Microbiology Quiz: Bacteria Identification
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Questions and Answers

What type of colonies does Bacillus anthracis produce?

  • Frosted glass colonies (correct)
  • Spherical colonies
  • Rough colonies
  • Clustered colonies

    • Which organism is commonly associated with food poisoning?

  • Lactobacillus
  • Corynebacterium
  • Erysiplothrix
  • Bacillus anthracis (correct)

    • Which method is necessary to visualize spirochetes effectively?

  • Silver stain (correct)
  • Gram stain
  • Electron microscopy
  • Bright-field microscopy

    • How are Borrelia species primarily transmitted?

    <p>By arthropods such as ticks and lice</p> Signup and view all the answers

    How many spirals do Borrelia typically have per organism?

    <p>3 to 10 spirals</p> Signup and view all the answers

    What type of organisms does Brucella blood agar best support?

    <p>Facultative and obligate anaerobes</p> Signup and view all the answers

    Which antibiotic in KVLB agar inhibits most gram-positive bacteria?

    <p>Vancomycin</p> Signup and view all the answers

    What color indicates an alkaline pH in CCFA due to Clostridium difficile metabolism?

    <p>Yellow</p> Signup and view all the answers

    What atmosphere is primarily used for anaerobic incubation?

    <p>Nitrogen gas</p> Signup and view all the answers

    What is the main purpose of the palladium pellets in anaerobic jars?

    <p>To catalyze hydrogen gas reaction</p> Signup and view all the answers

    How long should bags and jars be kept sealed after incubation?

    <p>48 hours</p> Signup and view all the answers

    Which Gram stain reaction results are associated with Peptostreptococcus?

    <p>Gram positive cocci</p> Signup and view all the answers

    What is a key identification test for presumptive anaerobic identification?

    <p>Colony morphology observation</p> Signup and view all the answers

    Which group of organisms is sensitive to kanamycin and colistin?

    <p>Fusobacterium</p> Signup and view all the answers

    What characteristic distinguishes Prevotella from Bacteroides ureolyticus?

    <p>Production of protoporphyrin</p> Signup and view all the answers

    Which organism is known for producing chartreuse fluorescence?

    <p>Fusobacterium</p> Signup and view all the answers

    Which organism requires hemin and vitamin K for growth?

    <p>Prevotella</p> Signup and view all the answers

    Which of the following is a characteristic of Bacteroides ureolyticus?

    <p>Resistant to vancomycin</p> Signup and view all the answers

    What is a distinguishing feature of Mobiluncus?

    <p>It is curved bacilli.</p> Signup and view all the answers

    Which of the following organisms is sensitive to bile?

    <p>Prevotella</p> Signup and view all the answers

    Which organism produces bread-crumb, speckled colonies?

    <p>Fusobacterium nucleatum</p> Signup and view all the answers

    What characteristic is commonly associated with clue cells?

    <p>Presence of bacteria clustered at edges</p> Signup and view all the answers

    Which of the following methods is NOT recommended for culturing vaginalis?

    <p>Culture analysis</p> Signup and view all the answers

    What is a key identifying feature of Nocardia spp. on a gram stain?

    <p>Beaded, branching filaments</p> Signup and view all the answers

    What is the main risk associated with Nocardia infection?

    <p>Pulmonary and cutaneous infections</p> Signup and view all the answers

    What type of colonies do Nocardia spp. form on nonselective media?

    <p>Waxy, chalky, and crumbly</p> Signup and view all the answers

    Which of the following is a method used to identify Tropheryma whipplei?

    <p>PCR or 16S rRNA</p> Signup and view all the answers

    What is a characteristic feature of spore-forming, non-branching bacilli?

    <p>Protective and resistant spores</p> Signup and view all the answers

    Which of the following statements about Bacillus is true?

    <p>Metabolically diverse and found in nature</p> Signup and view all the answers

    Which organism is known for its virulence factor Diphtheria toxin?

    <p>Corynebacterium diphtheriae</p> Signup and view all the answers

    What shapes do Gram-positive rods exhibit in a Gram stain for Corynebacterium?

    <p>Chinese letters or palisades</p> Signup and view all the answers

    What is a common laboratory diagnostic feature of C.diphtheriae?

    <p>Methylene blue stains showing metachromatic granules</p> Signup and view all the answers

    What type of infections can C.diphtheriae cause?

    <p>Cutaneous and respiratory</p> Signup and view all the answers

    Corynebacterium species are primarily classified as which type of bacteria?

    <p>Gram-positive rods</p> Signup and view all the answers

    What is the primary method for preventing Diphtheria?

    <p>Immunization with DIP/TET</p> Signup and view all the answers

    Which characteristic distinguishes non-spore-forming Gram-positive rods from spore-forming ones?

    <p>They do not have spores</p> Signup and view all the answers

    Which of the following is NOT a known feature of Corynebacterium species?

    <p>They are motile</p> Signup and view all the answers

    What color indicates a positive result for nitrate reduction?

    <p>Red</p> Signup and view all the answers

    Which species is distinguishable from others by its ability to reduce tellurite?

    <p>M.avium complex</p> Signup and view all the answers

    What is the effect of Tween 80 hydrolysis in mycobacterial culture media?

    <p>Turns media red</p> Signup and view all the answers

    Which medium allows growth for rapid growers in the absence of crystal violet?

    <p>Special MAC</p> Signup and view all the answers

    What is the primary transmission method for Mycobacterium tuberculosis complex?

    <p>Airborne droplets</p> Signup and view all the answers

    Which of the following factors distinguishes M.tuberculosis from M.bovis using T2H?

    <p>Sensitivity to inhibitors</p> Signup and view all the answers

    Which of the following is true about the colonies of Mycobacterium tuberculosis complex?

    <p>They are non-pigmented</p> Signup and view all the answers

    What is the result of adding zinc to the nitrate reduction test if red color does not appear?

    <p>No color change indicates complete reduction</p> Signup and view all the answers

    Study Notes

    Anaerobic Bacteria

    • Anaerobic bacteria do not need oxygen to grow
    • They vary in their ability to tolerate oxygen
    • Obligate (strict) anaerobes require anaerobic conditions for growth; oxygen is toxic to them and they are killed in its presence
    • Aerotolerant anaerobes can grow in an atmosphere with oxygen, but they grow best in anaerobic environments
    • Facultative anaerobes do not require oxygen, but they will use it if available

    Oxygen Reduction

    • Superoxide anion and hydrogen peroxide are toxic to strict anaerobes
    • Strict aerobes and facultative anaerobes have superoxide dismutase and/or catalase to neutralize these toxic substances

    Oxidation-Reduction (Redox) Potential

    • Anaerobic bacteria require a low redox potential; a high redox environment contains oxygen
    • Normal human tissue and aerobic culture media have a high redox potential
    • Reducing agents (thioglycollate, cysteine, dithiothreitol) are included in anaerobic media

    Normal Habitat

    • Anaerobic bacteria are found in soil, water, and animals
    • In humans, they are found in the oral cavity, upper respiratory tract (URT), intestinal tract, genitourinary tract, and skin
    • Facultative organisms in humans use up oxygen in protected areas and reduce the redox potential to inactivate harmful oxygen molecules

    Diseases

    • Anaerobic bacteria cause infections, commonly following trauma (deep or puncture wounds), animal or human bites
    • Exogenous anaerobic infections occur when organisms, spores, or toxins enter the body through ingestion or physical trauma
    • Examples include tetanus and botulism
    • Endogenous anaerobic infections occur near mucosal surfaces and examples include bacteremia, abscesses, gas gangrene, and pneumonia. 

    Clues to Anaerobic Diseases

    • Trauma (deep or puncture wounds)
    • Animal or human bite
    • Foul-smelling, gaseous discharge
    • Necrotic tissue, vascular stasis
    • Contains sulfur granules (actinomycosis)
    • Black or fluorescent red color
    • Previous therapy with aminoglycosides (aminoglycosides are ineffective against these microbes)
    • Failure to grow the organism on a Gram stain

    Specimen Collection

    • Appropriate specimens include blood, sterile body fluids (CSF, bone marrow, cavity fluid), urine (suprapubic aspirate), abscesses, ulcers, and draining wounds, and biopsied material.
    • Inappropriate specimens include those from sites with resident flora (oral, GI, GU tracts), superficial skin sites, voided or catheterized urine, expectorated sputum, throat or nasopharyngeal swabs, bronchial washings, vaginal, cervical and urethral swabs, and stool or rectal swabs

    Transport and Processing

    • Inoculate media with the specimen and transfer them to anaerobic conditions immediately

    • Limit exposure to the air

    • Do not let the specimen dry out

    • Do not refrigerate

    • Minimize time at room temperature

    • PRAS media (pre-reduced, anaerobically sterilized transport medium) using agar transport medium (modified Cary-Blair or Amies media) includes a rezasurin (oxygen indicator), reducing substances to protect against oxygen, and a sterile container in transport.

    Culture

    • Specimens are cultured aerobically on various media (BAP, CHOC, MAC) initially and then subsequently on anaerobic media to determine aerotolerance
    • Anaerobic bacteria require vitamin K, hemin, and yeast extract in culture media

    Nonselective Media

    • CDC or Anaerobic Blood Agar Plates (anaBAP) support growth of obligate and facultative anaerobes
    • Anaerobic Broth, Thioglycollate or chopped (cooked) meat support growth of obligate and facultative anaerobes, revealing aerobic, anaerobic, and microaerophilic bacteria from the growth patterns

    Selective Media

    • Phenylethyl alcohol blood agar supports growth of gram-positive and gram-negative obligate anaerobes, but it inhibits enteric gram-negative rods (GNR)
    • Colistin nalidixic acid blood agar is similar in function, supporting facultative anaerobic and obligate anaerobic GNRs, but it inhibits enteric gram-negative rods (GNR)
    • Bacteroides Bile-Esculin (BBE) agar is used for selective and is differential for esculin hydrolysis. It supports growth of bile-tolerant organisms like Bacteroides fragilis
    • Kanamycin-vancomycin laked blood agar selects for Bacteroides and Prevotella bacteria. Kanamycin inhibits most facultative GNRs, and vancomycin inhibits gram-positive bacteria.
    • Cycloserine-cefoxitin-fructose agar supports, is selective, and is differential for Clostridium difficile. 

    Anaerobic Incubation

    • Anaerobic cultures are typically incubated between 35–37 °C for up to 48 hours.
    • The anaerobic atmosphere contains nitrogen gas (80–90%), hydrogen gas (5–10%), and carbon dioxide (5–10%). Nitrogen and hydrogen remove oxygen while CO2 assists select anaerobes.

    Anaerobic Systems

    • Anaerobic jars, bags, or chambers are used to create an anaerobic environment (inert gas atmosphere) for microbial cultivation.

    Anaerobic Jars

    • The jar technique and GasPak jar employs a catalyst (palladium pellets), generating hydrogen (H2) and carbon dioxide (CO2) by the addition of water, sealing, and incubation at 35°C. Methylene blue or resazurin indicators turn white when reduced (anaerobic) and turn blue when oxidized (aerobic).

    Anaerobic Bags

    • Commercially available anaerobic bags hold 1–3 plates and contain an oxygen removal system, an indicator, and a gas impermeable plastic bag

    Anaerobic Chamber

    • Anaerobic chambers provide an oxygen-free environment using a palladium catalyst, desiccant, indicator, and anaerobic gas mixture

    Culture Examination

    • Examine the cultures in the anaerobic environment after a 48-hour incubation in jars or bags. Examine and process cultures quickly to minimize oxygen exposure.

    Anaerobic Identification Tests

    • Gram stains, morphology of colonies, and rapid tests (e.g., catalase, motility) are used in preliminary identification
    • This helps narrow down the possibilities to specific genera and species.

    Anaerobes Gram Stain RXN

    • A Gram stain is used initially to determine if the bacteria are gram-positive or gram-negative rods. 
    • This process is used to quickly separate bacteria initially based on their cell wall characteristics.

    Rapid Identification Tests

    • Aerotolerance, fluorescence, and disk tests are commonly used
    • Lecithinase, Naglar test, and lipase tests are also frequently used during rapid identification

    Aerotolerance

    • Aerotolerance tests determine whether the isolated organism is a strict anaerobe, aerotolerant anaerobe, or a facultative anaerobe. 
    • The isolation is cultured aerobically and anaerobically on both blood agar and chocolate agar

    Fluorescence

    • Organisms fluoresce under ultraviolet light with a wavelength of 366 nanometers (nm) in varying shades of red, orange, pink, or chartreuse.

    Antibiotic Disk Test

    • Confirmation of the Gram stain is performed by testing the susceptibility to antibiotics including kanamycin, vancomycin, and colistin

    Disk Tests

    • Sodium polyanethol sulfonate (SPS) disk can identify Peptostreptococcus, which are sensitive to this agent. 
    • Tests for nitrate reduction and bile tolerance can also be used

    Lecithinase

    • Lecithinase tests detect the presence of Clostridium using a medium (Egg Yolk Agar) with lecithin that produces insoluble fat that creates an opaque zone in positive reactions. 

    Nagler Test

    • The Nagler reaction is frequently used to detect alpha toxin of C. perfringens. An antiserum to this specific toxin neutralizes the alpha form of lecithinase. 

    Lipase

    • Lipase tests hydrolyze triglycerides in Egg Yolk Agar (EYA) producing glycerol and fatty acids.

    Reverse CAMP

    • The reverse CAMP test identifies S. agalactiae bacteria in association with C. perfringens by observing an arrowhead-shaped zone. 

    Definitive Identification Tests

    • Biochemical tests, commercial systems, gas-liquid chromatography, cellular fatty acid analysis, and 16S rRNA gene sequencing are used

    Biochemical and Commercial Systems

    • PRAS or non-PRAS biochemical test media and preexisting commercial biochemical and preexisting bacterial enzymes minisystems are used to quickly identify the species. 

    Gas-Liquid Chromatography

    • Cellular fatty acids or metabolic end products may be analyzed via GLC or HPLC, producing patterns unique to each bacterial species. 

    Gene Sequencing

    • 16S rRNA gene sequencing can identify the sequence and compare the sequence to other specimens in a database for confirmation and final identification

    Clostridium Species

    • These bacteria are catalase negative but are motile (except C. perfringens) and are gram-positive or gram-variable/gram-negative rods
    • They are susceptible to vancomycin and the spores appear as unstained or refractile structures with oval, round, or subterminal locations
    • Some Clostridia are aerotolerant. They tend to cause exogenous infections by entering the body via ingestion or wounds 
    • Common diseases from some Clostridia include tetanus, gas gangrene, botulism, and food poisoning

    Clostridium perfringens

    • C. perfringens are the most common Clostridium species. They are typically found in undercooked meat, soil, and water. 
    • They are gram-positive boxcar-shaped rods; often show a double beta hemolysis zone on anaerobe blood agar (anaBAP)
    • They show positive Reverse CAMP reactivity, lecithinase, and Nagler reactivity. 
    • Spores are not commonly observed in this species. 
    • C. perfringens infections lead to diseases such as myonecrosis (gas gangrene) or food poisoning 

    Clostridium difficile

    • C. difficile can be normal flora of stool
    • C. difficile infections arise when antibiotics kill normal flora. This leads to an increase in C. difficile, sometimes causing antibiotic-associated diarrhea or pseudomembranous colitis. 
    • C. difficile produces two toxins (A and B).
    • C. difficile produces characteristic yellow ground glass colonies on cycloserine-cefoxitin-fructose agar (CCFA). 

    Clostridium botulinum

    • C. botulinum produces botulism toxin. This causes neurotoxin-related paralysis. 
    • This may be caused by ingesting home canned veggies, cured meats, or honey.

    Clostridium tetani

    • C. tetani produces the neurotoxin tetanospasmin.
    • Individuals often contract the disease when the organism enters the body through a wound site.
    • Tetanus (lockjaw) is the primary disease caused by C. tetani.

    C. septicum

    • C. septicum are aerotolerant, produce swarming colonies with subterminal spores, and are associated with myonecrosis and bacteremia, commonly in individuals with leukemia, lymphoma, or large bowel carcinoma. 

    Non-Spore-Forming Anaerobic GPR

    • Actinomyces, Bifidobacterium, Propionibacterium, Eggerthella, and Eubacterium spp. are common, non-spore-forming, anaerobic gram-positive rods (GPR). These bacteria commonly cause actinomycosis.

    Actinomyces

    • Actinomyces spp. includes some aerobic and anaerobic bacteria. Nocardia is an aerobic form of Actinomyces. Actinomyces israelii is a common anaerobe responsible for actinomycosis. 
    • Actinomyces spp. are commonly found in human and animal mucosal surfaces. Nocardia spp. are not weakly acid-fast but Actinomyces spp. are.

    A. israelii Actinomycosis

    • Actinomyces israelii commonly causes actinomycosis, a condition typically in the jaw. 
    • It creates fistulae, draining pus with granules from the bacteria.

    A. israelii Cultures

    • The bacteria are associated with "lumpy jaw." Cultures may show pus and sulfur granules grown on anaBAP, demonstrating slow growth (7–9 days).
    • Colonies display a molar-tooth pattern.
    • Gram-positive rods displaying anaerobic branching are visible.

    Bifidobacterium

    • Bifidobacterium are anaerobic gram-positive rods with an Actinomyces-like morphology and sometimes bifurcated GPR shape. 
    • They are in the normal flora of the intestines and oral cavity and may cause actinomycosis.

    Propionibacterium

    • Propionibacterium are anaerobic, diphtheroid-like, gram-positive rods somewhat resembling corynebacteria.
    • A common component in specimens and the contaminants in blood cultures, they are in skin flora and linked to conditions like acne (P. acnes). 
    • Their identification often displays catalase and indole positivity. They may also cause subacute bacterial endocarditis (SBE).

    Eggerthella and Eubacterium spp.

    • Eggerthella, previously Eubacterium, are anaerobic diphtheroid-like gram-positive rods; they lack branching

    Anaerobic GPC

    • Peptostreptococcus are gram-positive cocci (GPC) in chains 
    • Peptostreptococcus anaerobius is susceptible to sodium polyanethol sulfonate (SPS) disks, whereas all other anaerobic cocci and resistant
    • Peptococcus are gram-positive cocci (GPC) in clusters

    Anaerobic GNR

    • Bacteroides, Prevotella, Prophyromonas, Fusobacterium, and Veillonella are anaerobic GNR commonly found in normal flora in the mucous membranes.

    Bacteroides fragilis Group

    • Bacteroides fragilis is the most common anaerobic gram-negative rod (GNR) in normal flora, often in the GI tract 
    • The group displays bile resistance and resistance to kanamycin, vancomycin, and colistin disks in laboratory settings
    • Bacteroides thetaiotaomicron is the second most common bacterium in this group. It is associated with intra-abdominal infections, bacteremia, and soft tissue infections

    Bacteroides ureolyticus Group

    • Bacteroides ureolyticus is an anaerobic gram-negative rod (GNR). It displays bile sensitivity and has a nonpigmented morphology.
    • Organisms in this group may display pitting of agar after growth, and they grow in formate and fumarate. They are resistant to vancomycin but sensitive to kanamycin and colistin

    Prevotella

    • Prevotella is an anaerobic gram-negative rod (GNR). 
    • It is bile-sensitive and resistant to kanamycin and vancomycin.
    • Growth occurs on KVLB agar, but not BBE agar. Sometimes it produces protoporphyrin, displaying dark-pigmented colonies fluorescing a "brick red" under UV light.

    Porphyromonas

    • Porphyromonas are anaerobic gram-negative rods that depend on hemin and vitamin K, are resistant to kanamycin and colistin, are bile and vancomycin-sensitive, and do not grow on KVLB

    Fusobacterium

    • Fusobacterium are anaerobic gram-negative rods that fluoresce chartreuse.
    • The bacterium, designated F. nucleatum, shows fusiform narrow rods, demonstrating a bread-crumb or speckled colony. F. necrophorum also displays lipase positivity.

    Mobiluncus

    • These organisms are associated with bacterial vaginosis (BV), pelvic inflammatory disease (PID), and abdominal infections.
    • Curved bacilli display gram variability and are characterized by motility, negative catalase and indole reactions, and inhibition by vancomycin

    Spirochetes

    • Spirochetes are long, slender, spiral-shaped bacteria that are motile. They are notable for their morphology, not seen on Gram stains, require silver stains, and are best observed on darkfield or phase microscopy

    Borrelia

    • Borrelia species are transmitted by arthropods, typically ticks and lice, and are associated with 3-10 spirals per organism.
    • Borrelia burgdorferi is a causative agent of Lyme disease. 

    Borrelia recurrentis

    • Borrelia recurrentis causes relapsing fever, characterized by recurring episodes of spirochetemia, fever, headache, and muscle pain. 
    • Relapses occur due to antigenic variation, a mechanism of evading the immune system.

    Relapsing Fever Lab Tests

    • Microscopic examination of blood samples when patients have a fever is used to detect organisms during the febrile periods with Giemsa or Wright’s stains. 
    • Culturing is rare and serology is impractical due to antigenic variation.

    Borrelia burgdorferi

    • Borrelia burgdorferi are transmitted by ticks (Ixodes), especially from deer or mice ticks, and cause Lyme disease in north-east regions of the US

    Ixodes

    • Ixodes are the vector for Borrelia, specifically B. burgdorferi, that is responsible for Lyme disease. The various life cycles of this tick (larva, nymph, female, male) lead to different human disease manifestations

    3 Stages of Lyme Disease

    • Stage 1 is characterized by the initial appearance of Erythema chronicum, migrans (ECM), with a target lesion or bull's eye appearance. Additionally, lymphadenopathy and symptoms resembling the flu are also experienced.

    • Stage 2 is the stage of dissemination and presents with fever, bone pain, and joint pain. In addition, splenomegaly and malaise are typical presentations.

    • Stage 3 is the later or persistent infection stage usually appearing months to years after the initial infection. This may lead to chronic arthritis, neurologic defects, meningioencephalitis, or cardiac problems

    Lyme Disease Lab Tests

    • Serologic tests are usually used, focusing on antibody detection using a rapid plasma reagin (RPR) test.
    • Microscopic examination of skin or blood, or cultures, are uncommon for determining this bacteria. 

    Leptospira

    • L. biflexa is a non-pathogenic species of spirochete, whereas L. interrogans is.
    • Animal pathogens can spread leptospires to humans via water contaminated with animal urine. 

    Leptospira interrogans

    • L. interrogans is characterised by spirals with hooked ends, tightly coiled morphology, and transmitted via various animal sources.

    Leptospirosis

    • Leptospirosis is a zoonotic infection transmitted from infected animals (dogs, rats, cattle) via urine-contaminated water.
    • Leptospires can enter the body via breaks in the skin or intact mucosa that contract the bacteria.
    • Manifestations of this disease vary in severity from asymptomatic to severe.

    Treponema

    • Treponemes are characterized by 4–14 spirals per organism. Four pathogenic organisms exist.

    Treponema pallidum subspecies pallidum

    • Venereal syphilis is caused by T. pallidum subsp. pallidum. These organisms appear as a variety of clinical presentations that are commonly transmitted sexually or via nongenital lesions.
    • Congenital syphilis may also occur and displays three stages of disease progression (primary, secondary, tertiary)

    Primary Syphilis

    • Primary syphilis is characterized by the development of painless chancres at the inoculation site appearing days to months after infection.
    • The lesion is infectious.

    Secondary Syphilis

    • Secondary syphilis appears 2–12 weeks after the primary lesion. It presents with systemic dissemination throughout the body with associated fever, lymphoadenopathy, and headache.
    • Secondary syphilis is characterized by the appearance of widespread unusual rashes on the skin and mucous membranes, and these lesions may include the palms and soles.

    Tertiary or Late Syphilis

    • Tertiary or late syphilis occurs in individuals untreated for up to one-third of the cases from the initial infection
    • Tertiary syphilis frequently infects various body sites, such as the skin, liver, and bones to cause granulomatous lesions (gummas)
    • Individuals may experience neurologic deficits such as deafness, blindness, partial paralysis, shuffling gait and/or mental disturbances
    • Cardiovascular issues may also appear, including syphilitic aortitis

    Early-Onset Congenital Syphilis

    • This occurs when the mother has early-stage syphilis and treponemes cross the placenta. 
    • Infection manifests in the mother and fetus with numerous body system infections, typically causing the death of the infected fetus.

    Late-Onset Congenital Syphilis

    • This occurs when a mother has untreated chronic syphilis and symptoms typically develop in children after two years of age. These symptoms may include deafness, blindness, mental retardation, bone deformities, or tooth malformations.

    Serologic Laboratory Diagnosis

    • Treponemal tests (e.g., EIA, TPPA, FTA-ABS) specifically identify treponemal-specific antibodies. Nontreponemal tests (e.g., VDRL, RPR) screen for antibodies against treponemal lipids such as cardiolipin-lecithin.

    Nontreponemal Tests

    • Nontreponemal tests screen using reagins (antibodies) to treponemal lipids such as cardiolipin-lecithin from released antigens during treponemal infection
    • These tests are fairly sensitive, but their lack of specificity leads to several false positives. False positives may occur in Lyme disease, certain viral infections, autoimmune diseases, or pregnancy.

    VDRL and RPR

    • Visual observations of rapid plasma reagin (RPR) and Venereal Disease Research Laboratory (VDRL) tests commonly display the presence or absence of treponemal infection

    Treponemal Tests

    • Treponemal tests detect treponemal-specific antibodies and confirm the presence of infection. Treponemal tests may remain positive even after treatment.

    TPPA

    • TPPA test results are used to distinguish the presence or absence of antibodies specific for Treponema pallidum bacteria in patients.

    Direct Microscopic Exam

    • Darkfield microscopy is used in the direct microscopic exam, focusing on looking for corkscrew-shaped treponemes from fluids of syphilitic lesions. 
    • Oral lesions are typically not examined because nonpathogenic treponemes may also appear with this morphology.

    Other Pathogenic Treponemes

    • Other treponemal infections, like endemic syphilis, yaws, and pinta, are associated with distinct clinical symptoms, geographic regions, and specific laboratory features that may not be distinguished by existing laboratory tests

    Mycobacteria

    • Mycobacteria are aerobic, non-spore-forming bacteria that are notable for their high lipid content (mycolic acids) in their cell walls, which interferes with their staining characteristics (gram-stain well);
    • cultures typically require increased CO2 and are held for 6 weeks before being considered negative. 
    • Mycobacteria are slim gram-variable rods; are not stainable via traditional methods but have beaded or rod-like structures visible after acid-fast staining.

    Mycobacteria Safety

    • Tuberculosis infections are associated with increased prevalence and should be considered dangerous
    • Laboratory safety and security protocols like separate labs, nonrecirculating ventilation, negative air pressure environments, and biosafety cabinets are needed. 
    • Specimens should be rigorously handled with appropriate procedures (double sealing, gloves) in BSL2 or BSL3 biological safety cabinets.

    Mycobacteria Species

    • The major pathogens include M. tuberculosis complex, nontuberculous mycobacteria (NTM), M. leprae (Leprosy)

    Specimen Collection

    • Samples may include sputum, bronchial washings, gastric contents, urine, stool, and tissue.
    • Collection protocols may vary based on the source site or patient factors; early morning samples are often preferred.
    • If sputum cannot be obtained, a bronchoscopy or biopsy can be conducted

    Digestive and Decontamination

    • Samples requiring digestion (specimens with normal flora such as urine or gastric aspirates) are usually decontaminated with N-acetyl-L-cysteine (NALC).
    • Contaminated samples/specimens may also be decontaminated with sodium hydroxide (NaOH) before analysis

    Specimen Processing

    • Mycobacterial organisms may be clumped and/or in clumps of protein, and NALC (mucolytic agent) is useful in digestion. Decontamination isolates normal flora for subsequent processing.
    • Concentration methods including centrifugation for 20 minutes at 3000 rpm are used to identify organisms.

    Staining for AFB Smear

    • Mycobacteria stains using a Ziehl Neelsen procedure (Heat-stable method)
    • In-vitro use of a Kinyoun method (Cold method) staining for AFB microbes; the carbolfuchsin staining procedure is employed for both approaches. A counterstain with methylene blue will visually confirm the presence of acid-fast bacteria.

    AFB Culture Media

    • Solid media are based on eggs (Lowenstein-Jensen) or agar media (Middlebrook 7H10 and 7H11) or fluid media (Tween 80 or instrumentation broth).

    Solid Egg-based Media

    • Lowenstein-Jensen media is typically 60% egg in a nutrient base, and it includes Malachite green to prevent Gram-positive bacteria growth

    Solid Agar-Based Media

    • Middlebrook 7H10, 7H11, and Mitchison’s 7H11, Mycobactosel-Middlebrook 7H11 media contain nutrients, albumin, and a low concentration of malachite green, with antibiotics added to make it a selective culture

    Culture Examination

    • Examined twice weekly for 4 weeks and once thereafter for agar-based specimens
    • Microscopic examination (ranging from 4x to 10x) and observation of microcolonies using hand-lens is recommended

    Liquid Media

    • Tween 80, a surfactant, disperses mycobacteria in liquid media, enabling them to grow faster

    Instrumentation - Liquid Media

    • Automated methods rapidly detect AFB growth. 
    • Common methods include the MB/BacT system using colorimetry or the ESP myco system via a manometric method that measures the pressure change from a gas mixture in the specimen as the bacteria consume or generate the gas.

    Approach to ID AFB

    • Positive AFB growth (liquid or solid media) is initially confirmed by performing an acid-fast stain
    • More cumbersome confirmation methods include biochemical tests and lipid analysis of the mycolic acid in the cell wall.

    Growth Parameters

    • Mycobacterial growth is often categorized as rapid growers (visible growth within 7 days), or slow growers (growth taking longer, ≥7 days), which assists in preliminary categorization and classification 
    • Colony morphology varies from smooth to buff and cords to “cauliflower-like” colonies grown on Lowenstein-Jensen (LJ).

    Photoreactivity

    • Light can influence color production in some Mycobacteria colonies.

    Identification Testing

    • Biochemical tests including catalase, niacin, nitrate, Tween-80 hydrolysis, and tellurite reduction 
    • Miscellaneous tests, such as N, AP growth on MAC, and growth temperature, are used to distinguish different bacteria based on a variety of factors.

    Catalase

    • Suspension of the specimen is tested for catalase activity at 68°C or at room temperature. If bubbles occur after the addition of Tween-80 and hydrogen peroxide, it is positive for catalase. 

    Niacin

    • M. tuberculosis lacks the ability to convert free niacin into niacin ribonucleotide, causing accumulation of niacin in the medium.
    • The conversion results in a color change; typically a visible yellow color upon reaction with cyanogen bromide 

    Nitrate Reduction

    • MTB reduces nitrate to nitrite or nitrogen gas via the addition of sulfanilamide and N-naphthylenediamine dihydrochloride after inoculation of isolates into a substrate. 
    • Colorimetric testing differentiates the results into negative (red color persisting) and positive (loss of red color, likely transformation into a colorless component) results

    Tween 80 Hydrolysis

    • Common nonpathogenic mycobacteria are capable of hydrolyzing Tween-80, which leads to a color change from amber (negative result) to red (positive result)

    Tellurite Reduction

    • The reduction of tellurite to tellurium produces black colonies for positive results. The reduction rate in mycobacteria can vary, and in some species, takes only three days .

    Growth Inhibition

    • Tests differentiating TB from other mycobacteria include reagents such as P-nitroacetylamino-β-hydroxypropiophenone (NAP) and Thiophene-2-carboxylic acid hydrazide (T2H). These reagents may inhibit mycobacterial growth.

    Growth on Special MAC

    • Media without crystal violet is employed to detect organisms capable of growing on Middlebrook Agar (MAC) medium

    Growth Temperatures

    • NTM and AFB bacteria growth may be observed at different temperatures including 24°C, 30°C, 37°C, and 42°C

    Classification of Mycobacteria

    • Mycobacteria can be classified into several groups including M. tuberculosis complex (MTC), nontuberculous mycobacteria (NTM), or M. leprae

    M. tuberculosis Complex

    • M. tuberculosis (most common), M. bovis, M. africanum, M. canettii, and M. microti are all components of the M. tuberculosis complex and cause tuberculosis (TB)
    • TB is commonly transmitted via airborne respiratory droplets, commonly nonpigmented colonies, and are slow growers

    M. tuberculosis Complex

    • Mycobacterium bovis is a component of the tuberculosis complex commonly transmitted via drinking unpasteurized milk, and it causes atypical tuberculosis, but it is rarely observed in modern times.

    M. tuberculosis Complex

    • Colony morphology shows buff, rough colonies with serpentine cords 
    • It likely demonstrates positive niacin/nitrate positivity tests, negative 68°C catalase test results, and susceptibility to reagents such as NAP and T2H for identification.

    Runyon NTM Classification

    • The Runyon NTM classification divides nontuberculous mycobacteria into four groups:
      • I Photochromogens
      • II Scotochromogens
      • III Nonphotochromogens
      • IV Rapid growers

    I Slow-Growing Photochromogens

    • M. kansasii, in the first group, commonly causes pulmonary disease

    I Slow-Growing Photochromogens

    • M. marinum is associated with swimming-pool granulomas, appearing as a light-sensitive species.

    II Slow-Growing Scotochromogens

    • M. scrofulaceum and M. gordonae are two species associated with mycobacterial cervical (neck) lymphadenitis.  M. gordonae is an example of a tap water bacillus.

    III Slow-Growing Nonphotochromogens

    • *M. avium complex (including M. avium and M. intracellulare) are associated with an increase in infections due to AIDS infection; 68°C catalase is positive in some cases

    III Slow-Growing Nonphotochromogens

    • M. ulcerans is often found in skin ulcers and is a slow-growing nonphotochromogen

    IV Rapid-Growers

    • M. fortuitum, M. chelonae, and M. smegmatis are all rapid growers associated with environmental organisms.

    Uncultivable

    • M. leprae, the organism causing leprosy, is uncultivable in standard laboratory procedures, and the identification is generally determined using clinical factors and microscopic observation

    Other Pathogenic Species

    • M. genavense, M. simiae, M. xenopi, and M. szulgai are examples of pathogenic, nontuberculosis (NTM) mycobacteria.

    Newer Identification Tests

    • GLC (gas-liquid chromatography) and HPLC (high-performance chromatography) are used to analyze the fatty acid composition in cell walls of unknown strains for distinguishing them from several types of bacteria based on their unique lipid profiles
    • Molecular identification techniques include nucleic acid hybridization and nucleic acid amplification that may improve detection speed and diagnostic accuracy.

    Antimicrobial Susceptibility Tests

    • Susceptibility tests for M. tuberculosis measure the effectiveness of a variety of antimicrobial drugs against the bacteria, including isoniazid, rifampin, ethambutol, and streptomycin

    Susceptibility Testing Methods

    • Resistance ratios are compared using the minimum inhibitory concentration (MIC), which determines the smallest concentration of a drug required to stop the growth of the bacteria, from test subjects in the laboratory study.
    • Proportional resistance methods predict resistance of a specific bacterial strain based on lab strain results for resistance. 

    Antimicrobial Susceptibility Tests

    • In cases of M. tuberculosis, drug therapy is used to attempt to prevent multi-drug resistant (MDR) strains by administering isoniazid (INH) and rifampin over a nine-month period. Susceptibility tests are not often done on nontuberculous mycobacteria (NTM) microbes; the tests may be completed only on rapid growers.

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