movement of microorganisms: the organism move in different directions and change their position in the field. - Non true movement of microorganisms: by  Passive drifting of the organisms in the same direction in a convectional current in the fluid.  Brownian movement, which is an oscillatory movement about a nearly fixed point. General Concepts for Specimen Collection and Handling : Specimen collection : Information for the clinicians should include the following in Sample collection: 1. Collect the specimen before administration of antimicrobial agents. 2. Selection of the appropriate anatomic site and specimen and in sufficient quantity. 3. Collection instructions, including the type of swab or transport medium 4. Transportation instructions, including time and temperature constraints 5. Labeling instructions, including patient demographic information (minimum of two patient identifiers) 6. Special instructions, such as patient preparation 7. Sterile versus nonsterile collection devices 8. Minimal acceptable quality and recommended quantity Specimen Transport : 1. ideally, specimens should be transported to the laboratory within 2 hours of collection. 2. All specimen containers should be leak-proof, and the specimens should be transported within leak-proof, plastic bags with a separate section for paperwork 3. Bags should be marked with a biohazard label Many microorganisms are susceptible to environmental conditions such as the presence of oxygen (anaerobic bacteria), changes in temperature (Neisseria meningitidis), or changes in pH (Shigella). Thus the use of special preservatives or temperature controlled or holding media for the transportation of specimens delayed for more than 2 hours is important to ensure organism viability (survival) Specimen Preservation Preservatives, such as boric acid for urine or polyvinyl alcohol (PVA) and buffered formalin for stool for ova and parasite (O&P) examination, are designed to maintain the appropriate colony counts (for urines) or the integrity of trophozoites and cysts (for O&Ps), respectively.  Other transport or holding media maintain the viability of microorganisms present in a specimen without supporting the growth of the organisms. Example for transport media: a. Stuart’s medium b. Amie’s medium Sometimes charcoal is added to these media to absorb fatty acids present in the specimen that could result in pH changes in the media and the killing of fastidious organisms such as Neisseria gonorrhoeae or Bordetella pertussis. Preparing of Specimens Many specimens require some form of initial treatment before inoculation onto primary plating media. Such procedures include homogenization (grinding or mincing) of tissue; concentration by centrifugation or filtration of large volumes of sterile fluids, such as ascites (peritoneal) or pleural (lung) fluids. Report and record : The report typically includes the following : Order entry, patient identification, and specimen identification , the result , antimicrobial therapy …  Media Preparation : The study of Microorganism requires techniques for isolating cells from a natural sources and growing them in the laboratory on synthetic media. Thus , Development of synthetic culture media and culture techniques have played important roles in the advance of this field. Media = Mixtures of nutrients that the microbes need to live. also provides a surface and the necessary moisture and pH to support microbial growth Microbiologists use Bacterial culture media for many purposes and applications : 1. Provide nutrients in order to grow and reproduce of bacteria. 2. Media are used to isolate and identify bacteria , according to metabolic properties. 3. Obtain pure culture Type of Bacteria according to constancy : a. Broth (Media provided in liquid phase ) - Positive result reading according to turbidity Clear – No Growth Turbid- Growth b. Solid Media (3 forms ) :  Media Classifications and Functions Media are categorized according to their function and use. In diagnostic bacteriology there are four general categories of media: enrichment, nutritive, selective, and differential. 1. Nutritive : Contain nutrients that support growth of most non fastidious organisms without giving any particular organism a growth advantage. Nutrient media include tryptic soy agar and nutrient agar plates for bacteria, or Sabouraud’s dextrose agar for fungi. 2. Selective :Inhibits the growth of some bacteria while selecting the other. contain one or two agent that inhibit some Bacteria. 3. Differential : distinguish the bacteria from other bacteria growing on the same agar plate. 4. Enrichment : contain specific nutrients required for the growth of particular bacterial pathogens. This media type is used to enhance the growth of a particular bacterial pathogen. One example of such a medium is buffered charcoal–yeast extract agar (BCYE), which provides l-cysteine and other nutrients required for the growth of Legionella pneumophila, the causative agent of legionnaires’ disease Many media used in diagnostic microbiology provide mor than one function such as :  MacConkey media Is both selective and differential Selective : Inhibits the growth of Gram positive bacteria while selecting for the growth of Gram negative , Bec.of presence of bile salts and crystal violet that inhibit gram positive bacteria. Differential : Differentiation between different organisms growing on the same plate.because neutral red (pH -sensitive dye ) and lactose (type of sugar ) have been added to media. Bacteria that used lactose for food (lactose fermenters ), produce acidic so this will change the color to pink. while non lactose fermenters will be colorless and clear. EMB (Eosin Methylene Blue ) is both selective and differential  - Selective : because it only grows gram negative bacteria. inhibits the growth of gram positive bacteria , Bec. Of the presence of Eosin AND Methylene blue. - Differential : Differentiation of lactose fermenters (dark purple colonies ) from non lactose fermenters (colorless colonies ) Note : E.coli is grown it will give a distinctive metallic green sheen )  MSA (Mannitol Salt Agar ) - Selective : because it has a high NaCL (7.5 % ) concentration that inhibits most Bacteria and few types of bacteria can grow on the hypertonic medium - Differential : because this medium contains a pH indicator to identify organisms that ferment mannitol. Organic acids wastes mannitol fermenters produce change the medium from red to yellow. - MSA works well for identifying pathogenic Staphylococci , such as Staphylococcus aureus , which will Ferment mannitol. Most non pathogenic staphylococci (Staphylococcus epidermidis )will not ferment mannitol.  Blood agar is enriched and differential media : - Most specimens received in a clinical microbiology lab are plated onto blood agar. it is an Enriched medium that will grow fastidious bacteria. Also contain 5%sheep blood. -This media is not selective. it is enriched and differential. Certain Bacteria produce enzymes (hemolysin) that act on red cells to produce either : 1-Beta hemolysis : Enzyme lyse the blood cell completely , producing a clear area around the colony. 2-Alpha hemolysis : Incomplete hemolysis produces a greenish discoloration around the colony. 3-Gamma hemolysis : No effect on the red cell  Chocolate agar : - it is a type of blood agar plate in which the blood cells have been lysed by heating the cells to 80 C to release X (Hemin ) and V (NAD+) Factors. chocolate agar is used for growing Haemophilus influenza , and Neisseria gonorrhoeae. Media with solidifying agents like agar ( is a natural polysaccharide produced by marine algae it is not degraded or metabolized by bacteria , It melts at 98 c and sets at 42 c ) Liquid media can be converted to solid media by adding (1-2% of agar. If you add (0.6% agar ) , the media is called semisolid. Media preparation : 1. Measure out the proper amount of media powder using the electronic Balance 2. Fill up your Flask with the proper volume of filtered water and place on the hot plate 3. Drop in a magnetic stirrer and turn the stirrer on. 4. Slowly pour in the media powder into water 5. Turn the hot plate on high (Agar will not dissolve in water that is hot , so DON’T heat the water until after adding the powder ) 6. Pour the media in the Bottle , then put it in auto clave with the autoclave indicator 7. After sterilized the media , let the media too cool ( about 40 c) 8. While the media is cooling , spray and wipe the bench with 95% ethanol. 9. Pour the media from the bottle into the plate until about half way full 10. Store the plate upside down inside the bag , to prevent them from drying out , and store at 4 c. Blood agar media preparation : It is enriched medium which is prepared by Measure out the proper amount of media powder (nutrient or blood agar ) and mix it with a proper D.W then put it in autoclaved , cooled to 50 c and finally add 5% of sheep blood with constant shaking. - If we want to prepare a Chocolate agar we add the blood when the media temperature approximately 72 c Checking the pH of a culture medium: The pH of most culture media is near neutral. The simplest way of testing the pH of a culture medium is to use narrow range pH papers or a pH meter. Media sterility testing: For ‘sterile’ media in screw-cap tubes or bottles, the simplest way to test for contamination is to incubate 5% of the batch at 35–37 °C overnight. Contamination by microorganisms capable of overnight growth, will be shown by a turbidity in a fluid medium and growth on or in a solid medium. Media in petri dishes are best examined for contamination immediately before use. Labelling and storage of culture media and additives: Dehydrated culture media and dry ingredients such as peptone, tryptone, an carbohydrates (solid form) should be stored at an even temperature in a cool dry place away from direct light. Container tops must be tight-fitting and in humid climates, tape-sealed. Additives such as blood, serum, antimicrobials in solid form, urea and carbohydrate solutions, require storage at 2–8 °C. All additives should be allowed to warm to room temperature before being used. Antimicrobial solutions should be stored frozen at -20 °C in the amounts required. Plates of culture media should be stored at 2–8 °C, preferably in sealed plastic bags. Most media in screw-cap tubes or bottles can be stored at room temperature (20–25 °C). Prepared media should be stored in the dark  Cultivation and isolation of Bacteria : (the process of growing microorganisms in culture by taking bacteria from the infection site) a) Streaking Plate ( inoculation bacteria in solid media\ plate agar by streaking method) The loop is used for preparing a streak plate. this involves the progressive dilution of bacteria over the surface of solidified agar medium in a petri dish in such a way that colonies grow well separated from each other. so the purpose of streaking is to obtain single isolated colonies. Principles : 1.Sterilize the inoculating loop in the bunsen burner by putting the loop into the flame until it is red hot. Allow it to cool. 2.Pick an isolated colony from the agar plate culture and spread it over the first quadrant (approximately 1/4 of the plate) using close parallel streaks or Insert your loop into the tube/culture bottle and remove some inoculum. 3.Immediately streak the inoculating loop very gently over a quarter of the plate using a back and forth motion. 4. Flame the loop again and allow it to cool. Going back to the edge of area 1 that you just streaked, extend the streaks into the second quarter of the plate (area 2). 5. Flame the loop again and allow it to cool. Going back to the area that you just streaked (area 2), extend the streaks into the third quarter of the plate (area 3). 6. Flame the loop again and allow it to cool. Going back to the area that you just streaked (area 3), extend the streaks into the center fourth c, the plate (area 4). 7. Flame your loop once more. 8. put it in incubator at 37 c for 24 h. 9. after 24 h , if there is more than one type of colony , each type should be streaked again on a separate plate to obtain a pure colony. Perfect streaking , pure colony B ) Inoculation of solid media in test tubes : 1- slant media: plunging the wire into the center of the medium to the bottom of the butt of the tube and withdraw and streaking the surface of the agar with loop in a S shaped or zig zag manner (this allow to grow both aerobically and anaerobically ) 2- stab media : inoculated by plunging the wire into the center of the medium. Labelling plates : All petri plates should be labeled as the following : 1. make certain that all plates are labeled on the bottom half ( the portion of the petri plate that contains the media ). 2. (Name , Data , type of specimen ) 3. Upside down position “upside down” means that ½ of the petri plate with media faces up. the empty ½ of the petri plate is down. 4. Plates will be incubated at 37c for 24 hrs - Aerobic incubation : at 37 °C in incubator. - Incubation in an atmosphere with add carbon dioxide.by using candle jar or anaerobic jar. - For prolonged incubation ,up to 8 weeks ,screw capped bottles should be used instead of petri dishes or tubes to prevent drying of medium. Preservation of pure culture : 1. Refrigeration (0-4 °C) : 2-3 weeks for bacteria and 3-4 months for fungi 2. Paraffin method.for several years 3. Cryopreservation (freezing in liquid nitrogen at -196 °C) 4. lyophilization (freezing at -70 °C and then dehydrated by vacuum  Stained Smears: Staining simply means coloring the microorganisms with a dye to see certain structures. Before the microorganisms can be stained, they must be fixed (attached) to the microscope slide. Fixing simultaneously kills the microorganisms and fixes them to the slide. How to prepare and fix smears for staining ? with the take one then let it Label date and Take colony form Drying and dry to see the the patient’s one the media. Fixation fixing Staining the MO slide colony or the smears undr name and organism Microsvope number Smears should be spread evenly covering an area of about 15–20 mm diameter on a slide. Drying and fixing smears: After making a smear, leave the slide in a safe place for the smear to air-dry, protected from dust, flies, and direct sunlight. The purpose of fixation is to preserve microorganisms and to prevent smears being washed from slides during staining. Smears are fixed by heat, alcohol. Microorganisms are not always killed by heat fixation, e.g. M. tuberculosis. a. Heat fixation: Allow the smear to air-dry completely. Rapidly pass the slide, smear uppermost, three times through the flame of a Bunsen burner or by placing it on a slide warmer (60° C) for at least 10 minutes. Allow the smear to cool before staining it. b. Alcohol fixation: This form of fixation is far less damaging to microorganisms than heat. Cells, especially pus cells, are also well preserved. Alcohol fixation is therefore recommended for fixing smears when looking for Gram negative intracellular diplococci. Alcohol fixation is more bactericidal than heat (e.g. M. tuberculosis is rapidly killed in sputum smears after applying 70% v/v alcohol). fix with one or two drops of absolute methanol or ethanol or 70% will be adequate, Leave the alcohol on the smear for a minimum of 2 minutes or until the alcohol evaporates. The techniques used to make smears from different specimens are as follows: 1. Purulent specimen: using a sterile wire loop, make a thin preparation. Do not centrifuge a purulent fluid, e.g. CSF containing pus cells. 2. Non-purulent fluid specimen: centrifuge the fluid and make a smear from a drop of the well-mixed sediment. 3. Culture: emulsify a colony in sterile distilled water and make a thin preparation on a slide. When a broth culture, transfer a loopful to a slide and make a thin preparation. 4. Sputum: use a piece of clean stick to transfer and spread purulent and caseous material on a slide. Soak the stick in a phenol or hypochlorite disinfectant before discarding it. 5. Swabs: roll the swab on a slide. This is particularly important when looking for intracellular bacteria such as N. gonorrhoeae (urethral, cervical, or eye swab). Rolling the swab avoids damaging the pus cells. Differential Stains😊 Differential stains react differently with different kinds of bacteria and thus can be used to distinguish them. The differential stains most frequently used for bacteria are the Gram stain and Ziehl–Neelsen (acid-fast) stain. 1. Gram stain: The Gram stain was developed by Hans Christian Gram. It is one of the most useful staining procedures because it classifies bacteria into two large groups: gram-positive and gram- negative. Component of gram stain: 1) Crystal violet (Primary stain) 2) Iodine (Mordent) 3) Decolorizing agent (alcohol 95% or an alcohol-acetone) 4) Safranin (Counter stain) Principal of gram stain: Gram positive cells have a thick layer of peptidoglycan in the cell wall that retains the primary stain, crystal violet. Gram negative cells have a thinner peptidoglycan layer that allows the crystal violet to wash out on addition of alcohol ,therefore they are stained pink or red by counterstain. Gram stain procedure: 1. A heat fixed smear is covered with crystal violet (1 minute). 2. After a short time, the purple dye is washed off, and the smear is covered with iodine (1 min.), a mordant. When the iodine is washed off, both gram-positive and gram-negative bacteria appear dark violet or purple. 3. Next, the slide is washed with alcohol or an alcohol-acetone solution (5 seconds), which removes the purple from the cells of some species but not from others. 4. The alcohol is rinsed off, and the slide is then stained with safranin (30 seconds), The smear is washed again, blotted dry, and examined microscopically.  Gram stain result: Gram positive bacteria............. Dark purple\Blue Yeast cells....................... Dark purple\Blue Gram negative bacteria.......... Pale to dark red Nuclei of pus cells....................... Red Epithelial cells........................ Pale red So according to this Picture which one is gram positive bacteria ? and which one is gram Negative Bacteria ? Reporting Gram smears: a. If no organisms or cells are detected in a smear of a clinical specimen, report “No organisms seen” or “No cells seen,” respectively. b. If there is organisms and cell report according to the table below. Gram stain is important test for the bacterial identification : a. Is it the bacteria negative or positive ? b. Shape of the bacteria (cocci , bacilli , spiral , coccobacillus c. Arrangements of bacterial cell (staph , strept , diplo ) Variations in gram reactions: Gram positive organisms may lose their ability to retain crystal violet and stain Gram negatively for the following reasons: 1) Cell wall damage due to antibiotic therapy or excessive heat-fixation of the smear. 2) Over-decolorization of the smear. 3) Use of an iodine solution which is too old, i.e. yellow instead of brown in colour (always store in a brown glass or other light opaque container). 4) Smear has been prepared from an old culture.  - Gram negative organisms may not be fully decolorized and appear as Gram positive when a smear is too thick. - Note: Always check new batches of stain and reagents for correct staining reactions using a smear containing known Gram positive and Gram negative organisms. 2. Ziehl–Neelsen (acid-fast) stain: This stain binds strongly only to bacteria that have a waxy material in their cell walls. Microbiologists use this stain to identify all bacteria in the genus Mycobacterium (mī’kō-bak- TI-r-e-um), including the two important pathogens Mycobacterium tuberculosis, the causative agent of tuberculosis, and Mycobacterium leprae (LEP-rī), the causative agent of leprosy. Ziehl–Neelsen (acid-fast) stain component: 1. Carbolfuchsin (primary stain) 2. Decolorizer (acid-alcohol or sulphuric acid) 3. Methylene blue or malachite green (counter stain) Principal of Ziehl–Neelsen (acid-fast) stain: The acid-fast microorganisms retain the pink or red color because the carbolfuchsin is more soluble in the cell wall lipids than in the acid-alcohol, in non–acid fast bacteria, whose cell walls lack the lipid components, the carbolfuchsin is rapidly removed during decolorization, leaving the cells colorless, when counter stain is applied the non–acid-fast cells appear blue. Ziehl–Neelsen (acid-fast) stain procedure: 1. The red dye carbolfuchsin is applied to a fixed smear, and the slide is gently heated for several minutes(8-10 min.). (Heating enhances penetration and retention of the dye). 2. Then the slide is cooled and washed with water. 3. The smear is next treated with acid-alcohol(0.5-1 min.), which removes the red stain from bacteria that are not acid-fast. 4. The smear is then stained with a methylene blue counter stain (1 min ). Non–acid-fast cells appear blue after the counter stain is applied. Ziehl–Neelsen (acid-fast) stain result: - AFB.... Red, straight or slightly curved rods, occurring singly or in small groups, may appear beaded. - Cells.... Green \ Background material...... Green - - Non acid fast ……………blue  Reporting Ziehl–Neelsen (acid-fast) stain: 1. When no AFB are seen after examining 100 fields: Report the smear as ‘No AFB seen’. 2. Positive: report as “Positive for acid-fast bacilli” and provide the count as following: a. More than 10 AFB/field........... report +4 1–10 AFB/field........ report +3 10–100 AFB/100 fields.. report +2 1–9 AFB/100 fields..... report +1 3. Albert staining of volutin granules: The Albert technique is used to stain the volutin, or metachromatic, granules of C. diphtheriae. The granules are most numerous after the organism has been cultured on a protein-rich medium such as Dorset egg or Loeffler serum, The presence of granules is of no significance regarding virulence. Reagents: 1. Toluidine blue-malachite green 2) Albert’s iodine Stain procedure: 1. Fix the dried smear using alcohol 2. Cover the smear with the toluidine blue malachite green stain for 3–5 minutes. 3. Wash off the stain with clean water. When the tap water is not clean, use filtered water or clean boiled rainwater. 4. Tip off all the water. 5. Cover the smear with Albert’s iodine for 1 minute. Wash off with water. 6. Wipe the back of the slide clean, and place it in a draining rack for the smear to air-dry. 7. Examine the smear microscopically, first with the 40x objective to check the staining and to see the distribution of material and then with the oil immersion lens to look for bacteria containing metachromatic granules. Stain result: Bacteria cells...................... Pale green Metachromatic granules............ Green-black  Organism Identification Using Phenotypic Criteria : However, most of the phenotypic characterizations used in diagnostic bacteriology are based on tests that establish a bacterial isolate’s morphology and metabolic capabilities. The most commonly used phenotypic criteria include the following: 1. Microscopic morphology and staining characteristics 2. Macroscopic (colony) morphology, including odor and pigmentation 3. Environmental requirements for growth 4. Resistance or susceptibility to antimicrobial agents 5. Biochemical reactions including enzymatic reactions or chemical profiles 1. Microscopic Morphology and Staining Characteristics Microscopic evaluation of bacterial cellular morphology (Shape – Bacilli or cocci , arrangement staph or strep or diplo.. ) and Gram stain (positive – blue and Negative – Red ) Gram stain morphology Gram positive Gram positive Gram negative Gram negative cocci bacill bacilli cocci Staphylococcus streptococcus Bacillus Enterobac psedomonas Neisseria 2. Macroscopic (colony) morphology, including odor and pigmentation Criteria frequently used to characterize bacterial growth (colony morphology) include the following: i. Colony size (usually measured in millimeters or described in relative terms such as pinpoint, small, medium, large) ii. Colony pigmentation example : Pseudomonas : Green colony in nutrient agar iii. Colony shape (includes form, elevation, and margin of the colony iv. Colony surface appearance (e.g., glistening, opaque, dull, dry, transparent , Mucoid) , Klebsiella , pneumonia has mucoid colony in media v. Changes in agar media resulting from bacterial growth (e.g., hemolytic pattern on blood agar, changes in color of pH indicators, pitting of the agar surface. a. vi. Odor (certain bacteria produce distinct odors that can be helpful in preliminary identification).Example , Pseudomonas : fruity \ Proteus that cause UTI has fish odor and swarming 3. Environmental requirements for growth : Environmental conditions required for growth can be used to supplement other identification criteria The ability to grow in particular incubation atmospheres. need aerobic or anaerobic ( need o2 or not ) An organism’s requirement, or preference, for increased carbon dioxide concentrations can provide hints for the identification of other bacteria such as Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria gonorrhoeae. In addition to atmosphere, the ability to survive or even thrive in temperatures that exceed or are well below the normal body temperature of 37°C may be helpful for organism identification. The growth of Campylobacter jejuni at 42°C and the ability of Yersinia enterocolitica to survive at 0°C are two examples in which temperature enrichment can be used to identify an organism. 4. Resistance or Susceptibility to Antimicrobial Agents Directly testing a bacterial isolate’s Sensitivity to a particular antimicrobial agent may be a very useful part of an identification scheme. Many gram-positive bacteria (with a few exceptions, such as certain Enterococcus spp., are sensitive to vancomycin( an antimicrobial agent ) In contrast, t gram-negative bacteria are resistant to vancomycin 5. Biochemical reactions including enzymatic reactions or chemical profiles (API) MRVP test , Indole test , Oxidase test , citrate test Nitrate reduction test , Urease test Coagulase test , Catalase test TSI / KIA , Sugar fermentation tests Gelatin liuefaction test , H2S production test  So Let's summarize what we talked about : The most frequent media that use in the lab is : 1. : that allow grow and bacteria (for all type). 2. : that allow Grow of Example , If the Result as the following : Growth (colony ) on Blood agar No Growth on MacConkey agar According to This Result , it is Gram positive bacteria. There are 6 Gram positive that cause disease in human : 1. Staphylococcus 2.Streptococcus 3.Bacillus 4. Clostridium 5. Corynebacterium 6. Listeria  So to identify the bacteria that caused infection we should do Biochemical test. On MSA (Fermented ) S.epidermidis On MSA (Non Fermental ) S.aureus CAMP TEST for aglactiae  Catalase test :  Many bacteria produce the enzyme catalase , which breaks down the hydrogen peroxide liberating oxygen.  The Hydrogen peroxide can be added directly to a slant culture or to bacteria smeared on a clean glass slide.  The test should not be performed with organisms growing on a blood containing medium because catalase is found in red blood cells. Purpose : To observe bacterial catalase activity. Materials : 3% hydrogen peroxide. Capillary pipettes. Clean glass slides. Nutrient agar slant cultures of Staphylococcus epidermidis (+ve) and Streptococcus (-e) Procedures : 1. small amount of growth from culture onto a clean microscope slide 2. Add a few drops of H2O2 onto the smear. if need , mix a toothpick. NOT use a metal loop needle with H2O2 ; it will give a false positive and degrade the metal. Coagulase Test : PURPOSE :  To determine the ability of the organism to produce coagulase which clots plasma.  To distinguish between staphylococcus species. Principle: Coagulase is an enzyme that convert soluble fibrinogen into insoluble fibrin. Two forms of coagulase: I. Bound coagulase (clumping factor) - Detected in the coagulase slide test , Can directly convert fibrinogen to Insoluble fibrin and causes the staphylococci to clump together 2. Free coagulase : Detected in the coagulase tube test , Reacts with a globulin plasma factor (coagulase reacting factor-CRF) to form a thrombin like factor, staphylothrombin --> catalyzes the conversion of fibrinogen to Insoluble fibrin. Interpretation Slide Coagulase test Positive — white fibrin clots in plasma; Negative —smooth suspension Tube Coagulase test Positive —formation of fibrin clot; Negative— no clot is formed Biochemical test for Gram negative Bacteria : Growth (colony ) on MacConkey agar It is Gram Negative bacteria : ( Enterobacteriaceae ; E.coli , Pseudomonas , Proteus , Klebsiella , Shigella , Salmonella ) And pseudomonas and Neisseria (diplococci ) So we should do the following step to identify which one caused infection. 1. If this bacteria Ferment lactose (pink) or not (colorless ) 2. The Morphology for this colony ( Mucoid , Odor , shape of colony , pigmentation , swarming 3. Biochemical test ….. Citrate test : Positive result Negative Result Indole Test : Negative result Positive Result MR test : Positive Negativeresult Result Positive NegativeResult Result Positive Result Positive Result Positive result VP TEST : Positive Result Negative Result Negative Result Negative Result Negative Result Motility : Positive Result Negative Result Positive Result Negative Result Positive Result H2S Test : Negative Result Negative Result Positive Result Negative Result Positive Result TSI Test : A\A ,GAS A\A , GAS K\A (mitralis) K\A K\A , GAS ,H2S A\A (vulgaris) gas H2S 1. Oxidase test : = The oxidase test identifies organisms that produce the enzyme cytochrome oxidase. = Cytochrome oxidase participates in the electron transport chain by transferring electrons Urease fromResult : Positive a donor moleculeNegative to oxygen. ResultThe oxidase reagent (N'-tetramethyl Positive Result Negative Result phenylenediamine Negative Result dihydrochloride) contains a chromogenic reducing agent, which is a compound that changes color when it becomes oxidized. If the test organism produces cytochrome oxidase, the oxidase reagent will turn blue or purple within 15 seconds. Interpretation : 1. Positive :blue/ dark purple/black color 2. Negative: no color development Citrate test : Indole Test : Negative result Positive Result MR test : Negative Result Positive Result Positive Result Positive Result Positive result VP TEST : Positive Result Negative Result Negative Result Negative Result Negative Result  2. IMViC : It that stands for indole, methyl red, Voges-Proskauer, and citrate. To obtain the results of these four tests, three test tubes are inoculated: indole test, methyl red - Voges Proskauer broth (MR-VP broth), and citrate. Simmon’s Citrate Agar : Principle: This is medium used to determine if an organism can use citrate as its sole carbon source. -In organisms capable of utilizing citrate as a carbon source, the enzyme citrase hydrolyzes citrate into oxaoloacetic acid and acetic acid. The oxaloacetic acid is then hydrolyzed into pyruvic acid and CO2. If CO2 is produced, it reacts with components of the medium to produce an alkaline compound (e.g.Na2CO3). The alkaline pH turns the pH indicator (bromothymol blue) from green to blue. This test is often used to differentiate between members of Enterobacteriaceae. Procedure: 1. Inoculate citrate agar on surface of the slant ( there is no need to stab the agar). 2. Incubate the agar at 35C for 24hrs 3. Observe the development of blue color. Expected result: - Citrate positive : The media turns from green to blue e.g Klebsiella pneumoniae and Proteus mirabilis. - Citrate negative: The media stays green. Escherichia coli and Shigella dysenteriae Indole test (Tryptophanase enzyme) Principle The metabolism of the amino acid tryptophan by organisms that possess the enzyme tryptophanase produces three major degradation products including indole (resulting from the deamination of tryptophan). Indole then can be detected by the addition of Kovac’s reagent ( pdimethylaminobenzaldehyde) to the Sulfide-indole-motility (SIM) medium. The indole test procedure is a commonly used for the differentiation of Enterobacteriaceae. procedure SIM media or tryptophan broth is inoculated using transfer needle with test bacteria for at least 24 hours. Then add five drops of kovac’s reagent down the inner wall of the tube. Interpretation : A-red-pink layer on the surface of media is positive result (the bacteria can breakdown the tryptophan to form indole ). B-no red layer is negative result. Motility agar The semi-solid medium (SIM) is used to determine whether an organism is motile and thus capable of swimming away from a stab mark. A sterile, cool inoculating wire is used to obtain inoculum from a pure culture of the test organism. The wire is stabbed into motility stab media approximately two-thirds of the depth of the media. The tube is incubated for approximately 24 hours and observed for evidence of motility. Generally, if the entire tube is turbid, this indicates that the bacteria have moved away from the stab mark (are motile). If, the stab mark is clearly visible and the rest of the tube is not turbid, the organism is likely non- motile  Note: (SIM) medium is a semisolid agar used to determine hydrogen sulfide (H2S) production (A black precipitate), indole formation, and motility. SIM medium is used to differentiate members of the family Enterobacteriaceae. - Nonmotile and indole-negative bacterium Klebsiella pneumoniae - Motile and indole-positive bacterium Escherichia coli - Motile, indole-negative, and H2S-producing bacterium Proteus mirabilis Methyl Red Test and Voges Proskauer (VP): This test is used to determine the ability of an organism to produce acid end product (e.g. lactic acid) or neutral end product (e.g. acetoin) from glucose fermentation. The methyl red (MR) and Voges Proskauer (VP) tests are read from a single inoculated tube of MR-VP broth (contains  glucose and peptone). After 24-48 hours of incubation the MR-VP broth is split into two tubes. One tube is used for the MR test; the other is used for the VP test. Methyl Red Test: to test an organism produces acid end product (e.g. lactic acid) from glucose fermintation. E. coli is one of the bacteria that produces acids, causing the pH to drop below 4.4. When the pH indicator (methyl red) is added to this acidic broth it will be cherry red (a positive MR test). For a negative result (the pH is above 6.0, MR turns yellow) Voges-Proskauer Test: to test if the organism products neutral end product (e.g. acetoin) from glucose fermentation. A reagents used for the VP test are Barritt's A (alpha-naphthol) and Barritt's B (KOH) are added to a broth (MR-VP broth). If bacteria(e.g. Enterobacter and Klebsiella) do produce more neutral products from glucose, they turn a “brownish-red to pink” color (a positive VP test). This color may take20 to 30 minutes to develop. If the culture is negative (e.g. E. coli ) for acetoin, it will turn “brownish green to yellow” o Note: A culture will usually only be positive for one pathway: either MR+ or VP+. Escherichia coli is MR+ and VP-. In contrast, Klebsiella pneumoniae is MR- and VP+. Pseudomonas aeruginosa is a glucose non fermenter and is thus MR- and VP-. Procedure : MR : Inoculate the MR/VP broth with a pure culture of the test organism. Incubate the broth at 35°C for 48–72 hours (no fewer than 48 hours). At the end of this time, add 5 drops of the methyl red reagent directly to the broth. VP : Inoculate a tube of MR/VP broth with a pure culture of the test organism. Incubate for 24 hours at 35°C. At the end of this time, aliquot 1 mL of broth to a clean test tube. Add 0.6 mL of 5% α-naphthol, followed by 0.2 mL of 40% KOH , allow it to stand for 10 to 20 minutes. UREASE TEST Purpose ❖To determine the ability of an organism to produce the enzyme, urease, which hydrolyzes urea. ❖To identify the rapid urease producers (Proteus and Morganella) and weak urease producers (Klebsiella pneumoniae and species of Enterobacter) Principle ❖Urease splits the urea molecule into ammonia (NH3), CO2 and water(H2O). Ammonia reacts in solution to form an alkaline compound, ammonium carbonate, which results in an increased pH of the medium and a color change in the indicator to pink red. Procedure : The broth medium is inoculated with a loopful of a pure culture of the test organism; the surface of the agar slant is streaked with the test organism. Both media are incubated at 35°C for 18–24 hours. Interpretation - Positive: Rapid urease activity; red throughout the medium - Positive: Slow urease activity: red in slant initially gradually converting the entire tube - Negative: No urease activity; medium remains yellow Stuart (urea) broth : Positive: Red color in the medium Negative: No color change (buff to pale yellow) A. Positive Proteus spp. B. Positive Klebsiella spp. C. Negative: Escherichia coil TSI (Triple Sugar Iron) and KIA (Kligler's Iron Agar) Triple Sugar Iron Agar (TSI) and Kligler's Iron Agar (KIA) are used to determine if bacteria can ferment glucose and/or lactose and if they can produce hydrogen sulfide or other gases. (If an organism can ferment glucose, it is "glucose positive". If it ferments lactose, it is "lactose positive".) In addition, TSI detects the ability to ferment sucrose. These characteristics help distinguish various Enterobacteriacae, including Salmonella and Shigella, which are intestinal pathogens. KIA ( kligler’s iron agar )resembles TSI (triple sugar iron )in all respects except that KIA contains two sugars (lactose and glucose) while TSI contain three sugars (lactose, glucose and  sucrose). Thus KIA tests for an organism’s ability to ferment glucose or lactose but not sucrose. COMPOSITION 1- Protein sources - beef extract, peptone, yeast extract, proteose peptone 2- Sugars (lactose, sucrose, glucose) 3- Indicators a. phenol red - carbohydrate fermentation b. ferrous sulfate - hydrogen sulfide production 4- Sodium thiosulfate - source of sulfur atoms 5- Sodium chloride - osmotic stabilizer BIOCHEMICAL REACTIONS Carbohydrate fermentation acid production : ✓ Yellow deep — glucose fermented ✓ Yellow slant — lactose and/ or sucrose fermented Gas formation: ✓ Bubble formation ✓ Cracking or splitting of the agar ✓ Upward displacement of the agar ✓ Pulling away of the medium from the walls of test tube H2S production: Blackening of the butt (FeS — black precipitate) Procedure: KIA and TSI tubes are inoculated with a long, straight wire, which is then stabbed into the deep of the tube,.When the inoculating wire is removed from the deep of the tube, the slant surface is streaked with a back-and-forth motion. Inoculated tubes are placed into an incubator at 35°C for 18 to 24 hours. If the gas being produced is hydrogen sulfide (H2S), it reacts with the ferrous sulfate and precipitates out as a black precipitate (ferric sulfide) in the butt. Organisms producing large amounts of hydrogen sulfide (e.g. Salmonella and Proteus) may produce so much black precipitate that it masks the yellow (acid) color of the butt Carbohydrate Fermentation broth with Durham tube Principle This is a differential medium tests an organism's ability to ferment the sugar. Fermentation products are usually acid (lactic acid, acetic acid etc.), neutral (ethyl alcohol etc.), or gases (carbon dioxide, hydrogen, etc.). To determine the products of sugar fermentation, a carbohydrate fermentation broth is prepared at pH 7.4. This broth contains 3 essential ingredients: 1.0% of the carbohydrate to be tested (e.g. glucose or lactose or mannitol), nutrient broth, and the pH indicator phenol red. If an organism is capable of fermenting the sugar, then acidic byproducts are formed and the pH indicator (phenol red) turns yellow. This is a test commonly used when trying to identify Gram-negative enteric bacteria, all of which are glucose fermenters moreover, Some organisms are capable of CO2 (gas) production due to fermentation. This gas is trapped in the Durham tube and appears as a bubble at the top of the tube. Procedure : 1. Obtain one tube for each sugar, usually one for glucose, lactose, sucrose, mannitol. Do not get them confused, they look the same. They should have an inverted Durham tube (they are to test for gas production). 2. Use your wire loop to aseptically inoculate each tube with your unknown bacteria. 3. Incubate the tube for 18-24 hrs at 37 C. 4. After incubation is complete, compare an inoculated tube to an uninoculated tube. Observe color changes and gas production (gas bubbles trapped in the Durham tube) Expected results: A color change to yellow in the fermentation media indicates a positive carbohydrate fermentation test (+ ) e.g. Escherichia coli, Proteus mirabilis and Shigella dysenteriae are capable of fermenting (glucose) On the other hand Pseudomonas aeruginosa is a non- fermenter. Note: If gas is present the result is recorded as +/gas. E.g. Escherichia coli and Proteus mirabilis are both gas producers. But Shigella dysenteriae ferments (glucose) does not produce gas.  A negative test carbohydrate fermentation test (-) is determined by a purple to gray color. Gas production is not assessed in negatives. Nitrate Reduction Test It is used to determine if an organism is capable of reducing nitrate (NO3) to nitrite (NO.) or other nitrogenous compounds via the action of the enzyme nitratase (also called nitrate reductase). - This test is Important in the identification of both Gram-positive and Gram-negative species. Media and reagents: 1. Nitrate Broth or Nitrate Agar (Slant). 2. Reagent A (α-Naphthylamine, Acetic acid (5 N), 30%). 3. Reagent B (Sulfanilic acid, Acetic acid (5 N), 30%). Procedure: 1. Inoculate the nitrate medium with a loopful of the test organism isolated in pure culture on agar medium, and incubate at 35°C for 18–24 hours. 2. At the end of incubation, add 1 mL each of reagents A and B to the test medium, in that order. GELATIN LIQUEFACTIon test Purpose: This test is to determine an organism's ability to produce proteolytic-like enzymes and liquefy gelatin. Principle: Gelatin is to large to enter a bacterial cell wall and thus extracellular enzymes must catabolize them into smaller components before they can be utilized. Possession of these extracellular gelatinases can aid in the differentiation of bacteria. This test Is used to differentiate between suedes in the genera Staphylococcus and Dort,Idiom as well as aid in the Identification of other species and genra. Positive : Proteus vulgaris Negative: Klebsiella aerogenes Procedure : 1. Inoculate a heavy inoculum of test bacteria on the tube containing nutrient gelatin medium. 2. Incubate the inoculated tube along with an uninoculated(control) medium at 35°C, for up to 2 weeks. 3. Remove the tubes daily from the incubator and place in ice bath or refrigerator (4°C) for 15-30 minutes (until control is gelled) every day to check for gelatin liquefaction.(Gelatin normally liquefies at 28°C and above, so to confirm that liquefaction was due to gelatinase activity, the tubes are immersed in an ice bath or kept in refrigerator at 4°C). 4. observe if gelatin has been hydrolyzed. API system (Analytical Profile Index) The API system represent another type of kit for rapid identification of bacteria. this system provides , in a single strip a series of 20 microtubules. The test included in the strip containing dehydrated media determine whether the organism ferments glucose , mannitol ,etc ; produce indole and H2S , splits urea , break down the amino acid tryptophan , produces gelatinase , forms acetylmethylcarbinol from glucose (VP test ). API (Analytical Profile Index) 20E is a biochemical panel for identification and differentiation of members of the family Enterobacteriaceae Principle : A bacterial suspension is used to rehydrate each of the wells and the strips are incubated. During incubation, metabolism produces color changes that are either spontaneous or revealed by the addition of reagents. All positive and negative test results are compiled to obtain a profile number, which is then compared with profile numbers in a commercial codebook (or online) to determine the identification of the bacterial species. 1 2 4 1 2 4 1 2 4 1 2 4 1 2 4 1 2 4 1 2 4  Colony Count Estimate the number of microorganisms There are three methods for determining bacterial numbers : 1- Plate count method( dilution plating): is an indirect measurement of cell density and give information related only to live bacteria. 2- Spectrophotometric analysis :based on turbidity and indirectly measures all bacteria , dead or alive. 3- Hemocytometry : is method where every cell ( dead or alive ) is counted.  Diluting a sample : The standard plate count method consists of diluting a sample with sterile saline or phosphate buffer diluent until the bacteria are dilute enough to count accurately. That is, the final plates in the series should have between 25 and 250 colonies. Fewer than 25 colonies are not acceptable for statistical reasons, and more than 250 colonies on a plate are likely to produce colonies too close to each other Colony-forming unit (CFU, cfu, Cfu) is a unit used in microbiology to estimate the number of viable bacteria cells in a sample. Dilution = sample volume total volume of (sample +dilution) CFUs per ml = number of colonies per plate x dilution factor  Antimicrobial Agents (Kirby-Bauer Method ) An important function of the diagnostic microbiology laboratory is to help the physician select effective antimicrobial agents for specific therapy of infectious disease Materials : 1. Sterile saline in 2-ml tubes 2. 0.5 McFarland standard : 3. Mueller-Hinton agar plates 4. Antibiotic disks 5. Forceps Mueller-Hinton agar MH agar is considered the best medium to use for routine susceptibility testing of non fastidious bacteria. for fastidious bacteria we add blood to Mueller Hinton agar The McFarland Standards are commonly used to standardize the approximate number of bacteria in a liquid suspension or broth culture of the bacterial cell by comparing the turbidity of the cultured test suspension with that of the McFarland Standard Procedure : A. allow disks to come to room temperature before opening the container. B. touch 4 to 5 colonies of bacteria with your sterilized and cooled inoculating loop. C. Emulsify the colonies in 5 ml of Toth or saline until the turbidity is approximately equivalent to that of the McFarland No. 0.5 turbidity standard. D. Dip a sterile cotton swab into the bacterial suspension and inoculated as follows: first streak the whole surface of the plate closely with the swab; then rotate the plate through a 60rangle and streak the whole surface again; finally rotate the plate another 60'and streak once more. As a final step, the rim of the agar is swabbed. E. Discard the swab in disinfectant. F. Heat the forceps in the Bunsen burner flame, and allow to cool. G. Using a disk dispenser or sterile forceps, apply the appropriate Antimicrobial disks onto the agar. Press the disk gently into full contact with the agar, using the tips of the  forceps. Place no more than 6-7 disks. Discs must not be relocated once they have made contact with the agar surface. H. Heat the forceps again and cool. I. Incubate plates as follows: - Campylobacter species - at 35°C x 18 hours - Hemophilus species - CO, 35°C x 18 hours - S. pneumoniae - CO2, 35°C x 20 to 24 hours Interpretation : After incubation, measure the diameters of the zone of complete inhibition with a ruler with millimeter markings using transmitted light and compared for size with values listed in a standard chart. Results a three-category classification is often adopted.  Sensitive : An organism is called "sensitive " to a drug when infection caused by it is likely to respond to treatment with this drug, at recommended dosage.  Intermediate: Infection may respond to therapy higher dosage.  Resistant: This term implies that the organism is expected not to respond to a given drug Minimum Inhibitory Concentration (MIC) of the antibiotic is the lowest concentration at which it prevents the visible growth of a given organism. MICs can be determined by agar or broth dilution methods. The tube dilution say is done by a series of tubes containing increasingly dilute preparations (two fold) of the antibiotic is introduced into a broth a ah a standard flambe, of rest organisms and incubated. The lowest concentration in the series to show no microbial growth is the MIC. Bacteriologic Methods (Diagnostic microbiology ) 1. Diagnostic of Urinary tract infection UTI: Bacterial infection of urinary tract infection are very common. The most frequent cause of Urinary tract infection is E.coli , Other common agents are Proteus , Pseudomonas , Klebsiella , Enterocooci , S.aures , Yeast Specimen collection and transport : Specimen type for culture : Urine 1. Urine should be collected in sterile , screw cap container 2. Clean catch(voided after washing the external orifice ) 3. Midstream (to minimize the contamination of urine specimen by normal flora of the urethra , vagina and perineal regions ). 4. 24 hours urine specimen are unacceptable for culture due to bacterial over growth. 5. In general, specimens must be collected before the administration of antibiotics. Transport : 1. Specimen should be processed within 2 hrs because (over growth of bacteria is common) 2. In case of delay the specimen can be refrigerated for up to 6-18 hrs. 3. In the absence of refrigeration 1.1 boric acid maintains bacterial count for up to 24 hrs. Specimen processing : o Standardized volumetric wire loops are used for inoculation of urine (0.01 , 0.001) , mean that this calibrated loop deliver 0.01ml or 0.001ml of urine volume a) Routine culture ,Routinely are inoculated onto blood , MacConkey ,CLED media b) After culture , Colony count Colony count : Quantitative culture is performed for all urine specimen in order to discriminate between contamination and real infection , for this purpose the following procedures are performed : (1) Serial 10 fold dilution as we previously mentioned.( Streaking methodology ) (2) Inoculation of urine specimens using standardized calibrated loops (0.01 or 0.001) meaning that the number of colonies on the agar should be multiplied by 100 or 1000 respectively (e.g if you used 0.01 m  13 colonies seen * 100 = 1300 CFU/ml ) Procedure : 1. Mix the Urine sample. 2. Sterilize the wire loop by flaming. 3. With the calibrated sterile loop. insert the loop vertically in to the urine sample to transfer 0.01 ml of the urine specimen to the center of a Blood and MacConkey / or in EMB agar plate and streak across the drop in several planes so that the specimen is- distributed evenly across the plate. 4. Incubate the plate at 37C for 24 hours. 5. Read your plates, count the colonies on each, and then the number of colonies if present is multiplied by the dilution factor (100 or 1000 depending on the loop diameter used), and report the numbers of organisms per milliliter present in the urine specimen. 6. Do differential diagnosis of the isolated organism. 7. Antibiotic sensitivity test is done. Term used are: a) Insignificant growth : number of colonies on blood agar is < 103 CFU /ml of a single type of colony ( contamination by normal flora ) b) Moderately significant growth 103105 CFU of a single organism on blood agar. d) if you have only one potential pathogen with a colony count of 103 CFU /ml in this case look for the symptomatic male or female with pyuria (of 10 WBC/HPF) then complete the work. otherwise report as insignificant. Exception: Pure cultures of S.aureus are always significant regardless of the count. presence of yeasts in any number should be reported to reported to the physician  Diagnostic of the digestive System infection : The most common bacterial pathogens causing diarrhea are Shigella, Salmonella and Campylobacter. Specimen collection , Transport and Handling : (1) Stool : For microbiological examination should be collected during the acute phase of diarrhea. Collected in clean , wide mouth plastic containers Should be delivered within an hour and should process immediately , in case of delay, specimen should be transported in transport media, Specimen containing urine should be rejected. refrigeration of sample is not recommended (shigella are very sensitive to lower temperature and will die ) (2) Rectal Swab: - are used only if it is not possible to obtain stool - Swab should be placed in transport medium, immediately to avoid drying. Specimen processing A. Direct visual examination : -Try to take your specimen for the region of pus and blood - Wet mount preparation is the fastest method for the detection of parasites (Entamoeba and Giardia) - Wright's stain (is) used to describe the presence of WBCs and RBCs. - Gram staining is not routinely performed for stool specimens (Due to the large amount of fecal bacterial flora) B. Culture Setup : For culture of Salmonella and Shigella, a selective, differential medium such as Salmonella Shigella agar (SS agar) or Deoxycholate Citrate Agar (DCA) is used. These media are selective because they allow Gram-ve rod to grow but inhibit many Gram +ve organisms. Their differential because salmonella and Shigella do not ferment at lactose whereas many other enteric gram-ve rods do. We can differentiate between salmonella and Shigella by H2S , Salmonella produce H2S appear in Black colony while Shigella not Selective agar for Campylobacter (Campy blood agar ) :Antibiotic mixture that includes Vancomycin which inhibits normal enteric flora , at 42 c in incubator. Procedure : 1. Using a swab, take up a small amount of feces and inoculate it on DCA plate.. Streak for isolation, using a loop. 2. Incubate the plate at 37°C for 24 hours. 3. Observe for pathological growth on DCA. If growth present, do differential tests for identification and antibiotic sensitivity test. If no pathological growth present, Inoculate the swab with the fecal sample, by putting the swab in selenite broth and incubate the broth at 37C for 24h. Selenite F broth \ Thioglycolate broth (Enrichment broths are used for enhanced recovery of salmonella m Shigella and other enteric pathogens. these Broths are plated onto selective media in the next days if the primary culture from stool specimens shows negative results. 4. Do subculture from selenite into DCA or SS agar and incubate the plate at 37°C for 24 hours. 5. Look for any pathological growth, if growth present, do different tests for identification and antibiotic sensitivity test. 6. If no pathological growth present, write that in the report.. In case of Vibrio cholera is expected, rice-colored liquid stool should be cultured in alkaline peptone water and on TCBS agar. Diagnostic of the Respiratory tract infection : Prepare culture of a throat swab and a sputum specimen , each simulating material that might be obtained from a sick patient. Significant organism that may be isolated must be identification and reported. If organism that you consider to be part of the normal flora are isolated , report as normal flora Throat Culture : Throat swab: swab areas that have pus or are red and swollen (from tonsils, posterior pharyngeal wall and other area that is inflamed). Avoid touching tongue, inside of cheeks or roof of mouth. Are used to detect the presence of Streptococcus pyogenes that cause (Tonsilitis ) and when diphtheria and Candida is suspected and Bordetella Transport Of specimen : Swab should be sent to the lab within 4 hrs. In case of delay they should be refrigerated or sent in a transport media (Aimes transport media ) Gram stain not done on a throat swab since many gram positive cocci are part of the normal upper respiratory tract flora. Procedure : 1. Using the swab in the "specimen” tube , inoculate a small area of the blood agar plate. 2. The swab should be rolled over the inoculation area to maximize transfer of microorganisms. 3. Discard the swab in disinfectant solution. 4. With a sterilized inoculating loop, streak the remainder of the plate to obtain isolated colonies. 5. Incubate the plate at 37 c for 24 hours. 6. After the plate has incubated m examine it carefully for the Presence of hemolysis and record type of hemolysis ,Record the colonial morphology 7.To Know the type if is it Group A Streptococcus use Bacitracin antibiotic disk in blood media and incubated 24 hrs. if growth is inhibited around the disk it is group A. if not , it is anon group A 8.Do sensitivity test. Sputum Culture (Lower Respiratory Tract ) : Sputum is processed in the laboratory for diagnosis of bacterial and fungal infections of the lower respiratory tract. E.g : pneumonia or tuberculosis. The most frequent cause of pneumonia is.S. pneumoniae , S. aureus and gram negative rods, such as ,K. pneumoniae and P.aeruginosa , M.tuberculosis Laboratory diagnosis is often made by isolating the causative agent from sputum sent for culture. However, because sputum specimens pass through the oropharynx, contaminating members of the normal throat flora (NTF) may interfere with culture results. Specimen collection: Sputum is Collected by cough. If the patient Cannot cough and the need for a microbiologic diagnosis is strong, induction of sputum m tracheostomy aspirate Bronchial lavage, or lung biopsy ,may be necessary. Because these procedures bypass the normal flora of the upper airway, they are more likely to provide an accurate microbiologic The specimen for culture really be sputum not saliva , and Collect sputum before antibiotics are given. Gram stain is performed on all sputum specimens before plating to determine acceptability of the specimen (presence of many squamous epithelial cells indicates poorly collected specimen containing spit rather than lung secretions). Acceptable sputum