Questions and Answers
What is the optimum temperature range for mesophiles?
Which type of bacteria is negatively affected by oxygen?
What is the typical CO2 requirement for bacterial incubation?
What pH range is optimized for most bacteria?
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What technique effectively sterilizes using moist heat?
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What is the role of catalase in bacteria?
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At what temperature and pressure does the autoclave operate to ensure sterilization?
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Which nutrient is specifically required by some bacteria, but not all?
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What is the main outcome of adding water to the components of a gas pak?
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Which method is NOT typically used for sterilization?
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Which antibiotic is particularly effective against Clostridium difficile and MRSA?
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What is the mechanism of action for aminoglycosides?
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Which antibiotic class includes Aztreonam?
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Which of the following antibiotics can cause aplastic anemia as a side effect?
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Which of the following actions is characteristic of sulfa drugs?
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What distinguishes disinfection from sterilization?
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Which antibiotic is specifically noted for its use against Pseudomonas aeruginosa?
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Which antibiotic can affect bones and teeth in children?
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What action does bactericidal antibiotics perform?
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Which of the following best describes the action of trimethoprim when combined with sulfonamides?
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Linezolid is effective against which type of bacteria?
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Which type of antibiotic is characterized by a narrow spectrum of activity?
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What principle does selective toxicity refer to in antimicrobial therapy?
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Which of the following defines bacteriostatic action?
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What is the term for a drug interaction where the combined effect is less than expected?
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Quinupristin/dalfopristin is primarily utilized for treating which type of infection?
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What is the primary purpose of microaerophiles in relation to oxygen levels?
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What is the role of capnophiles in bacterial growth?
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Which of the following best describes complex media used for bacterial growth?
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What happens during refrigeration regarding bacterial growth?
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What is the effect of osmotic pressure from a hypertonic solution on bacteria?
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What is the function of reducing agents in anaerobic media?
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Which of the following methods forms hydroxyl radicals that can damage DNA?
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What is the primary action of phenol as a disinfectant?
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What is the primary purpose of the Kirby-Bauer method in susceptibility testing?
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Which condition must be maintained for the Mueller-Hinton agar used in susceptibility testing?
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What does a 'D'-shaped zone of inhibition indicate in inducible strains?
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What is the significance of determining the minimum bactericidal concentration (MBC)?
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What characteristic does the Modified Hodge test assess in bacteria?
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Which bacterial species is notably associated with carbapenem resistance?
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What does the detection of MRSA typically involve?
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Which component is crucial for the growth of certain resistant bacterial strains in culture media?
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What is the primary mechanism of action for quinupristin/dalfopristin?
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Which of the following statements correctly differentiates between bactericidal and bacteriostatic actions?
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Which class of antibiotics is effective against Gram-positive bacteria resistant to other antibiotics?
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What type of spectrum does an antibiotic with narrow spectrum activity have?
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What does the term 'synergism' refer to in the context of drug combinations?
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Which of the following is a characteristic of broad-spectrum antibiotics?
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What is the effect of selective toxicity in antimicrobial therapy?
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Which type of action do bacteriostatic antibiotics perform?
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What should be reported as resistant if an extended-spectrum beta-lactamase (ESBL) is detected?
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Which statement accurately describes the enzyme activity related to resistance?
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What could lead to a methicillin zone decrease over time in control organisms?
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What is a potential reason for observing a 'zone within a zone' phenomenon during antibiotic susceptibility testing?
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Which organism is especially noted as a potential ESBL producer?
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What is likely the cause if colonies are found within the zone of inhibition?
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What could explain the observed small zones universally on control plates?
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Which statement best describes a method for evaluating potential methicillin resistance?
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Which method is utilized to find the first plate with no bacterial growth when testing near the minimum inhibitory concentration (MIC)?
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What additional methods can be used for testing fastidious bacteria?
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What could be an effect of having a low pH on agar during disk diffusion testing?
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What is a potential source of error in disk diffusion related to inoculum density?
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What condition can cause an aminoglycoside zone to appear too large during testing?
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Which of the following is NOT a modified method for testing slow-growing bacteria?
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What is a characteristic error linked to tetracycline zone sizes in disk diffusion testing?
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Which test method is described as utilizing strips to determine MIC values for antimicrobials?
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What is the primary grouping of antibiotics that inhibits cell wall synthesis?
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Which antibiotics primarily target the 30S ribosomal subunit to inhibit protein synthesis?
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What is the action of Ciprofloxacin, a member of the quinolines class?
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What is a common effect of using Tetracyclines in young children?
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What distinguishes antimicrobial actions of Sulfa drugs from other antibiotics?
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Which antibiotic is primarily used in combination with penicillin for Enterococcus infections?
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What is the major mechanism of action for Chloramphenicol?
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Which of the following antibiotics is known to be effective against anaerobes and Gram-positive organisms?
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What is the result of using an autoclave for sterilization?
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What is a characteristic feature of the action performed by antibiotics like Erythromycin?
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What type of bacteria is primarily identified using Bordet-Gengou Agar?
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Which medium is used to selectively cultivate Gram positive cocci?
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What is the primary purpose of using Thiosulfate Citrate-Bile Sucrose (TCBS) agar?
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CAMPY-Blood Agar is particularly effective for isolating which of the following bacteria?
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Which agar is enriched with hemoglobin and is typically used to grow Haemophilus and Neisseria species?
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Which medium is specifically used for the cultivation of Leptospira?
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What type of bacteria does Phenylethyl Alcohol Agar (PEA) mainly select for?
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What purpose does Buffered Charcoal Yeast Extract (BCYE) serve in microbiology?
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Which medium is specifically enriched with Hemin, Cystine, and Vitamin K for the growth of anaerobes?
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What color change occurs on Cooked or Chopped Meat Medium due to deaminase activity following the addition of 10% FeCl3?
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Which organism is specifically mentioned in the context of anaerobic media?
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What characteristic is observed in 6.5% NaCl Broth when incubating certain bacteria?
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Which of the following options correctly identifies the appearance of the surface on CDC Anaerobic Blood Agar after incubation?
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What does the presence of a turbid medium in 6.5% NaCl Broth indicate?
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Which component is essential for culturing anaerobes effectively in enriched media?
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What type of surface color change is observed on Cooked or Chopped Meat Medium due to certain metabolic activities?
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What is the optimal time for collecting a blood culture specimen for Salmonella typhi?
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Which of the following methods is NOT appropriate for microscopy as stated in the diagnostic methods?
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Which condition is critical for blood culture collection?
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What should NEVER be done to spinal fluids when preparing specimens?
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What is a criterion for the rejection of specimen samples?
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In what temperature range are most culture plates incubated?
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What is the minimum requirement of cultures to draw within a 24-hour period for blood cultures?
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Which type of microscopy is specifically useful for observing spirochetes?
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Which medium is specifically designed to enhance the pigment production of Bacteroides species?
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What biochemical reaction occurs with the carbohydrate fermentations in the Bacteroides Bile Esculin Agar?
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Which medium is utilized for the selection of Mycobacterium species?
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Which indicator is used in carbohydrate fermentation tests within anaerobic media?
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What characteristic do black colonies signify in Bacteroides Bile Esculin Agar?
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Which medium is particularly noted for enhancing grouping and the visibility of metachromatic granules in C. diphtheriae?
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Which metabolic reaction in anaerobic media is indicated by the formation of a blurred stab?
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Which type of colonies are typically associated with the B. fragilis group in anaerobic cultures?
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Which type of anaerobic media supports most organisms?
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What is the primary purpose of a gas pak jar in anaerobic culture?
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Which of the following is a characteristic trait of selective agar?
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What dilution of blood to broth is recommended for anaerobic cultures in adults?
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Which environment composition is typically used in anaerobic gas generation?
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What type of media includes chemicals or dyes to differentiate between organisms?
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Which type of container is deemed best for culturing fungi and acid-fast organisms?
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What is the primary characteristic of pre-reduced media used for anaerobic culture?
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What is the preferred action to take before collecting a blood culture specimen?
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Which of the following is NOT a criterion for rejection of a specimen?
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Which type of microscopy is useful for identifying spirochetes?
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What is the optimal temperature for culturing Campylobacter species?
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What is the recommended action to ensure integrity in spinal fluid specimen handling?
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Why should specimens be collected prior to antibiotic therapy?
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What should never be done with anaerobic or GC specimens during handling?
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Which of the following dyes is used in fluorescence microscopy for staining?
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What is the optimal humidity level required for bacterial culture in specific conditions?
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Which condition is necessary for supporting anaerobic bacteria growth?
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In collecting blood cultures, what is the recommended minimum dilution of blood to broth for adults?
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Which media type supports the growth of most organisms without selective inhibition?
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How many cultures should be drawn within a 24-hour period?
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Which type of organisms is best isolated using the Isolator® system?
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For the growth of anaerobic microbes, which environmental gas composition is optimal?
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What is the role of broth media containing thioglycollate or cysteine?
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What is a common symptom of food poisoning caused by enterotoxins?
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Which exotoxin is NOT typically associated with Staphylococcus infections?
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Which group of patients is most at risk for infections caused by coagulase negative Staphylococcus?
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What characteristic allows Staphylococcus species to show resistance to penicillin?
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Which of the following antibiotics is often used against penicillin-resistant Staphylococcus aureus?
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What is the primary target of the exotoxin TSST-1 in toxic shock syndrome?
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What is the primary reason for using 10% KOH in conjunction with Calcofluor White?
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Which characteristic is NOT associated with Staphylococcus aureus?
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Which of the following infections is NOT typically caused by Staphylococcus aureus?
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What is the drug of choice for treating methicillin-resistant Staphylococcus aureus (MRSA)?
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What is the primary function of the iodine mordant during Gram Staining?
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Which statement best describes the colony morphology of Staphylococcus aureus on Blood Agar Plate (BAP)?
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What does the presence of catalase positive indicate about a bacterial species?
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Which of the following stains is specifically designed for Mycobacteria?
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What role does the decolorizer play in the Gram Staining process?
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Which of the following is a common feature of infections caused by Staphylococcus aureus?
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In identification tests, which characteristic is unique to Staphylococcus aureus as compared to other staphylococci?
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Which loop calibration is conventionally used for urine colony count plates?
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Which result is expected from a Gram-positive organism after performing a Gram Stain?
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How long should stained bacteria be read and reported after the inoculation process?
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What is the outcome when using Auramine and Rhodamine for staining?
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Which of the following is a primary stain in microbiological methods?
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What is the purpose of using a calibrated 0.001 ml loop in microbiological procedures?
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Which primary stain in microbiology is used alongside a decolorizer to differentiate between Gram-positive and Gram-negative bacteria?
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What is the result obtained when performing the Kinyoun or Ziehl-Neilson stain on Mycobacteria?
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What characteristic differentiates the staining results of Auramine-Rhodamine from traditional methods?
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In the Gram staining process, what role does the decolorizer play?
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Why is methanol or heat used in the Gram staining procedure?
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What does the use of potassium permanganate in the Auramine-Rhodamine stain primarily accomplish?
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How long should the colonies be read and reported after inoculation?
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What is a distinguishing trait of Staphylococcus aureus related to its pathogenicity?
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What is the primary role of catalase in bacteria?
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What characteristic does Staphylococcus saprophyticus have among urinary tract pathogens?
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What type of infections is Staphylococcus aureus primarily associated with?
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Which group of bacteria is primarily associated with opportunistic infections in immunocompromised patients?
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What is the primary mechanism of action of vancomycin against bacteria?
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Which statement is true regarding Staphylococcus epidermidis?
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Which of the following best describes the role of Streptolysin O?
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Which of the following is a feature of Staphylococcus aureus in relation to antibiotic resistance?
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What does a positive coagulase test indicate about the bacteria?
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What type of hemolytic activity does Streptococcus pyogenes (Group A) exhibit?
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What is the main consequence of methicillin resistance in Staphylococcus aureus?
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What is the expected reaction of the catalase test for Staphylococcus species?
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What is a common characteristic of coagulase-negative Staphylococci in relation to infections?
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Which Staphylococcus species is most commonly associated with community-acquired infections?
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What effect does storing bacterial cultures at 35-37°C have on Staphylococcus species?
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Which bacterium is identified by its growth in 6.5% NaCl and is commonly associated with UTIs and bacteremia?
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What characteristic colony morphology distinguishes Streptococcus pneumoniae?
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Which method can be used to identify antibiotic sensitivity of Streptococcus pneumoniae?
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What laboratory feature is commonly used to identify Enterococcus in culture?
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Which bacterial species is primarily associated with color cancer?
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Which specific hemolytic pattern is typically observed in viridans group streptococci associated with subacute bacterial endocarditis?
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What is the hemolysis reaction indicated by the greening around a colony?
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What key attribute differentiates Staphylococcus from Streptococcus in terms of catalase activity?
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Which of the following conditions is NOT typically associated with Streptococcus pneumoniae?
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Which condition defines a positive screening for penicillin resistance in S.pneumoniae?
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What type of infection is rarely caused by S.viridans?
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What does a 'rusty sputum' indicate in patients and which pathogen is commonly responsible for this symptom?
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The shaded areas in laboratory results signify what?
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Which characteristic is incorrectly associated with alpha hemolysis?
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What is the main clinical implication of identifying S.pneumoniae with the mentioned characteristics?
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Which colony morphology is associated with S.pneumoniae?
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Which organism is characterized by the presence of metachromatic granules and a 'Chinese Letter' arrangement?
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What characteristic movement is associated with Listeria monocytogenes at 25°C?
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Which species causes infections in individuals such as fishermen and veterinarians?
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What distinguishes Bacillus from other Gram-positive rods regarding hemolysis?
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The Elek test is used to determine the production of which of the following?
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Which Gram-positive rod is associated with causing neonatal meningitis and sepsis?
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Which Gram-positive rod is catalase positive and can form spores?
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What is a significant characteristic of Erysipelothrix when grown in gelatin?
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What is a key characteristic of Neisseria gonorrhoeae in terms of its growth conditions?
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Which statement accurately describes Moraxella catarrhalis?
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What distinguishes the colony appearance of Moraxella catarrhalis?
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Which of the following statements is true regarding Neisseria gonorrhoeae?
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What type of infections can Acinetobacter species cause?
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What is the reaction of Neisseria gonorrhoeae on culture media?
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Which of the following properties is true about Acinetobacter species?
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In the context of bacterial meningitis, what is a common finding in the CSF for infections caused by Gram-negative cocci?
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What condition must be met for the organism S.enterica to cause infection?
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Which biochemical test indicates a positive result for H2S production?
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Which of the following correctly describes the structure related to Plesiomonas?
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Which biochemical test result indicates a strong preference for lysine?
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What is the expected result of the ONPG test in the presence of B-d-galactosidase?
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Which serotype of S.enterica is primarily associated with septicemia?
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What reagent is used to confirm positive nitrate reduction in bacteria?
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What does a positive urea test indicate in bacterial cultures?
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Which selective media uses Eosin Y as a differentiating agent?
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In Hektoen-Enteric (HE) agar, what is the H2S indicator that produces a black precipitate?
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Which of the following is a key reaction that differentiates Enterobacter from Citrobacter?
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What is the color of lactose-positive colonies on MacConkey Agar?
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In which agar is Salicin used as a differentiating agent?
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Which reaction is variable (v) in the biochemical tests for Escherichia coli?
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Which of the following exhibits a negative reaction for hydrogen sulfide production on KIA tests?
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Which agar has a transparent appearance for lactose-negative colonies?
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What is the acid-base indicator used in MacConkey Agar?
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Which organism shows a positive reaction for deaminase activity?
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What characteristic distinguishes Escherichia coli from Enteroinvasive E. coli (EIEC)?
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Which strain of Shigella is associated with the most severe cases of bacillary dysentery?
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What type of toxin is produced by Enterotoxigenic E. coli (ETEC)?
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Which characteristic applies solely to the strain 0157:H7 of Enterohemorrhagic E. coli (EHEC)?
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What is the minimum number of organisms needed for the disease caused by Bacillary dysentery?
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Which statement correctly describes the lactose fermentation characteristic of Shigella species?
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Qual es le resultato positive in le agar de ferro de lysine (LIA)?
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Qual dye es utilizate como indicatore in le detection de acetylmethylcarbinol?
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Qual es le percentuale de glucose in le media MIO?
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Le agar de lysine iron (LIA) ha un indicatore de quale gas?
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Qual es le colore indicative de un reaction positive in le media MIO?
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Qual de le sequente non es un resultato positive pro acetoin detection?
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Le que type de resultato in KIA representa un aumento de fermento de glucose?
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Qual es le resultato in KIA quando H2S es presente?
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Quo tipo de fermentatio resulta in un butt jaune in un medio de Kligler Iron Agar?
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Qual es le significato de un slant jaune in un test de Kligler Iron Agar?
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Quale caracteristica es specific a Klebsiella pneumoniae?
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Qual de le sequentes bacterias es indole positive?
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Qual test verifica la capacitate de bacteria a decarboxylate amino acidos?
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Quale de le sequente afirmations es ver de Citrobacter?
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In un test de indole, qual es le resultato positive?
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Quale bacterium non es un motile?
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Qual es le resultato de un test de citrate quando le medio es positive?
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Qual resulta indicara la production de H2S in un medio de Kligler Iron Agar?
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Quale bacterium es asociated con la enfermedad de cuela mesenterica?
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Qual es le resultato positive in un test de urease?
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Quales caracteristicas distingue la specie Yersinia enterocolitica?
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Quo es le reagent in le test de indole?
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Quale de le sequentes affirmations es vera pro Proteus mirabilis?
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Quale substantia non es considerata un caracteristica de Serratia?
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Quo es le resultado positive quando un organism realmente produce phenylalanine deaminase?
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Qual de istas caracteristicas es typic per organismos gram-negativ non-fermentadores?
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Qual es le interpretazione de un tub macconkey con coloration verde?
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Qual organismo es general in relation a infecciones in pacientes con fibrose cystica?
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Qual pigmento non es typic per Pseudomonas aeruginosa?
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Quo indica un tub semi-solide incubate in un modo con oxygeno restrictate, donde le parte aperte es yellow e le parte claude es green?
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Quo es le resultato de un tige OF media que es color yellow in le tige aperte e verde in le tige claude?
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Qual de iste organismos es oxydase positive e cuje crescimento es tipic in le cultura de MacConkey?
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Quale de iste bacterias es associato con odore de sulfuro?
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Quale action es le conde de protection indicale quando urgen in le deserto?
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Le que bacteria es notabile pro su association con geysers?
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Quale statement es ver si considerante H2S?
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Quale de iste bacterias non produce H2S?
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Quale descrition correcte reflecte un caso de 'firecracker' in un desert?
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Which organism is characterized by being motile with polar flagella and having hemolytic colonies on BAP?
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What color is Vibrio cholerae on TCBS agar?
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Which biochemical test is positive for Campylobacter jejuni?
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Which characteristic differentiates Vibrio species from enteric bacteria?
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Which type of growth medium is used for isolating Campylobacter jejuni?
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What is a typical clinical presentation of V. cholerae infection?
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What treatment is commonly effective for infections caused by Campylobacter jejuni?
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Which compound do Vibrio species test positive for that aids in their classification?
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Which species of Brucella is known to cause brucellosis in cows?
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Which Brucella species demonstrates a positive result for the presence of H2S?
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What unique characteristic regarding nutrient consumption is associated with B. abortus?
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Which species of Brucella is primarily associated with causing disease in goats?
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Which of the following Brucella species has an uncertain result for Thionine consumption?
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What is the incubation period during which brucellosis can be confirmed through blood culture?
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Which characteristic differentiates Yersinia pestis from Francisella tularensis?
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What is a common feature of Pasteurella multocida?
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Which organism requires Ascitic Fluid Sample for diagnosis?
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What kind of media supports the growth of Francisella tularensis?
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Which pathogen is primarily associated with the disease known as 'Rabbit Fever'?
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What is the biosafety level of Yersinia pestis?
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What characteristic is used to distinguish the colonies of Streptobacillus moniliformis?
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Which organism is NOT primarily transmitted through animal bites?
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What is the purpose of using chocolate agar in bacterial culture?
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Which statement best describes the Regan/Lowe medium?
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What is a key characteristic of S. aureus in relation to satellite colonies?
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What is the recommended incubation period for bacterial colonies in this context?
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Which factor is necessary for growing certain bacterial species in a hemolytic area?
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What is the main reason for moving away from cough plates for specimen collection?
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What is the role of PCR in the context of bacterial identification?
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Which type of bacteria is effectively grown using Bordet-Gengou medium?
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What is a common clue indicating an anaerobic infection?
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Which of the following techniques is utilized for anaerobic culture?
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What is indicated by the presence of sulfur granules in a specimen?
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Which of the following is one of the classic indications of anaerobic infection?
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In anaerobic culture, what role do indicators like methylene blue or resazurin play?
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What might be a characteristic finding in blood containing exudates from an anaerobic infection?
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What is a potential problem if an organism fails to grow when cultured aerobically?
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Which substance is used to determine bacterial sensitivity to ampicillin in urethritis cases?
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How is Spirillum minor primarily diagnosed in clinical settings?
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Which organism is recognized for its characteristic spreading, corroding colonies?
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What is a common environmental source for L.pneumophila infections?
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Which antibiotic is known to be resistant in treating Mycoplasma hominis infections?
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What is a significant clinical complication associated with M.hominis colonization?
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Which factor contributes to the corkscrew motility observed in spirilla bacteria?
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What is the primary culture requirement for the microorganism discussed in the characteristics section?
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For collecting sputum specimens, which procedure is advised?
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Which organism is characterized by molar tooth colonies of gram-positive branching rods?
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Which organism produces a weakly positive lipase reaction?
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What method is used to confirm infection by Coxiella burnetti?
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Which Rickettsial infection is characterized by a rash on the palms of the hands and soles of the feet?
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What growth requirement medium is used for culturing Mycobacteria?
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Which group is characterized as obligate intracellular parasites?
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Which type of microorganism produces pigment only in the presence of light?
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Which classification describes bacteria that do not produce any pigment?
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Which of the following bacteria is associated with the presence of Neisseria in the throat?
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Which type of pigment-producing bacteria generates pigment in both light and dark conditions?
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In a throat culture, what organism would NOT be expected as part of the normal flora?
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What differentiates a scotochromogen from a non-photochromogen?
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Which organism is associated with Rickettsial Pox and is primarily spread by house mites?
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What vector is involved in the transmission of Typhus Fever caused by R. prowazekii?
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Which of the following organisms is confirmed to cause Q Fever and requires a CF test for confirmation?
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Which of these tests is utilized for detecting Rickettsial antibodies through the use of Proteus antigens?
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Which species is characterized by a rash present on the palms of the hands and soles of the feet?
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Which bacteria is primarily associated with peptic ulcers?
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In what unique environmental condition does Mycobacterium leprae primarily grow?
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Which condition is primarily associated with infections from Mycobacterium avium complex?
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What is the primary reason mycobacteria do not gram stain well?
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Which component is NOT used in the Lowenstein-Jensen growth medium?
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How long does it typically take for mycobacteria to grow in culture?
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What staining method is specifically associated with the presence of mycolic acids in bacteria?
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Which virus is primarily associated with chronic fatigue syndrome?
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Which of the following viruses is associated with serious congenital abnormalities and is contraindicated in pregnancy?
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What type of virus is known to cause severe respiratory infections predominantly in young children and leads to the formation of giant multinucleated cells?
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Which of the following viruses is naturally occurring only in humans and can lead to aseptic meningitis?
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Which of the following diseases is not caused by a DNA virus listed in the content?
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The virus associated with acute infectious infantile diarrhea, which can lead to serious complications like death in infants, is:
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Which virus is most commonly associated with congenital infections?
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What is the primary cause of smallpox outbreaks in laboratory settings today?
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What is the primary reservoir for Rhabdovirus, which is known for causing rabies?
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Which of the following viruses utilizes EIA techniques and is confirmed by Western Blot for testing?
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Which type of infections are primarily caused by Herpes Simplex Virus type II?
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What organism is indicated by a 'rice water stool' in patients?
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Which organism is likely to be isolated from bluish purulent head lesions?
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Which virus is primarily associated with the development of acute infectious infantile diarrhea characterized by 'rice water stools'?
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What is typically observed in the brain tissue for diagnosing rabies in infected animals?
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What typical result is demonstrated by Campylobacter jejuni?
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Which organism is most likely responsible for a vaginal specimen showing a 'fishy' odor when mixed with 10% KOH?
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A photochromogenic mycobacterium isolated at 30°C is most likely which species?
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Which specimen type is least effective for detecting the hallmark of rabies?
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Which acute infectious condition is not caused by a virus listed in the options?
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When testing for possible Streptococcus pneumoniae, which specific test should be performed?
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Which group of antibiotics is commonly considered in the combination therapy for endocarditis caused by Streptococcus Group D?
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Which organism is correctly identified as producing a red pigment?
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Which characteristic distinguishes Streptococcus pneumoniae from other Streptococci?
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What is the primary cause of gas gangrene?
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Which organism is resistant to novobiocin and is known to cause urinary tract infections?
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What separates Staphylococcus from Streptococcus in terms of catalase activity?
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What type of media is C. difficile primarily grown on?
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Study Notes
Bacterial Growth Requirements
- Temperature influences bacterial growth:
- Psychrophiles thrive at low temperatures (optimum = 15°C).
- Mesophiles prefer moderate temperatures (optimum = 37°C; most pathogens).
- Thermophiles grow at high temperatures (optimum = 50-60°C).
- Optimal pH levels:
- Bacteria typically thrive at pH 6.5-7.5.
- Fungi prefer pH 5.0-6.0.
- Oxygen requirements vary:
- Aerobes need oxygen to grow.
- Facultative anaerobes can utilize oxygen but can survive without it.
- Obligate anaerobes are harmed by oxygen.
- Microaerophiles prefer lower oxygen levels than atmospheric concentration.
- Capnophiles prefer higher CO2 concentrations.
- Aerotolerant organisms do not require oxygen but are not harmed by it.
Typical Incubation Conditions
- Requires 5-10% CO2, achievable in incubators or candle jars.
- Temperature needed is generally between 35-37°C.
- Humidity levels should be maintained at 50-70%.
Culture Media
- Growth media must meet the specific needs of bacteria.
- Agar, a polysaccharide from marine algae, is commonly used (melts at 100°C, solidifies at ~45°C).
- Complex media are frequently used, containing peptones and extracts.
- Anaerobic media includes reducing agents to eliminate dissolved oxygen.
Sterilization and Inhibition Techniques
- Heat denatures proteins:
- Moist heat via autoclave (121°C, 15 psi for 15 minutes).
- Dry heat methods like incineration and hot air ovens (170°C for 2 hours).
- Pasteurization uses temperatures like 140°C for short durations.
- Filtration removes microorganisms (pore size 0.22 µm - 0.45 µm).
- Refrigeration slows microbial growth while desiccation maintains organism viability.
- Hypertonic solutions (osmotic pressure) can result in plasmolysis.
- Radiation can damage microbial DNA by forming hydroxyl radicals.
- Disinfection methods include:
- Phenols, halogens (iodine, chlorine), alcohols for microbial cell disruption.
Antibiotics and Their Actions
- β-lactams (e.g., penicillins, cephalosporins) inhibit cell wall synthesis.
- Glycopeptides (e.g., vancomycin) are effective against Clostridium difficile and MRSA.
- Aminoglycosides (e.g., gentamicin) inhibit protein synthesis but are ineffective against anaerobes.
- Tetracyclines (e.g., doxycycline) target protein synthesis affecting bone and teeth development.
- Chloramphenicol can cause aplastic anemia; it intervenes in protein synthesis.
- Macrolides (e.g., erythromycin) affect 50S ribosomal subunits in protein synthesis.
- Quinolones (e.g., ciprofloxacin) inhibit nucleic acid synthesis, particularly effective against certain aerobes.
- Sulfa Drugs (e.g., sulfamethoxazole) serve as folic acid synthesis intermediates; used in combination with trimethoprim for enhanced efficacy.
- Streptogramins and Oxazolidinones target protein synthesis, useful against resistant Gram-positive bacteria.
Antimicrobial Therapy Principles
- Narrow-spectrum antibiotics target specific groups, while broad-spectrum cover both Gram-positive and Gram-negative bacteria.
- Selective toxicity ensures microbial destruction without harming host cells.
- Antimicrobial action categorization:
- Bactericidal: kills the bacteria outright.
- Bacteriostatic: halts bacterial growth; dependency on host immune response for eradication.
- Drug interactions can exhibit:
- Synergism: combined effect greater than individual actions.
- Antagonism: one drug diminishes the effect of another.
Sterilization and Disinfection
- Sterilization kills all microorganisms, including spores and viruses.
- Disinfection inactivates or inhibits microorganisms but may not affect spores.
- Autoclave example: 121°C at 15 psi for 15 minutes.
- Other sterilization methods: Incineration and Filtration.
Antibiotics and Their Actions
-
B-lactams (e.g., Penicillins, Cephalosporins): Inhibit cell wall synthesis.
- Notable examples include Ceftriaxone and Imipenem.
-
Glycopeptides (e.g., Vancomycin): Target cell wall synthesis and are effective against Clostridium difficile and MRSA.
-
Aminoglycosides (e.g., Gentamicin, Tobramycin): Inhibit protein synthesis, specifically act on the 30S ribosomal subunit, not effective against anaerobes.
-
Tetracyclines (e.g., Tetracycline, Doxycycline): Also inhibit protein synthesis on the 30S subunit; may affect bone and teeth development in children.
-
Chloramphenicol: Inhibits protein synthesis on the 50S subunit and may cause aplastic anemia.
-
Macrolides (e.g., Erythromycin, Clindamycin): Act on the 50S ribosomal subunit; Clindamycin is used for Gram-positive and Gram-negative anaerobes.
-
Quinolones (e.g., Ciprofloxacin): Inhibit nucleic acid synthesis, effective against Pseudomonas aeruginosa and other aerobes.
-
Sulfa Drugs (Sulfonomides) (e.g., Sulfamethoxazole): Analogues of PABA, used for UTIs and enteric infections; often combined with trimethoprim.
-
Streptogramins (e.g., Quinupristin/dalfopristin): Inhibit protein synthesis; effective against vancomycin-resistant Enterococcus faecium.
-
Oxazolidinones (e.g., Linezolid): Inhibit protein synthesis; used for Gram-positive infections, resistant to other antibiotics.
Antimicrobial Therapy Principles
- Narrow spectrum: Target specific groups of bacteria.
- Broad spectrum: Effective against both Gram-positive and Gram-negative bacteria.
- Selective toxicity: Only targets microbes without harming host cells.
- Bactericidal action: Kills bacteria without the need for host immune response.
- Bacteriostatic action: Reversible inhibition; depends on the host's defenses for ultimate effectiveness.
Susceptibility Testing
-
Kirby-Bauer Method: Disk diffusion test using Mueller-Hinton agar; determines antibiotic susceptibility.
- Critical parameters include agar depth, pH, and incubation conditions.
-
Broth Methods:
- MIC (Minimum Inhibitory Concentration): Lowest concentration preventing bacterial growth.
- MBC (Minimum Bactericidal Concentration): Lowest concentration resulting in >99.9% killing.
-
E-test: Uses a strip with a gradient of antibiotic concentrations to determine MIC.
-
Automated AST: Includes systems such as BD Phoenix and Vitek for susceptibility testing.
Extended Spectrum Beta-Lactamase (ESBL)
- Enzymes provide resistance to extended-spectrum cephalosporins and monobactams.
- Common in Escherichia and Klebsiella species.
- Detection requires reporting all penicillin, cephalosporin, and aztreonam as resistant if ESBL is present.
Sources of Error in Disk Diffusion Testing
- Incorrect pH or nutrient deficiencies in the agar can lead to abnormal results.
- Inoculum size and agar depth can significantly impact inhibition zone sizes.
- Methicillin degradation in storage affects test reliability.
Detection of Resistance
- MRSA detected with a zone of ≤10 mm using an oxacillin disk.
- Vancomycin-resistant Enterococci (VRE) and carbapenem-resistant Enterobacteriaceae (CRE) are significant clinical concerns.
Media Types and Purposes
- Routine Media is common and assesses general bacterial presence.
- Blood Agar (BA) detects hemolytic reactions in most bacteria.
- Phenylethyl Alcohol Agar (PEA) selects for Gram-positive cocci and certain anaerobic Gram-negative bacilli.
- Columbia CNA Agar specifically selects for Gram-positive cocci.
- Thayer-Martin Agar is used for culturing Neisseria gonorrhoeae and N. meningitidis.
- CAMPY-Blood Agar is optimal for isolating Campylobacter sp..
- Thiosulfate-Citrate-Bile Salts-Sucrose (TCBS) Agar identifies Vibrio sp..
- Buffered Charcoal Yeast Extract (BCYE) Agar is cultured for Legionella sp..
Special Media
- Bordet-Gengou Agar isolates Bordetella pertussis.
- Cystine-Glucose Agar is specific for Francisella tularensis.
- Fletcher's Medium is used for Leptospira isolation.
- Skirrow Agar is selective for Helicobacter pylori.
- Loeffler's Medium is utilized for Corynebacterium diphtheriae.
- Thiosulfate-Citrate-Bile Salts-Sucrose (TCBS) Agar is tailored for Vibrio sp.
Anaerobic Media
- Bacteroides Bile Esculin Agar (BBE) selects for Bacteroides fragilis group.
- Kanamycin-Vancomycin Laked Blood Agar (KVLB) enhances pigment production of Bacteroides sp..
- Cycloserine-Cefoxitin Fructose Agar (CCFA) is selective for Clostridium difficile.
- CDC Anaerobic Blood Agar enriched with hemin, cystine, and vitamin K supports anaerobes.
- Cooked or Chopped Meat Medium is used to culture anaerobes.
Specimen Collection and Handling
- Collect material directly from the infection site for accurate results.
- Timing is critical; for example, collect blood for Salmonella typhi during the first week and stool in subsequent weeks.
- Utilize appropriate collection devices to avoid contamination.
- Avoid refrigerating spinal fluids, anaerobic specimens, or GC samples.
- Collect samples before antibiotic therapy for optimal culture results.
- Set up cultures within 2 hours post-collection to ensure viability.
Criteria for Rejection
- Avoid preservatives that can interfere with results.
- Insufficient sample quantity or dry swabs are unacceptable.
- Leaky containers may lead to contamination and pose biohazard risks.
Blood Culture Collection
- Prep skin with alcohol (70%) and iodine to reduce contamination.
- Ideal time to draw blood is prior to fever spikes.
- Limit to 2-3 cultures in a 24-hour period to prevent overflow.
- Utilize an antibiotic removal device if the patient is on antibiotics.
- Ideal blood-to-broth dilution ratio is 1:10, with a minimum of 10 ml for adults.
Culture Conditions
- Most media incubated at 35-37°C, with exceptions for Campylobacter (42°C) and Yersinia (25-30°C).
- Maintain anaerobic conditions using media like thioglycollate.
- Utilize gas pak jars or anaerobic chambers for optimal growth.
Microscopy Techniques
- Light Microscopy offers resolving power of 0.2 µm, using ocular and oil immersion lenses.
- Darkfield Microscopy highlights spirochetes via reflected light.
- Fluorescence Microscopy employs stains like auramine-rhodamine for enhanced visualization.
- Electron Microscopy achieves high-resolution imaging, effective for viral identification.
Specimen Collection and Handling
- Collect material from the infection site promptly.
- Optimal timing for specific cultures, e.g., Salmonella typhi: blood in the first week, stool in the second and third weeks.
- Use appropriate collection devices for different specimens.
- Do not refrigerate spinal fluids, anaerobic, or gonococcal specimens.
- Collect specimens before the initiation of antibiotic therapy.
- Set up cultures within two hours of collection to ensure viability.
Criteria for Rejection of Specimens
- Presence of preservatives in collected samples.
- Insufficient specimen quantity impacts diagnostic utility.
- Dry swabs will not yield viable organisms.
- Leaky containers may lead to contamination and pose biohazard risks.
Blood Culture Collection
- Prep the skin with 70% alcohol and iodine before collection.
- Best to draw blood cultures right before fever spikes for accurate results.
- At least two cultures should be collected, but limit to three within a 24-hour window.
- Utilize antibiotic removal devices (ARD) if the patient is on antibiotics.
- Isolator® systems are most effective for fungi and acid-fast organisms.
- Blood should be diluted at a ratio of 1:10 into broth; for adults, collect at least 10 mL.
Culture Conditions
- Most plates incubate at 35-37°C; Campylobacter requires 42°C, Yersinia between 25-30°C.
- Maintain anaerobic conditions using broths with thioglycollate or cysteine and gas pak systems.
- Media types include non-selective (blood agar) and selective (EMB, MAC) which inhibit certain organisms.
Inoculation Techniques
- Use nichrome or platinum loops for streaking to isolate cultures.
- Employ calibrated loops for urine colony counts, with a standard reading after 18-24 hours.
Common Stains in Microbiology
- Gram Stain: Crystal Violet, decolorized with alcohol/acetone, counterstained with Safranin; identifies gram-positive (purple) and gram-negative (pink) organisms.
- Kinyoun & Ziehl-Nielson Stains: Used for mycobacteria, pink results indicate acid-fast organisms.
- Auramine-Rhodamine Stain: Fluorescent stain for mycobacteria with an orange color indicating positive results.
- Calcofluor White Stain: Identifies yeast and fungi; produces a bluish-white fluorescence when combined with 10% KOH.
Gram Positive Cocci - Staphylococcus
- Staphylococcus forms "grape-like" clusters; common pathogen includes Staphylococcus aureus.
- S. aureus: Coagulase positive; causes furuncles, impetigo, and post-surgical infections.
- Incidence of intoxications like toxic shock syndrome and food poisoning are notable.
- Resistant strains (MRSA) pose challenges; vancomycin is the treatment of choice.
- Coagulase Negative Staphylococcus: Opportunistic infections in immunocompromised individuals and those with prosthetic devices.
Resistance and Susceptibility
- Staphylococcus is often resistant to penicillin due to plasmid-mediated β-lactamase production.
- Susceptibility to penicillinase-resistant penicillins (e.g., methicillin) can vary among strains.
Inoculation Techniques
- Streak for isolation using nichrome, platinum, or disposable loops.
- Use calibrated loops (0.01 ml or 0.001 ml for urine colony count plates).
- For colony counting: Multiply the number of colonies by 100 for a 0.01 ml loop or by 1000 for a 0.001 ml loop.
- Read and report results after 18-24 hours.
Common Stains in Microbiology/Mycobacteriology
-
Gram Stain
- Primary: Crystal Violet
- Decolorizer: Alcohol/Acetone
- Counterstain: Safranin
- Results: Positive (Purple), Negative (Pink)
- Principle: Iodine mordant forms a complex with violet dye; washes out from gram-negative cells.
-
Kinyoun & Ziehl-Nielsen Stain
- Primary: Carbol Fuchsin
- Decolorizer: Acid Alcohol
- Counterstain: Methylene Blue
- Results: Positive (Pink), Negative (Blue)
- Principle: Specifically for Mycobacteria.
-
Auramine-Rhodamine Stain
- Primary: Auramine and Rhodamine
- Decolorizer: Acid Alcohol
- Counterstain: Potassium Permanganate
- Results: Positive (Orange), Negative (No Fluorescence)
- Principle: Quick detection of Mycobacteria.
Catalase and Coagulase Tests
- Catalase Test: H2O2 breaks down to water and oxygen; positive results show bubbling (Staphylococcus positive, Streptococcus negative).
- Coagulase Test: Detects coagulase and Protein A; determines Staphylococcus aureus from other staphylococci.
Staphylococcus (Catalase Positive)
-
S. aureus
- Coagulase Positive
- Associated Infections: Carbuncles, furuncles, paronychia, and bacteremia.
- Intoxications: Scalded Skin Syndrome, Toxic Shock Syndrome, Gastritis (enterotoxin).
- Characteristics: Most beta-lactamase producers; many are MRSA (Methicillin-resistant).
-
S. epidermidis
- Coagulase Negative
- Associated Infections: Endocarditis, prosthetic device infections.
- Characteristics: Most are methicillin-resistant; sensitive to novobiocin.
-
S. saprophyticus
- Coagulase Negative
- Associated Infections: Urinary Tract Infections (UTIs) in young women.
- Characteristics: Resistant to novobiocin.
Streptococcus Overview
-
Spherical or oval bacteria; can form chains or pairs.
-
Lancefield classification based on specific carbohydrate characteristics.
-
S. pyogenes (Group A)
- Beta hemolytic.
- Produces Streptolysin S (stable) and Streptolysin O (oxygen-labile).
- Causes scarlet fever rash due to erythrogenic toxin.
-
Enterococcus
- Grows on bile esculin medium (BEM) and in 6.5% NaCl.
- Associated Infections: UTIs, bacteremia.
-
S. pneumoniae
- Alpha hemolytic. Characterized by crater-like colonies.
- Associated Infections: Primary lobar pneumonia, meningitis, bacteremia.
- Laboratory Diagnosis: Quellung reaction and sensitivity to optochin; bile soluble.
Other Notable Streptococci
-
S. viridans
- Associated with subacute bacterial endocarditis; typically alpha hemolytic.
Key Points for Practitioners
- Perform daily quality control (QC) tests for reliable results.
- Wash hands and wear lab coats to prevent contamination.
- Remember the key morphology and biochemical characteristics for accurate identification and diagnosis.
Gram Negative Cocci
- Neisseria and Moraxella are key genera characterized as diplococci (kidney bean shape) and oxidase positive.
- Neisseria gonorrhoeae:
- Grows on chocolate and Thayer-Martin agar requiring 5-10% CO2; may take up to 48 hours for growth.
- Ferments glucose and is associated with gonorrhea, often asymptomatic and commonly mixed with Chlamydia.
- Gram stain is sensitive in males but not in females.
- Moraxella catarrhalis:
- Causes respiratory infections, grows well on chocolate and blood agar but not on MacConkey's agar.
- Colonies are hard, motile, asaccharolytic, and usually beta-lactamase positive.
Acinetobacter Species
- Emerging pathogen causing respiratory infections, UTIs, or acts as a colonizer.
- Appears as a coccobacillus and displays resistance to many drugs requiring antibiotic susceptibility testing.
- Can cause neonatal meningitis and sepsis.
Gram Positive Rods
- Corynebacterium:
- Catalase positive and displays a "Chinese letter" arrangement; metachromatic granules may be observed.
- Growth on Loeffler's slants and capable of Tellurite hydrolysis.
- Toxin production is determined by the Elek test.
- Listeria:
- Catalase positive with tumbling motility at 25°C; may cause neonatal meningitis and sepsis.
- Can survive in cold conditions (cold enrichment).
- Erysipelothrix:
- Catalase negative, associated with infections in fishermen, butchers, and veterinarians.
- Demonstrates "test tube brush" growth in gelatin.
Bacillus Species
- Bacillus species are spore formers, catalase positive, with "ground glass" hemolytic colonies.
- Capable of producing exotoxins and linked with bacillary dysentery.
Escherichia coli
- Most common cause of UTIs in females and responsible for various intestinal infections.
- Key characteristics include being indole and lactose positive with IMViC reactions of ++--.
- Types of pathogenic E. coli:
- Enterotoxigenic (ETEC) produces heat labile (LT) and heat stable (ST) toxins.
- Enteroinvasive (EIEC) penetrates epithelial cells of the large intestine, may be lactose negative.
- Enterohemorrhagic (EHEC), notably O157:H7, causes severe illness with Shiga-like toxin production.
Shigella
- Comprised of four groups:
- S. dysenteriae (Group A, most severe),
- S. flexneri (Group B),
- S. boydii (Group C),
- S. sonnei (Group D, most common in the US).
Common Gram Negative Selective Media
- MacConkey Agar differentiates lactose fermenters (dark pink) from non-fermenters (transparent).
- Eosin Methylene Blue (EMB) agar indicates lactose fermentation with green sheen.
- Hektoen-Enteric (HE) agar and Salmonella-Shigella (SS) agar differentiate species based on H2S production and lactose fermentation.
Enterobacteriaceae: Primary Differentiating Tests
- Triple Sugar Iron (TSI) tests indicate fermentation patterns.
- Various biochemical tests assess urease, VP, and motility to differentiate genera.
- Notable reactions include oxidase tests and ONPG tests to detect beta-galactosidase.
Salmonella
- Requires a large inoculum for infection (>100,000).
- Key biochemical characteristics include H2S positive and lysine positive reactions.
- S. enterica has over 2000 serotypes, including pathogenic strains leading to typhoid fever and septicemia.
Biochemical Tests Summary
- Oxidase test: positive = purple; Nitrate test: positive = pink.
- TSI slant can provide a complete metabolic profile for identification of bacteria.
Key Reminders
- Differentiate bacteria based on catalase, H2S, and hemolytic activity.
- Recognize the importance of resistance patterns in emerging pathogens like Acinetobacter.
Klebsiella and Related Genera
- Klebsiella is an opportunistic pathogen commonly associated with urinary tract infections (UTIs) and pneumonia; often resistant to ampicillin.
- Characterized as non-motile, encapsulated, with variable urease activity, VP positive, and negative for ornithine.
- Notably includes Klebsiella granulomatis, previously known as Calymmatobacterium granulomatis, linked to sexually transmitted infections (STIs).
- Citrobacter is an opportunistic pathogen, lysine negative, and biochemically similar to Salmonella.
- Enterobacter species are motile and positive for ornithine, distinguishing them from Klebsiella.
- Serratia can produce red pigment and is positive for DNase and gelatinase.
Key Characteristics of Proteus Species
- Proteus mirabilis is highly sensitive to penicillins and indole negative.
- Proteus vulgaris is indole positive and produces hydrogen sulfide (H2S).
- Both species exhibit urea positive activity, which aids in their identification.
Yersinia Characteristics
- Yersinia enterocolitica grows optimally at room temperature with cold enrichment, known for being invasive and toxigenic.
- Yersinia pseudotuberculosis causes acute mesenteric lymphadenitis and resembles true tuberculosis.
- Yersinia arizonae tests positive for ONPG and ferments glucose with characteristic fermentation patterns on KIA media.
Biochemical Tests
- Kligler Iron Agar (KIA) distinguishes carbohydrate fermentation; no sucrose is present unlike TSI.
- Citrate test utilizes green media, turning blue for positive results indicating citrate utilization.
- Decarboxylase tests measure the ability to decarboxylate amino acids, with lysine, ornithine, and arginine indicators.
- Indole test indicates the presence of tryptophan breakdown; Kovac's reagent turns pink if positive.
Various Tests and Indicators
- Voges-Proskauer (VP) test detects acetylmethylcarbinol (acetoin); a pink color indicates a positive result.
- Urease test measures the breakdown of urea into ammonia and CO2, indicated by a pink phenol red color change.
- TSI test reactions use a mnemonic for visualization of results, distinguishing between acid and alkaline reactions.
- Pigment production can be tested with ferric chloride for phenylalanine deaminase, positive reaction yielding a green color.
Gram-Negative Non-Fermenters
- Oxidase-positive organisms often show weakly positive results or exceptions.
- Rarely grow on MacConkey's agar, do not ferment glucose, resulting in alkaline/alkaline reaction (alk/alk).
- Pseudomonas aeruginosa is a common pathogen, oxidase positive, and typically does not utilize lactose.
- Burkholderia cepacia is a polymyxin B-resistant opportunistic pathogen particularly concerning patients with cystic fibrosis.
Gram-Negative Oxidase Positive Fermenters
-
Vibrio Characteristics
- Curved rod-shaped bacteria with polar flagella
- Selectively cultured on Thiosulfate-Citrate-Bile Salts-Sucrose (TCBS) agar
- Blood Agar Plate (BAP) shows hemolytic colonies
- Lactose negative, differentiating them from enteric bacteria
- Nitrate reduction test: positive (NO3 to NO2)
Vibrio cholerae (Serogroup 01)
- Causes rapid onset gastroenteritis within 3-10 hours
- Results in profuse diarrhea often described as "rice water" stools, with mucus flecks
- Requires a high concentration of organisms for infection unless the host is hypochlorohydric
- Appears yellow on TCBS agar
Vibrio parahemolyticus
- Appears green on TCBS
- Associated with enteritis, often from consuming contaminated seafood
Vibrio vulnificus
- Also appears green on TCBS
- Can cause wound infections and other severe diseases, particularly in immunocompromised individuals
Aeromonas
- Motile with polar flagella, resembling Vibrio
- Majority are indole positive, differentiating them from other pathogens
- Capable of growing on MacConkey agar (MAC)
- Causes cellulitis, wound infections, and diarrhea
Other Organisms
-
Campylobacter jejuni
- Small, curved rods with a "seagull" appearance
- Microaerophilic, takes 2-4 days for growth
- Cultured on CAMPY agar
- Linked to raw poultry and contaminated water, similar to Salmonella
- Treatment usually involves erythromycin or tetracycline
- Biochemical characteristics include:
- Catalase positive
- Oxidase positive
- Hippurate positive
Zoonotic Diseases Overview
- Zoonotic diseases are infections that can be acquired directly or indirectly from animals to humans.
Brucella
- Gram-negative coccobacilli associated with brucellosis.
- Blood culture is positive within the first two weeks; cultures should be held for 21 days.
- Different species have varying features:
- B. abortus (cows): H2S positive, thionine positive, basic fuchsin negative.
- B. suis (pigs): H2S variable, thionine negative, basic fuchsin positive.
- B. melitensis (goats): H2S negative, thionine negative, basic fuchsin negative.
- Mnemonic: "Cows eat Thionine. Pigs eat Basic Fuchsin. Goats eat neither."
Francisella tularensis
- Faintly staining gram-negative coccobacillus that causes tularemia, also known as "rabbit fever."
- Infection through tick bites, posing a high risk to laboratory personnel.
- Characterized by tiny pinpoint colonies, requiring cystine-glucose media for growth.
- Tests positive for H2S using lead acetate.
- Operates under biosafety level 3 due to its infectious potential.
Yersinia pestis
- Gram-negative bacterium responsible for plague.
- Notable for bi-polar staining, resembling a safety pin under microscope.
- Transmitted primarily by fleas, rats, and other mammal reservoirs.
- Managed at biosafety level 3 due to its pathogenicity.
Pasteurella multocida
- Gram-negative rod associated with infections from cat and dog bites.
- Exhibits a "mousy" odor and demonstrates bi-polar staining.
- Oxidase and indole positive, indicating specific biochemical reactions.
- Ferments glucose and sucrose, useful for identification.
- Operates under biosafety level 2 precautions.
Streptobacillus moniliformis
- Long filamentous gram-negative rods presenting a pleomorphic shape with "puffball" or "string of pearls" colonies in thioglycolate broth.
- Causes "rat bite fever" and Haverhill fever, infections resulting from contact with infected species.
- Ascitic fluid sample is necessary for diagnosis; sodium polyanethol sulfonate (SPS) inhibits growth of other bacteria in cultures.
Bacterial Growth Requirements and Culture Techniques
- Red blood cells need heat to break down and release necessary growth factors (X and V factors).
- Special media is required for certain bacteria; common media includes:
- Chocolate Agar: Used for growth of fastidious organisms.
- Bordet-Gengou: Contains potato infusion with glycerol and 20% sheep red blood cells.
- Regan/Lowe: Oxoid charcoal agar with 10% horse blood; has a longer shelf life than other media.
- Satellitism: Phenomenon where Haemophilus species grow in the hemolytic zone surrounding S. aureus colonies, as S. aureus produces V factor and hemolyzes blood to release X factor.
Infections Caused by Haemophilus Species
- Meningitis in children predominantly affects 2-4 year-olds, usually preceded by nasopharyngeal colonization.
- Classic symptoms and diagnosis often involve blood culture over throat cultures, especially in epiglottitis cases in children.
Characteristics of HACEK Group
- Aggregatibacter aphrophilus: Associated with endocarditis, weakly oxidase-positive, indole negative.
- Aggregatibacter actinomycetemcomitans: Linked to periodontal and jaw abscesses; shows distinctive colony morphology in plaque.
- Cardiobacterium hominis: Can cause endocarditis and may result in false positive Gram reactions; oxidase positive, indole positive.
- Eikenella corrodens: Notable for its "bleachy" odor and pitting of agar; involved in periodontal infections.
- Kingella kingae: Causes septic arthritis and osteomyelitis primarily in children; presents with corroding colony appearance.
Mycoplasma and Ureaplasma
- Mycoplasma are the smallest free-living microorganisms, lacking a cell wall, making them resistant to Gram staining.
- M. pneumoniae is known for causing atypical pneumonia ("walking pneumonia") and leads to a positive cold agglutinin titer.
- Ureaplasma urealyticum is associated with non-gonococcal urethritis and urease production; treated with tetracycline or spectinomycin.
- M. hominis can colonize the genitourinary tract and is linked to postpartum fever.
Legionella pneumophila
- Causes Legionnaires' disease, a severe form of pneumonia; milder form called Pontiac fever.
- L. pneumophila serogroups 1 and 6 account for approximately 75% of cases.
- Found in potable water and faulty air conditioning systems.
- Identification relies on sputum, bronchial washings, or pleural fluid; Dienes stain used for specificity.
Anaerobic Infections
- Indicators of anaerobic infections include foul odor, close proximity to mucosal surfaces, and previous antibiotic therapy.
- Culture techniques include:
- Classic anaerobic culture using Gas Pak jars with palladium pellets for hydrogen generation.
- Methylene blue or resazurin act as indicators in cultures, changing color based on oxidation state.
Gram-Negative Anaerobes
- Fusobacterium nucleatum: Characterized by spear-shaped, gram-negative slender fusiforms, and shows bread crumb or speckled colonies at 36°C in anaerobic conditions.
- Bacteroides fragilis: Produces dark colonies greater than 1 mm on BBE and catalase positive. May fluoresce brick-red and is commonly found in polymicrobial infections.
- Resistance Susceptibility Testing: Notable resistance patterns include Bacteroides fragilis resistant to vancomycin and sensitive to kanamycin, while Fusobacterium genera show variable reactions.
Gram-Positive Anaerobes
- Clostridium difficile: Associated with Pseudomembranous Colitis (PMC) and recognized by the 'horse stable' odor on CCFA agar; known for emerging resistant strains.
- Clostridium perfringens: Exhibits a double zone of hemolysis and is a well-known cause of gas gangrene and food poisoning, though spores are seldom observed.
- Clostridium tetani: Notable for terminal spores shaped like racquets and is the causative agent of tetanus.
Actinomyces
- Actinomyces israelli: Identified by "molar tooth" colonies, branching gram-positive rods, and linked with lumpy jaw; associated with sulfur granules.
Treponema and Leptospira
- Treponema pallidum: Causes syphilis, with lesions appearing 2-10 weeks post-infection; chancre heals without treatment.
- Leptospira: Hooked spirals that are animal pathogens spread through contaminated water; darkfield microscopy useful for diagnosis, culturable in Fletcher’s media.
Chlamydia
- Chlamydia trachomatis: An obligate intracellular parasite responsible for sexually transmitted infections, treated with penicillin or tetracycline; diagnosed through Giemsa stain.
Borrelia
- Borrelia recurrentis: Known for "relapsing fever," transmitted by ticks or lice, characterized by looser coils visible under Giemsa or Wright's stain, and capable of relapsing due to antigenic variation.
Rickettsial Infections
- Infection vectors and notable Rickettsiae include:
- Coxiella burnetti: Transmitted via inhalation; confirmed with Complement Fixation Test.
- Rickettsia rickettsii: Transmitted by ticks; characterized by a rash on palms and soles.
- Rickettsia typhi: Flea-borne and associated with murine typhus.
Mycobacteria
- Mycobacteria Characteristics: Slim, gram-variable rods resistant to normal staining due to high lipid content; distinguished through acid-fast staining techniques such as Ziehl-Neelsen and Kinyoun stains.
-
Growth and Culture:
- Optimal growth conditions include incubation at around 30-36°C and can take 3-6 weeks for visibility; special media like Lowenstein-Jensen and Tween 80 may be employed for enhanced growth.
Specimen Collection Guidelines
- Recommended specimens include sputum (three consecutive mornings), bronchial washings, and urine; must be collected sterilely and stored properly to prevent contamination.
Rickettsiae
- Small gram-negative coccobacilli, obligate intracellular parasites.
- Transmitted primarily by arthropod vectors (insects).
- Common symptoms include fever, headache, and rash; notable exception is Q fever which presents without a rash.
- Best visualized using Giemsa stain.
- Weil-Felix test detects Rickettsial antibodies using Proteus serotypes OX-19, OX-2, and OX-K; a titer of 1:160 or a four-fold rise indicates infection.
Common Rickettsiae and Associated Vectors
- Rickettsial Pox (R. akari): spread by house mites; no Proteus reactions.
- Q Fever (Coxiella burnetti): airborne transmission; confirmed via complement fixation test.
- Typhus Fever (R. prowazekii): transmitted by lice; positive for OX-19, variable for OX-2.
- Rocky Mountain Spotted Fever (R. rickettsiae): ticks as vectors; characterized by rash on palms and soles; positive for OX-19 and OX-2.
- Murine Typhus (R. typhi): transmitted by rat fleas; positive for OX-19 and OX-2.
Mycobacteria
- Slim, gram-variable rods with high lipid content, resulting in poor Gram staining.
- Staining methods include Ziehl-Neilsen (hot) and Kinyoun (cold) acid-fast stains; Auramine-Rhodamine for fluorescent microscopy.
- Cultivation requires specific conditions: Lowenstein-Jensen media, temperature at 36°C (some at 30°C), and takes 3-6 weeks for growth.
- Automated cultures can expedite growth and testing for antibiotic susceptibility.
- Significant pathogen: Mycobacterium avium complex, associated with pulmonary and disseminated disease, especially in immunocompromised patients.
Mycobacterium Leprae
- Etiological agent of leprosy (Hansen disease), affects skin, mucous membranes, and nerves.
- Characterized by progressive disease manageable with treatment.
- Optimal growth in the footpads of armadillos.
Differentiating Mycobacteria
- Notable characteristics for identifying species: niacin production, nitrate reduction, Tween 80 hydrolysis, and tellurite utilization.
- Mycobacterium tuberculosis: rapid grower and positive for key biochemical reactions; forms a distinct identification profile.
Specimen Collection and Pathogen Association
- Throat/Oropharynx: normal flora includes alpha-streptococci; pathogens can be Group A Streptococcus and Neisseria meningitidis, especially in children.
- Deep Wounds/Abscesses: typically contain anaerobes and aerobes; collection methods should bypass normal flora.
- Superficial Wounds: often colonized by Staphylococcus aureus and Streptococcus Group A.
- Blood: typically sterile; any organism found in immunocompromised individuals or those with prosthetic heart devices is seen as pathogenic.
- Peptic Ulcers: associated organisms vary; specimen collection may involve NP swabbing procedures.
Virology Overview
- Viruses contain either RNA or DNA, but not both; they lack structural elements for protein synthesis and replicate exclusively within host cells.
- Viral specimens, including pre- and post-convalescent sera, should be shipped on dry ice.
- Specimens for viral culture require transport media enriched with nutrients (such as fetal calf serum or albumin) and antibiotics.
Laboratory Methods
- Enzyme Immunoassay (EIA) detects viral antibodies or antigens, like HBsAg and anti-HBsAb.
- Viral culture methods assess live virus presence.
- Electron microscopy allows visualization of viral particles.
- Molecular techniques include DNA probes and Polymerase Chain Reaction (PCR):
- PCR amplifies nucleic acid sequences in cycles, doubling the target each time.
RNA Viruses and Associated Diseases
- Flavivirus: Causes Yellow Fever, Dengue, and St. Louis Encephalitis; primarily transmitted by mosquitoes.
- Hantavirus: Linked to Pulmonary Syndrome and Hemorrhagic Fever; transmitted by rodents.
- Hepatitis A Virus (HAV): Causes Hepatitis A; commonly associated with shellfish; among the most stable viruses infecting humans.
- Hepatitis C Virus (HCV): Previously known as non-A, non-B hepatitis; causes Hepatitis C.
- Influenzavirus: Responsible for influenza outbreaks.
- Morbillivirus: Causes measles; more severe in adults.
- Mumps Virus: Causes mumps.
- Poliovirus: Leads to poliomyelitis and aseptic meningitis; naturally infects only humans.
- Respiratory Syncytial Virus (RSV): Causes serious respiratory infections in young children, leading to giant multinucleated cells.
- Rhabdovirus: Causes rabies; identified by Negri bodies in the brain; wildlife is a reservoir.
- Rhinovirus: Common cause of the cold.
- Rotavirus: Causes acute infantile diarrhea, potential fatality in infants.
- Rubivirus: Causes rubella; linked to serious congenital abnormalities; vaccination available but contraindicated during pregnancy.
Retroviruses and Associated Diseases
- Human Immunodeficiency Virus (HIV): Causes AIDS; detected via EIA, confirmed by Western Blot.
- Human T-Cell Leukemia Virus (HTLV-I): Causes T-cell leukemia and tropical spastic paraparesis; confirmed by EIA techniques.
- Human T-Cell Leukemia Virus (HTLV-II): Associated with hairy-cell leukemia; confirmed by EIA.
DNA Viruses and Associated Diseases
- Adenovirus: Common cause of respiratory infections.
- Cytomegalovirus (CMV): Primary viral cause of mental retardation; significant risk for immunocompromised patients.
- Epstein-Barr Virus (EBV): Causes infectious mononucleosis and chronic fatigue syndrome; associated with Burkitt's lymphoma.
- Hepatitis B Virus (HBV): Causes Hepatitis B; detectable via ELISA; vaccination available.
- Herpesviruses: Includes HSV-1 (oral infections) and HSV-2 (genital infections).
- Human Papillomavirus (HPV): Causes genital and cutaneous warts; some types associated with cervical carcinoma; vaccine available.
- Poxvirus: Smallpox, eradicated in the general population but noted for lab outbreaks.
- Varicella-Zoster Virus: Causes chickenpox in children and shingles in adults; diagnosis based on clinical observation.
Important Notes
- Recognize that viruses are non-cellular entities that reproduce only in host cells, consisting of nucleic acid enveloped in a protein coat.
- Evaluate laboratory results carefully to distinguish viral diseases and assess disease association, notably Negri bodies in rabies diagnoses.
Microbiology Sample Questions
- "Rice water stool" is linked to Vibrio cholerae infections.
- Bluish purulent lesions with a gram-negative, motile rod suggest Pseudomonas aeruginosa.
- Small gram-negative rods in genital soft chancre indicate a possible infection by Haemophilus ducreyi.
- Phenomenon of alpha hemolysin observed on blood agar often relates to Streptococcus pneumoniae.
- Lysine decarboxylase production differentiates Citrobacter from Salmonella.
- Autoclave sterilization requires 15 psi, at 121 °C for 15 minutes.
- Clinical rabies diagnosis relies on the observation of Negri bodies in brain tissue.
- Catalase positive, coagulase negative gram-positive cocci suggest Staphylococcus saprophyticus in suspected UTI specimens.
- Gardnerella vaginalis is associated with a "fishy" odor when mixed with KOH, indicating bacterial vaginosis.
Gram Stain and Biochemical Tests
- A sputum specimen showing gram-positive cocci in chains may suggest Streptococcus pneumoniae, confirmed via optochin susceptibility.
- Serratia marcescens is recognized for producing a red non-water soluble pigment.
- For urinary tract infections, the presence of > 10 epithelial cells indicates specimen contamination.
- Campylobacter jejuni demonstrates oxidase positivity and optimal growth at 42 °C.
Specific Pathogens and Media
- Loeffler’s medium is optimal for isolating Corynebacterium diphtheriae.
- The drug of choice for methicillin-resistant Staphylococcus infections is vancomycin.
- Clostridium perfringens is linked to gas gangrene, identifiable by its stormy fermentation.
- Photochromogenic mycobacterium isolated at 30 °C is likely Mycobacterium marinum.
- Ebola virus causes hemorrhagic fever, while HIV is responsible for AIDS, and rabies virus presents differently.
Diagnosis and Clinical Presentation
- Endocarditis due to Streptococcus Group D necessitates a synergistic relationship between penicillin and aminoglycosides.
- Gram variable rods in a wet prep from a patient with vaginitis indicate a possible Gardnerella vaginalis infection.
- Diagnosis of Clostridium difficile requires examination of CCFA agar, while C. tetani displays "drumstick" shaped endospores.
- Textural differences in TSI (triple sugar iron) can help identify unknown organisms, particularly H2S production.
Additional Notes
- Endotoxins from Neisseria meningitidis can lead to DIC (disseminated intravascular coagulation) and adrenal gland destruction.
- Mycobacterium xenopi is a non-photochromogenic organism grown typically at 37 °C.
- The specimen of choice for epiglottitis is collected via spinal fluid analysis.
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