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Autoclave ➢ Principle: heat under steam pressure ➢ It is at this temperature in which all microorganisms (except for prions) and their endospores are destroyed within approximately 15 minutes ➢ 121 °C, 15 psi for 15 minutes – recommended for media, liquids, utensils, glass pipettes ➢ 132 °C, 15 psi for 30-60 minutes – recommended for decontaminating medical wastes

Parts of an Autoclave

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Pressure chamber – the main component of a steam autoclave, it is where the materials to be sterilized are placed Lid – the purpose of the lid is to seal the chamber and maintain the sterilizing conditions within the autoclave. Other components can be found in the lid such as: o Pressure gauge – it indicates the pressure inside the autoclave o Pressure releasing unit/Whistle – pressure within the autoclave can be controlled by lifting the whistle and releasing certain amount of vapor Steam generator – it is present underneath the chamber to heat the water and generate steam

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Sterilize the materials inside the chamber using moist heat and pressure the high pressure ensures the rapid penetration of heat into deeper parts of the material The moisture causes denaturation of microbial proteins Once sterilization is completed, pressure inside the chamber is released through the whistle

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Step in running an Autoclave ➢ Steps depend on the autoclave type and the manufacturer. The ff. are the general procedures 1. Check the inside of the autoclave if there are items left from the previous cycle 2. Place sufficient amount of water inside the chamber 3. The materials to be sterilized are then placed inside 4. The lid is closed using screws to ensure airtight condition, and the electric heater is switched on 5. The water then boils and the pressure is allowed to reach the designated level 6. Once the pressure is reached, the whistly blows to remove excess pressure from the chamber 7. After the whistle, the autoclave is run for a holding period (15 minutes or more) 8. The heater is switched off and the autoclave is allowed to cool until the pressure gauge indicates the pressure inside has lowered down 9. The lid is opened and the sterilized materials are taken out of the chamber

Uses of an autoclave ➢ Used in order to sterilize materials in the laboratory such as culture media, glassware and other instruments ➢ Used in order to sterilize medical wastes that may contain dangerous microbes

Precautions in using the Autoclave ➢ he autoclave should not be overcrowded in order to ensure that all parts of the material can be penetrated by the steam ➢ Wastes and clean items should be autoclaved separately ➢ Do not attempt to open the lid when the autoclave is running ➢ Avoid placing materials that might melt inside the autoclave (plastic trays, etc.)

Dry heat sterilization ➢ Direct flaming – involves the direct application of high heat in materials such as inoculating loops. This can be accomplished using an open flame (a) or an incinerator (b) ➢ Oven heating – 160 to 170 degrees Celsius for 1.5 to 2 hours Radiation ➢ Radiation creates free radicals that causes intracellular damage ➢ Ionizing radiation – causes mutation in the DNA; destroys vegetative cells and endospores o Used to “pasteurize” meat products o Utilizes gamma rays and x-rays ➢ Non-ionizing radiation – damages cellular DNA o Used on exposed surfaces and rooms o Utilizes ultraviolet rays Chemical infection ➢ Phenols – first widely used antiseptic and disinfectant; destroys plasma membranes and denature cell proteins ➢ Alcohol – denatures proteins and causes dissolution of lipid membranes; effective at 60-90% concentration ➢ Chlorine (Hypochlorite) – used at 1:10 dilution; 3 minutes contact time Preparation of disinfectants ➢ Use the dilution formula C1V1=C2V2 Where: C1 is the initial concentration, V1 is the initial volume, C2 is the final concentration, and V2 is the final volume

➢ Ex. Prepare 20 mL of 80% alcohol from absolute (100% alcohol) ➢ The initial volume (V1) is calculated by manipulating the formula V1 = C2V2 / C1

➢ Substitute the given V1 = C2V2 / C1 V1 = (80%) (20%) / (100%) V1 = 16 mL of 100% alcohol is needed Amount of water needed = 4 mL

ANTIMICROBIAL SUSCEPTIBILITY TESTING When to perform susceptibility testing? ➢ Antimicrobial susceptibility testing should be performed on a bacterial isolate from a clinical specimen if the isolate is determined to be a probable cause of the patient’s infection and the susceptibility of the isolate to particular antimicrobials cannot be reliably predicted based on previous experience with the bacteria at a specific health care facility. ➢ Susceptibility tests are not performed on bacteria that are predictably susceptible to the antimicrobial agents commonly used to treat infections caused by these bacteria. Group A βhemolytic Streptococcus, for example, is not routinely tested because it is universally susceptible to penicillin, the drug of choice in treating infections caused by this bacterium. ➢ In contrast, the recommended agent for treating Staphylococcus aureus infections is oxacillin, but not all S. aureus may be susceptible to oxacillin. Consequently, susceptibility testing is indicated for a S. aureus isolate that is the suspected cause of an infection.

Factors to Consider When Determining Whether Testing Is Warranted ➢ In addition to the unpredictable susceptibility of a potential pathogen, other important factors must be considered when determining whether antimicrobial susceptibility testing is warranted, including: o Body site from which the bacterium was isolated o Presence of other organisms and quality of the specimen from which the organism was grown o Host’s status

Traditional Antimicrobial Susceptibility Test Methods

Inoculum Preparation ➢ Inocula are prepared by adding cells from four to five isolated colonies of similar colony morphology growing on a noninhibitory agar medium to a broth medium and then allowing them to grow to the log phase. Four to five colonies, rather than a single colony, are selected to minimize the possibility of testing a colony that might have been derived from a susceptible mutant. ➢ Inoculum can also be prepared directly by suspending colonies (16- to 24-hour colonies) grown overnight on an agar plate directly in broth or saline Inoculum Standardization ➢ To standardize the inoculum, the inoculated broth or direct suspension is vortexed thoroughly. Then, under adequate lighting, the tube is positioned side by side with the McFarland 0.5 standard against a white card containing several horizontal black lines (Fig. 13.1). The turbidities are compared by looking at the black lines through the suspensions. ➢ Once standardized, the inoculum suspensions should be used within 15 minutes of preparation. ➢ A convenient and more precise alternative to visual adjustment to match the McFarland standard is the use of a nephelometric or spectrophotometric device McFarland Turbidity Standards ➢ The inoculum concentration of bacteria to be tested must be standardized. False-susceptible results may occur if too few bacteria are tested, and false-resistant results may be the outcome of testing too many bacteria. The most widely used method of inoculum standardization involves comparing the turbidity of the inoculum preparation with McFarland turbidity standards. ➢ McFarland standards can be prepared by adding specific volumes of 1% sulfuric acid and 1.175% barium chloride to obtain a barium sulfate solution with a specific optical density. The most commonly used is the McFarland 0.5 standard, which contains 99.5 mL of 1% sulfuric acid and 0.5 mL of 1.175% barium chloride. This solution is dispensed into tubes comparable with those used for inoculum preparation, which are sealed tightly and stored in the dark at room temperature. ➢ The McFarland 0.5 standard provides turbidity comparable with that of a bacterial suspension containing approximately 1.5 × 108 CFU/mL. Recently suspensions of latex particles have been used as a simpler, more stable alternative to barium sulfate to achieve turbidity comparable with that of the McFarland standard.

Dilution Susceptibility Testing Methods ➢ Principle ➢ Dilution antimicrobial susceptibility test methods are used to determine the MIC, or the lowest concentration of antimicrobial agent required to inhibit the growth of the bacterium. ➢ Generally serial twofold-dilution concentrations are tested (expressed in micrograms per milliliter). Once the MIC has been determined, the organism is interpreted as nonsusceptible,

susceptible, intermediate, or resistant to each agent with the use of a table provided in the CLSI dilution testing document or in the FDA-approved package insert for the antimicrobial. ➢ Organisms with MICs at or below the breakpoint are susceptible, and those with MICs above that breakpoint are intermediate or resistant. The CLSI documents describe the details of performing MIC tests by broth macrodilution, broth microdilution, and agar dilution methods.

Broth Macrodilution (Tube Dilution) Tests ➢ Broth dilution MIC tests performed in test tubes are referred to as broth macrodilution MIC or tube dilution MIC tests. ➢ Generally, a twofold serial dilution series, each containing 1 to 2 mL of antimicrobial agent, is prepared. Mueller-Hinton broth is the medium recommended for broth dilution MIC tests of nonfastidious bacteria. A standardized suspension of test bacteria is added to each dilution to obtain a final bacterial concentration of 5 × 105 CFU/mL. A growth control tube (broth plus inoculum) and an uninoculated control tube (broth only) are used with each test. ➢ After overnight incubation at 35° C, the MIC is determined visually as the lowest concentration that inhibits growth, as demonstrated by the absence of turbidity. ➢ Broth macrodilution is impractical for use as a routine method when several antimicrobial agents must be tested on an isolate. ➢ Also, this method can be used when minimum bactericidal concentration (MBC) end points are to be determined subsequently.

Broth Microdilution Tests ➢ Plastic trays contain between 80 and 100 (usually 96) wells. Wells are filled with small volumes (usually 0.1 mL) of two-fold dilution concentrations of antimicrobial agent in broth. ➢ The inoculum suspension is prepared and standardized, as described earlier. An intermediate dilution of this inoculum suspension is prepared in water or saline, and a multipronged inoculator or other type of inoculating device is used to inoculate the wells to obtain a final concentration of approximately 5 × 105 CFU/mL (5 × 104 CFU/0.1-mL well). ➢ The actual dilution factor used for preparation of the intermediate dilution depends on the volume of inoculum delivered to each well by the inoculating device and the organism being tested. ➢ An example of this calculation is illustrated in Table 13.3. ➢ After overnight incubation at 35° C, the tray is placed on a tray-reading device to facilitate visual examination of each well (Fig. 13.4). ➢ The MIC for a particular drug is the lowest concentration showing no obvious growth. Growth may be seen as turbidity, a haze, or a pellet in the bottom of the well

Disk Diffusion Testing ➢ Principle

➢ The disk diffusion test, also commonly known as the Kirby-Bauer test ➢ Briefly, a McFarland 0.5 standardized suspension of bacteria in MuellerHinton broth is swabbed over the surface of a standardized MuellerHinton agar plate, and paper disks containing specific concentrations of antimicrobial agent are placed onto the inoculated surface. After incubation of 16 to 18 hours, the diameters of the zones produced by antimicrobial inhibition of bacterial growth are measured, and the result is interpreted as nonsusceptible, susceptible, intermediate or resistant to a particular drug according to preset criteria. Disk Storage. ➢ For long-term storage, disks are stored at −20° C or below in a non–frost-free freezer. ➢ A working supply of disks can be stored in a refrigerator at 2° to 8° C for at least 1 week. Disks should always be stored in a tightly sealed container with desiccant. Inoculation and Incubation. ➢ ➢

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Inoculum suspensions are prepared using a log phase or direct colony suspension standardized to match the turbidity of a McFarland 0.5 standard, as described earlier. A sterile cotton swab is dipped into the suspension, pressed and rotated firmly against the side of the tube to express excess liquid, and then swabbed evenly across the surface of a MuellerHinton agar plate. The plate should be swabbed two more times, turning the plate 60 degrees each time, using the same swab (without going back into the suspension) to ensure an even “lawn” of bacteria on the plate. At the same time, it is recommended that a “purity plate” (usually sheep blood agar, or chocolate agar for more fastidious organisms) be inoculated from the swab, so that the next day when results are checked, it can be visually determined with certainty that the isolate was pure on the lawn plate. Usually, a plate 150 mm in diameter is used; it can accommodate testing of as many as 12 different antimicrobial disks with most bacteria—placement of more than 12 disks on the plate may result in overlapping zones, which are difficult to measure and may produce erroneous results. Within 15 minutes of inoculation, the antimicrobial disks are applied to the agar individually with sterile forceps, or with a multiple-disk dispenser (Fig. 13.8). The disks are pressed firmly to ensure contact with the agar. Within 15 minutes of disk placement, the plates are inverted and placed in a 35° C ambient air incubator for 16 to 18 hours

Reading Plates and Test Interpretation. ➢ After incubation, the plate is examined to ensure that the test organism has grown satisfactorily. The lawn of growth must be confluent or almost confluent. The appearance of individual colonies is unacceptable (Fig. 13.9). ➢ Provided that growth is satisfactory, the diameter of each inhibition zone is measured using a ruler or calipers. Plates are placed a few inches above a black, nonreflecting surface, and zones are examined from the back side (agar side) of the plate illuminated with reflected light (Fig. 13.10).

➢ Tiny colonies at the zone edge and the swarm of growth into the zone that often occurs with swarming Proteus spp. are ignored; the obvious zone is measured. ➢ Obvious colonies within a clear zone should not be ignored. These colonies may occur as a result of contamination or testing of a mixed culture; however, these colonies sometimes represent a minority resistant subpopulation. When such colonies are noted, the original isolate should be retested.

Swarming ➢ Read inhibition and ignore swarming (most often seen for Proteus spp.) ➢ Transmitted light (plate held up to a light source; Fig. 13.11) rather than reflected light will increase the accuracy of tests with the penicillinase-resistant penicillins, linezolid, and vancomycin when testing staphylococci and for vancomycin when testing enterococci. ➢ Tests performed on media containing blood are examined from the top of the plate with the lid removed. For plates containing blood, it is important to read the zone of inhibition of growth and not the zone of inhibition of hemolysis. ➢ Once zone measurements have been made, the millimeter reading for each antimicrobial agent is compared with that specified in the interpretive tables of the CLSI documents or FDA drug package insert and results are interpreted as susceptible, nonsusceptible, intermediate, or resistant.

Identification of Staphylococcus and Streptococcus Culture ➢ Staphylococcus spp. o The colonies are round; are at least 1 to 2 mm in diameter; are convex, opaque, and glistening with a complete edge; and are soft or butter -like in consistency. Some varieties are surrounded by zones of beta hemolysis. The color of the colony varies from an off - white to cream color to golden yellow. o Many selective media, such as phenylethyl alcohol agar (PEA), Mannitol Salt agar (MSA), and colistin -nalidixic acid agar (CNA), are used for the isolation of staphylococci from mixed cultures.

➢ Streptococcus spp. o The type of hemolysis that the organism produces when grown on sheep blood agar is used initially to identify the streptococcus species. There are three types of hemolysis: α, or incomplete green hemolysis; β, or complete clear hemolysis; and γ, or no hemolysis.

Catalase Test ➢ Principle ➢ Members of the staphylococci produce an enzyme, catalase, which reacts with hydrogen peroxide to liberate oxygen. When a colony is emulsified in 3% hydrogen peroxide, the resultant bubbles on the slide may be visualized, which constitutes a positive test result.

SLIDE COAGULASE TEST ➢ Principle o Staphylococci produce many extracellular products, but only one is generally accepted as a marker of pathogenic strains: the production of the enzyme coagulase, which clots rabbit and human plasma. Coagulase production is also one of the major criteria used to identify S. aureus. All strains of S. aureus are, by definition, coagulase positive. There are two kinds of coagulase enzyme: the cell-bound “clumping factor,” which is bound to the bacterial cell wall and causes clumping when the bacterial suspension is mixed with plasma, and free coagulase, which is an extracellular enzyme that causes a clot to form when bacterial cells are incubated with the plasma. ➢ Specimen o A well-isolated colony from a blood agar plate ➢ Procedure o Emulsify a well-isolated colony in a drop of water on a glass slide to produce a dense, uniform suspension. If any evidence of autoagglutination is noted before the plasma is added, the culture is not suitable for the slide test. o Add one drop of plasma to the suspension and mix; then rotate for 5 seconds.

TUBE COAGULASE TEST ➢ Principle o The tube coagulase test detects the presence of free coagulase enzyme produced by S. aureus. This extracellular enzyme forms a clot when bacterial suspension is incubated with the plasma. The screening test by the slide method detects only the cell-bound coagulase, and therefore any negative slide coagulase test result must be confirmed with the tube coagulase test. ➢ Specimen o A broth culture of the organisms to be tested or a single colony from a blood agar plate ➢ Procedure o To 0.5 mL of undiluted rabbit plasma, add one loopful of growth from 18- to 24-hour agar culture, 0.1 mL of broth culture, or a single colony from a blood agar plate. • o Incubate in a water bath at 35 C and examine for clotting at 30-minute intervals for 4 hours. o If no clot is observed at the end of 4 hours, let stand at room temperature for 18 to 24 hours. Observe for clotting.

NOVOBIOCIN SUSCEPTIBILITY TEST ➢ Principle o Staphylococcus saprophyticus is a significant isolate in urine samples of young females. A coagulase-negative staphylococcus (CoNS), S. saprophyticus must be differentiated from other CoNS, such as S. epidermidis, in urine cultures from young females with significant colony counts. Whereas S. saprophyticus is resistant to 5-μg novobiocin disk, other CoNS are susceptible. Other CoNS that are resistant to 5-μg novobiocin are Staphylococcus xylosus, Staphylococcus cohnii, and Staphylococcus sciuri, which are uncommon isolates from the urine. ➢ Procedure o Divide a sheep blood agar into two sections and label one half with the positive control and the other with the negative control. Inoculate the control organisms onto their respective sections. Place a 5-μg novobiocin disk at the center of the inoculum. Repeat the procedure using the second sheep blood agar with the test or unknown organism. o Incubate the cultures at 35C in an ambient incubator for 18 to 24 hours. o Determine if the test organism is susceptible (>17 mm) or resistant (17 mm—susceptible o 15 mm—S. pneumoniae • 2. Zone diameter of inhibition...