Micro II Lab SQ

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Robyn Schufford Medical Microbiology Lab Study Questions Unit 1 - Acid-Fast Bacteria 1. What does the term "acid-fast" mean? "Acid-fast" refers to a property of certain bacteria, particularly the Mycobacterium genus, which retain a dye (such as carbol fuchsin) even after being treated with an acid-alcohol solution. 2. What causes Mycobacteria to be acid-fast? Mycobacteria are acid-fast due to the high lipid content in their cell walls, specifically mycolic acids. These waxy substances make the cell wall impermeable to many stains and chemicals, including the acid-alcohol solution used in staining procedures. 3. Define photochromogen, scotochromogen, and nonphotochromogens. ● Photochromogen: Photochromogens are mycobacteria that produce pigment when exposed to light, typically after prolonged incubation. The pigment production is influenced by light but does not require it for growth. Examples include Mycobacterium kansasii and Mycobacterium marinum. ● Scotochromogen: Scotochromogens are mycobacteria that produce pigment both in the light and in the dark, although the pigment may be more pronounced when grown in the dark. Examples include Mycobacterium scrofulaceum and Mycobacterium szulgai. ● Nonphotochromogens: Nonphotochromogens are mycobacteria that do not produce pigment regardless of light exposure. Examples include Mycobacterium tuberculosis and Mycobacterium avium complex (MAC). 4. Describe the incubation temperatures and atmospheres optimal for recovery of AFB. ● Temperature: Most AFB, including Mycobacterium tuberculosis, grow best at temperatures between 35-37°C (95-98.6°F). This temperature range mimics the human body temperature, which is why these bacteria are often associated with infections in warm-blooded animals. ● Atmosphere: AFB typically require a slightly higher level of carbon dioxide (CO2) than atmospheric levels (5-10%) for optimal growth. Additionally, some species may require increased humidity to promote growth. 5. Name and describe three decontamination and concentration methods necessary to recover acid-fast bacilli from respiratory specimens. ● N-acetyl-L-cysteine (NALC)-NaOH Digestion: This method involves treating the respiratory specimen with a decontaminating solution containing NALC and sodium hydroxide (NaOH). NALC helps in liquefying mucus and reducing the viscosity of the specimen, while NaOH helps in killing contaminating organisms. After digestion, the sample is neutralized and centrifuged to concentrate the AFB. This method is commonly used for sputum specimens.

● Sodium Hypochlorite (NaOCl) Sedimentation: In this method, the respiratory specimen is treated with a solution containing sodium hypochlorite (bleach) to decontaminate it. NaOCl kills non-mycobacterial organisms while preserving AFB. The specimen is then allowed to settle, and the sediment containing AFB is concentrated by centrifugation. This method is suitable for concentrated or induced sputum specimens. ● Centrifugation-Concentration Technique: This method involves centrifuging the respiratory specimen to concentrate AFB. Prior to centrifugation, the specimen may be treated with decontaminating agents such as NALC-NaOH or NaOCl to reduce contamination. After centrifugation, the supernatant is discarded, and the pellet containing AFB is resuspended in a smaller volume of saline or phosphate buffer. This concentrated sample is then used for smear microscopy or culture 6. Name 3 methods for staining for acid-fast bacilli and give the stains used in each. ● Ziehl-Neelsen (ZN) Staining Method: ○ Primary Stain: Carbol fuchsin (basic fuchsin) - stains acid-fast bacteria red. ○ Decolorizer: Acid-alcohol - removes the stain from non-acid-fast bacteria. ○ Counterstain: Methylene blue or brilliant green - stains non-acid- fast bacteria blue or green. ● Kinyoun Staining Method (Cold Acid-Fast Staining): ○ Primary Stain: Kinyoun's carbolfuchsin - a more concentrated form of carbol fuchsin. ○ Decolorizer: Acid-alcohol - removes the stain from non-acid-fast bacteria. ○ Counterstain: Methylene blue - stains non-acid-fast bacteria blue. ● Fluorochrome Staining Method (e.g., Auramine-Rhodamine Staining): ○ Primary Stain: Auramine O or auramine-rhodamine - stains acid- fast bacteria fluorescent yellow or orange under UV light. ○ Decolorizer: Acid-alcohol or potassium permanganate - removes excess stain from the background. 7. Name three types of media used to isolate acid-fast bacilli. Give an advantage and disadvantage of each. ● Lowenstein-Jensen (LJ) Medium: ○ Advantage: LJ medium is inexpensive, easy to prepare, and can be stored for a long time. It supports the growth of most mycobacterial species and allows for the observation of colony morphology. ○ Disadvantage: LJ medium has a slow turnaround time, typically taking 2-6 weeks for visible colony growth. Additionally, it lacks selectivity, allowing the growth of contaminants alongside mycobacteria. ● Middlebrook 7H10 and 7H11 Agar: ○ Advantage: Middlebrook agar provides a faster turnaround time compared to LJ medium, with visible colony growth typically

observed within 7-21 days. It contains nutrients that support the growth of fastidious mycobacterial species. ○ Disadvantage: Middlebrook agar is more expensive and labor- intensive to prepare compared to LJ medium. It may also require supplementation with antibiotics or selective agents to inhibit the growth of contaminants. ● Liquid Media (e.g., Middlebrook 7H9 Broth): ○ Advantage: Liquid media allow for the rapid detection of mycobacterial growth, with visible turbidity observed within days to weeks. They provide a higher yield of mycobacteria compared to solid media, making them suitable for specimens with low bacterial loads. ○ Disadvantage: Liquid media require careful monitoring and subculturing to prevent contamination and clumping of mycobacterial aggregates. They may also require additional processing steps for species identification or drug susceptibility testing. 8. Differentiate the organisms termed "rapid growers" from other Mycobacteria species. Rapid growers, also known as rapidly growing mycobacteria (RGM), are a group of mycobacteria species that exhibit relatively fast growth rates compared to other mycobacteria species. Here's how they differentiate from other mycobacteria species: 1. Growth Rate: ○ Rapid growers have shorter incubation periods and typically grow within 7 days on solid media, such as Middlebrook agar. In contrast, other mycobacteria species, including the slow-growing Mycobacterium tuberculosis complex, may take weeks to months to produce visible colonies. 2. Colony Morphology: ○ Rapid growers often produce smooth, moist, and pigmented colonies with rapid expansion. In contrast, slow-growing mycobacteria species may produce rough, dry, and non-pigmented colonies. 9. What is the purpose of using a biological safety cabinet when working with acid-fast specimens? The primary purpose of using a biological safety cabinet (BSC) when working with acid- fast specimens is to protect laboratory personnel, the environment, and the integrity of the specimens being handled. 10. Name and describe three tests used to definitively identify M. tuberculosis. ● Acid-fast Bacilli (AFB) Smear Microscopy ● Mycobacterial Culture ● Molecular Tests (e.g., Nucleic Acid Amplification Tests, NAATs) Unit 2 - Anaerobes

1. Describe primary set-up media for recovery anaerobes. Growth of which anaerobes are supported on each? ● Anaerobic Blood Agar (ABA): ○ Description: ABA is a solid medium containing 5-10% defibrinated sheep blood and a reducing agent (e.g., thioglycolate or cysteine) to create an anaerobic environment. The agar base may contain nutrients such as peptones, beef extract, and agar. ○ Supported Anaerobes: A wide range of anaerobic bacteria can grow on ABA, including obligate anaerobes such as Clostridium species, Bacteroides species, and Prevotella species, as well as facultative anaerobes and aerotolerant anaerobes. ● Brucella Blood Agar (BBA): ○ Description: BBA is a solid medium containing Brucella agar supplemented with 5-10% defibrinated sheep blood. It may also contain hemin and vitamin K1 to support the growth of fastidious anaerobes. ○ Supported Anaerobes: BBA supports the growth of a wide range of anaerobic bacteria, including members of the Bacteroides fragilis group, Fusobacterium species, Clostridium species, and other anaerobic cocci. ● Schaedler Agar: ○ Description: Schaedler agar is a solid medium containing a complex mixture of nutrients, including peptones, beef extract, and starch. It may also contain hemin and vitamin K1. The agar is heated to inactivate oxygen-binding proteins and create an anaerobic environment. ○ Supported Anaerobes: Schaedler agar supports the growth of a diverse array of anaerobic bacteria, including Bacteroides species, Clostridium species, Prevotella species, and anaerobic streptococci 2. Explain in detail the principle of the GasPak jar. The GasPak jar is a laboratory apparatus used to create an anaerobic environment for the growth of anaerobic bacteria or for the maintenance of anaerobic conditions during microbiological procedures. The principle behind the GasPak jar involves the generation of an oxygen-free atmosphere through the chemical reaction of a sachet containing specific chemicals. 3. Describe the characteristic hemolytic pattern of Clostridium perfringens. Clostridium perfringens is known for its characteristic hemolytic pattern on blood agar plates, which is often used for its identification. The hemolytic pattern of Clostridium perfringens is described as "double zone" or "double hemolysis" due to its distinct appearance: 1. Alpha Hemolysis (Partial Hemolysis):

○ Clostridium perfringens exhibits a narrow zone of partial hemolysis surrounding the colony on blood agar plates. This zone appears greenish or brownish in color due to the partial breakdown of red blood cells (erythrocytes). The alpha hemolysis is caused by the action of C. perfringens' alpha-toxin, also known as phospholipase C, which hydrolyzes phospholipids in the red blood cell membranes. 2. Beta Hemolysis (Complete Hemolysis): ○ Outside the zone of alpha hemolysis, there is a larger, clear zone of complete hemolysis surrounding the colony. This zone appears transparent or colorless because it represents the complete lysis of red blood cells in the agar, leaving a clear area around the colony. The beta hemolysis is caused by the action of other toxins produced by C. perfringens, such as perfringolysin O, which forms pores in the red blood cell membranes, leading to their rupture. 4. If there is an arrow-head shaped zone of hemolysis present on the reverse camp test, how would the organism appear on egg yolk agar? if an organism produces an arrowhead-shaped zone of hemolysis on the reverse Camp test, indicating phospholipase C activity, it would appear as having a characteristic opacity zone surrounding the colony on egg yolk agar due to its lecithinase activity. This combination of test results is consistent with the identification of Clostridium perfringens. 5. Name two most often isolated Fusobacterium species. Name and describe in detail the disease process it is most identified with? ● Two of the most frequently isolated Fusobacterium species are Fusobacterium nucleatum and Fusobacterium necrophorum. Among these, Fusobacterium necrophorum is particularly associated with a specific disease process known as Lemierre's syndrome. ● One of the hallmark features of Lemierre's syndrome is the development of septic thrombophlebitis, particularly involving the internal jugular vein. Fusobacterium necrophorum can cause inflammation and thrombosis (clot formation) within the internal jugular vein, leading to a condition known as internal jugular vein thrombophlebitis. ● Metastatic Infections: As the septic thrombophlebitis progresses, septic emboli (clots containing bacteria) can break off and spread to other parts of the body, causing metastatic infections. Common sites of metastatic infection include the lungs, joints, liver, and other organs. ● Clinical Presentation: Patients with Lemierre's syndrome often present with a triad of symptoms, including a sore throat, fever, and neck swelling or pain. As the disease progresses, they may develop symptoms of septicemia, such as rapid heart rate, hypotension, and organ dysfunction. Imaging studies, such as CT scans or ultrasound, may reveal evidence of internal jugular vein thrombosis and septic emboli. 6. Provide reactions for the presumptive identification of anaerobic gram-negative rods

Kanamycin Vancomycin Colistin Bile Esculin Bacteroides fragilis Prevotella Porphyromonas Fusobacterium R: resistant S: susceptibility 7. Describe the identification of other anaerobic bacteria from the lab exercise. ● Specimen Collection and Processing: ○ Clinical specimens, such as abscess aspirates, wound swabs, or tissue biopsies, are collected aseptically and transported to the laboratory for processing. ○ Specimens are plated onto selective and differential media appropriate for the growth of anaerobic bacteria, such as anaerobic blood agar, Schaedler agar, or Brucella agar supplemented with appropriate antibiotics or selective agents to inhibit the growth of aerobic or facultative bacteria. ● Primary Culture and Incubation: ○ Plates are incubated in an anaerobic environment using methods such as GasPak jars, anaerobic chambers, or specialized anaerobic incubators. This creates conditions conducive to the growth of anaerobic bacteria while inhibiting the growth of oxygen- sensitive organisms. ○ Incubation periods vary depending on the type of specimen and the suspected pathogens but typically range from 24 to 72 hours. ● Colonial Morphology and Gram Staining: ○ After incubation, colonies with different morphologies (size, shape, color, texture) are observed on the agar plates. ○ Gram staining is performed to determine the Gram reaction and cellular morphology of the isolated colonies. Anaerobic bacteria are typically gram-negative rods, gram-positive rods, or gram-positive cocci. ● Biochemical Assays: ○ Various biochemical tests are performed to further characterize the isolated bacteria and identify them to the genus or species level. ○ Common biochemical assays used for anaerobic bacteria identification include catalase test, indole test, nitrate reduction test, carbohydrate fermentation tests, and API systems (e.g., API 20A for anaerobes). 8. How can you tell if an isolate is a true anaerobe and not a facultative anaerobe?

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