Clinical Bacteriology: Aerobic Non-Spore Forming Gram-Positive Bacilli (Mycobacteria)

Comprehensive practice flashcards covering clinical bacteriology of aerobic non-spore forming Gram-positive bacilli (Mycobacterium tuberculosis complex, nontuberculous mycobacteria, staining, decontamination, culture media, automated systems, biochemical identification, and leprosy).

Which four bacterial genera are closely related to the genus Mycobacterium?

Rhodococcus, Nocardia, Tsukamurella, and Gordonia (Gordonica). RN TG

What are the cellular dimensions and general morphology of Mycobacterium species?

Slender, slightly curved or straight, rod-shaped bacilli measuring $0.2\text{ to }0.6\,\mu\text{m} \times 1\text{ to }10\,\mu\text{m}$.

What are the motility, spore-forming, and capsule characteristics of mycobacteria, and what is the single exception regarding spore formation?

They are nonmotile, non-encapsulated, and non-spore-forming, with the exception of Mycobacterium marinum, which demonstrates spore or spore-like structures.

Which specific cell wall components are responsible for the acid-fastness of mycobacteria?

N-glycolylmuramic acid and an exceptionally high lipid content consisting of mycolic acids.

What are the oxygen requirement and catalase status shared by all mycobacteria?

They are strictly aerobic and catalase positive.

What are MUCH's granules, and how do they behave during staining?

They are non-acid-fast granules that stain Gram-positive, giving the mycobacterial bacillus a beaded appearance.

What microscopic cell arrangements are typically formed by mycobacteria?

Palisades or snapping formations resembling letters such as X, Y, V, and L.

What is the optimal pH requirement for culturing mycobacteria?

$6.5\text{ to }6.8$.

What is the general generation time for mycobacteria, and specifically for Mycobacterium tuberculosis?

The general generation time is greater than $12\,\text{hours}$, and for Mycobacterium tuberculosis it is $20\text{ to }22\,\text{hours}$.

What is the recommended container for collecting and transporting mycobacterial specimens?

A sterile, wide-mouth cup equipped with a tightly fitted lid.

What protocol must be followed if processing of a mycobacterial specimen is delayed for more than $1\,\text{hour}$, and which specimen is the exception?

The specimen must be refrigerated at $4^\circ\text{C}$, with blood being the sole exception.

What is the preferred routine specimen and collection schedule for diagnosing pulmonary tuberculosis?

An early-morning, spontaneously expectorated sputum specimen collected on 3 consecutive days.

What volume of sputum is required for diagnostic mycobacterial processing?

$5\text{ to }10\,\text{mL}$.

What microscopic criteria indicate an acceptable sputum specimen quality for AFB testing?

Fewer than $10$ squamous epithelial cells ($<10\,\text{SEC}$) and greater than $25$ pus cells ($>25\,\text{pus cells}$) per low-power field.

What smear microscopy outcome from the first three sputum direct smears is needed to confirm a diagnosis of tuberculosis?

At least two of the first three sputum direct smears must be positive.

For which patient group is gastric lavage or aspirate the specimen of choice, and what is its physiological basis?

Children younger than 3 years of age, as well as senile or non-ambulatory patients who swallow respiratory secretions rather than expectorating sputum.

What volume, collection timing, and chemical treatment are required for gastric aspirate specimens?

A volume of $20\text{ to }25\,\text{mL}$ collected early in the morning following an overnight fast before exertion empties the stomach, neutralized immediately with sodium carbonate or buffer to pH $7.0$.

What is the collection regimen and minimum required volume for urine mycobacterial culture?

First-morning midstream urine collected daily for at least 3 days, with a minimum volume of $15\,\text{mL}$ (or the entire volume up to $40\,\text{mL}$).

What is the clinical utility of submitting a stool specimen for mycobacterial evaluation, and what pre-analytical condition must be observed?

It identifies patients with AIDS who are at risk for or suffering from disseminated Mycobacterium avium complex (MAC); it must be collected without preservative and strictly free of urine contamination.

Which blood culture systems and methods are recommended for isolating mycobacteria in cases of disseminated MAC infection?

The Isolator lysis-centrifugation system, BACTEC MGIT 960 system, BacT/ALERT 3D (or BacT/Alert MB Blood medium), and MYCO/F bottle.

What are the required diagnostic specimen volumes for cerebrospinal fluid (CSF), synovial fluid, and peritoneal fluid?

$2\,\text{mL}$ for CSF; $3\text{ to }5\,\text{mL}$ for synovial fluid; and $10\text{ to }15\,\text{mL}$ for peritoneal fluid.

What is the specimen of choice for a cutaneous tuberculous lesion or wound, and how is dehydration prevented during transport?

An aspirate is the specimen of choice, maintained by adding $10\text{ to }15\,\text{mL}$ of sterile normal saline solution (NSS).

What are the two primary objectives of the digestion and decontamination procedure?

To liquefy proteinaceous material (mucin) so mycobacteria can access medium nutrients, and to chemically eliminate contaminating non-mycobacterial flora.

Which three variables directly govern the efficacy of digestion and decontamination?

Concentration of the chemical agent, duration of exposure (time), and temperature.

What dual role does $2\%\text{ to }4\%\,\text{NaOH}$ serve in specimen processing, and in what ratio is it routinely added to sputum?

It acts as both a digestant and a decontaminating agent, routinely mixed in equal parts ($2\,\text{mL}$ sputum to $2\,\text{mL}$ NaOH).

What are the distinct functions of each component in the N-acetyl-L-cysteine-sodium hydroxide (NALC-NaOH) method?

NALC (or dithiothreitol) acts as the mucolytic liquefying agent, while NaOH acts as the chemical decontaminant.

In the Zephiran-trisodium phosphate (Z-TSP) method, what are the roles of trisodium phosphate and benzalkonium chloride?

Trisodium phosphate (TSP) rapidly liquefies sputum, and benzalkonium chloride (Zephiran) shortens exposure time while destroying contaminants.

Which decontaminating reagent is specifically indicated for specimens known to contain Pseudomonas aeruginosa?

$5\%\,\text{Oxalic acid}$.

What chemical reagent can preserve sputum specimens for up to $8\,\text{days}$ during transport?

$1\%\,\text{Cetylpyridinium chloride}$.

Within what specific gravity (SG) range must centrifugation be calibrated during mycobacterial specimen concentration?

$0.79\text{ to }1.07$.

What is the minimum concentration threshold of acid-fast bacilli required per milliliter to yield a positive smear on concentrated specimens?

At least $10^4\,\text{AFB/mL}$ (or $5,000\text{ to }10,000\,\text{AFB/mL}$).

What are the decolorizer and counterstain used in the Auramine-rhodamine fluorochrome staining procedure?

The decolorizer is acid alcohol, and the counterstain is potassium permanganate.

What mandatory confirmation must be performed following a positive fluorochrome smear result?

Confirmation using the Ziehl-Neelsen staining method performed directly on the same smear.

What laboratory precautions must be observed during AFB staining and microscopy to prevent cross-contamination?

Smear slides must never touch each other, staining jars must not be used, and the oil immersion objective (OIO) must be wiped between slides.

List four recognized causes of false-positive acid-fast smear microscopy results.

Cell wall alterations, insufficient decolorization, laboratory contamination, and delayed processing allowing overgrowth of saprophytic organisms.

List four recognized causes of false-negative acid-fast smear microscopy results.

Overzealous decontamination, loss of organisms during concentration, overly thick smears obscuring bacilli, and over-decolorization.

Under the CDC/standard reporting scale using the Ziehl-Neelsen method ($1000\times$ magnification), what is the interpretation of observing $1\text{ to }2\,\text{AFB}$ across $300\,\text{fields}$?

'Doubtful; request another specimen.'

Under the CDC/standard Ziehl-Neelsen ($1000\times$ magnification) scale, what quantitative findings correspond to reports of $1+, 2+, 3+,$ and $4+$?

$1-9\,\text{AFB}/100\,\text{fields} = 1+$; $1-9\,\text{AFB}/10\,\text{fields} = 2+$; $1-9\,\text{AFB}/\text{field} = 3+$; and $>9\,\text{AFB}/\text{field} = 4+$.

In Xpert MTB/RIF reporting, what does the notation 'RR' indicate?

'MTB detected, Rifampicin resistance detected.'

In what ink color must Xpert MTB/RIF results indicating 'MTB Detected' or 'Rifampicin resistance detected' be documented?

Red ink.

What are the standard incubation parameters (temperature, atmosphere, light) for recovering mycobacteria on culture media?

$35^\circ\text{C}$, in the dark, under an atmosphere of $5\%\text{ to }10\%\,\text{CO}_2$ with high humidity.

How must tubed slant media be handled during the first week of incubation, and why?

Incubated in a slanted position with screw caps loosened for $1\,\text{week}$ to allow excess moisture to evaporate and permit entry of $\text{CO}_2$.

What are the basal components of egg-based mycobacterial media, and what specific role does egg yolk play?

Fresh whole eggs, potato flour, and glycerol; egg yolk serves as a vital lipid source that stimulates mycobacterial growth.

What is the concentration and inhibitory role of malachite green in Lowenstein-Jensen (L-J) medium?

$0.025\%$, which suppresses the growth of contaminating Gram-positive and non-mycobacterial bacteria.

How does Petragnani medium differ from standard Lowenstein-Jensen medium, and when is it indicated?

It contains a higher concentration of malachite green ($0.052\%$), egg yolks, and whole milk, making it ideal for heavily contaminated specimens.

What medium modification is required to enhance the recovery of Mycobacterium bovis, and what additive must be excluded?

Supplementation with pyruvic acid enhances growth; glycerol must be omitted because it selectively inhibits Mycobacterium bovis.

What specific growth factors and culture conditions are required for Mycobacterium haemophilum?

It requires hemin, hemoglobin, or ferric ammonium citrate (e.g., chocolate agar or X-factor disks) and an incubation temperature of $28^\circ\text{ to }32^\circ\text{C}$ ($25^\circ\text{ to }33^\circ\text{C}$).

What unique nutritional supplement is added to Middlebrook 7H11 agar, and what clinical strain does it support?

$0.1\%\,\text{casein hydrolysate}$, which permits the recovery of isoniazid-resistant strains of Mycobacterium tuberculosis.

What is the detection principle utilized in radiometric BACTEC 12B and 13A broth cultures?

Metabolism of $^{14}\text{C}$-labeled palmitic acid by mycobacteria, which liberates radioactive $^{14}\text{CO}_2$ gas detected by the BACTEC 460TB instrument.

What antibiotics constitute the PANTA mixture used in BACTEC broth media?

Polymyxin B, amphotericin B, nalidixic acid, trimethoprim, and azlocillin.

What are the average detection times for Mycobacterium tuberculosis versus NTM when grown in the BACTEC system?

$9\text{ to }14\,\text{days}$ for Mycobacterium tuberculosis, and fewer than $7\,\text{days}$ ($<7\,\text{days}$) for NTM.

What components make up the Septi-Chek AFB biphasic culture system?

A liquid phase containing $20\,\text{mL}$ of Middlebrook 7H9 broth under $20\%\,\text{CO}_2$, and a solid phase slide paddle holding modified L-J, Middlebrook 7H11, and chocolate agar.

How does the automated Mycobacteria Growth Indicator Tube (MGIT 960) system detect microbial growth?

Using a fluorescence-quenching oxygen sensor embedded in the bottom of a tube of modified Middlebrook 7H9 broth, which fluoresces as growing organisms consume oxygen.

What detection principles are employed by the MB/BacT Alert 3D and ESP Culture System II systems, respectively?

MB/BacT Alert 3D uses a colorimetric carbon dioxide sensor; ESP Culture System II (VersaTREK) detects head-space pressure changes.

What biochemical test for mycobacterial identification is demonstrated in this image, and what does a positive result (yellow color) indicate?

Niacin accumulation test; a positive result (yellow color) indicates the accumulation of nicotinic acid (niacin), characteristic of Mycobacterium tuberculosis.

What specific culture requirements must be met before performing a niacin accumulation test?

The culture must be grown on Lowenstein-Jensen medium, be at least $3\,\text{weeks}$ old, and have at least $50\,\text{colonies}$.

Which biochemical test is depicted in this tube series showing varying shades of pink and red, and which major mycobacterial species yield a positive reaction?

Nitrate reduction test; positive (red color) reactions are seen in Mycobacterium tuberculosis, Mycobacterium kansasii, Mycobacterium szulgai, and Mycobacterium fortuitum.

In the nitrate reduction test, what is the role of adding zinc dust when no red color develops after adding reagents?

Zinc dust serves as a reducing agent to confirm a true negative; if unreduced nitrate is present, zinc forces reduction to nitrite, turning the broth red (confirming the test was negative).

What test is pictured here, and what is the height cutoff in millimeters used to define a strongly positive reaction?

Semiquantitative catalase test; a bubble column height of greater than $45\,\text{mm}$ defines a strongly positive reaction.

What are the incubation temperature and duration for the heat-stable catalase test, and which species are inactivated (negative)?

Heating at $68^\circ\text{C}$ for $20\,\text{minutes}$; Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium gastri, Mycobacterium haemophilum, and Mycobacterium marinum are inactivated (negative).

What is the principle and positive color indicator of the Tween 80 hydrolysis test?

Mycobacterial lipase cleaves Tween 80 into oleic acid and polyoxyethylated sorbitol, releasing neutral red dye which changes from amber to pink.

What is the principle of the iron uptake test, and which rapid grower is uniquely distinguished by a negative result?

Colonies convert ferric ammonium citrate into iron oxide, producing rusty brown colonies; Mycobacterium chelonae is negative, whereas other rapid growers are positive.

What biochemical test is demonstrated in these tubes, and what substance produces the bright pink positive color upon addition of sodium bicarbonate?

Arylsulfatase test; phenolphthalein released from the breakdown of tripotassium phenolphthalein disulfate turns bright pink upon addition of sodium bicarbonate.

Which rapid growers and slow growers yield a positive arylsulfatase test at $3\,\text{days}$ and $14\,\text{days}$, respectively?

$3\,\text{days}$: Mycobacterium fortuitum complex, Mycobacterium chelonae, Mycobacterium xenopi, and Mycobacterium triviale; $14\,\text{days}$: Mycobacterium marinum and Mycobacterium szulgai.

What enzymatic reaction and visual color change define a positive pyrazinamidase test?

Pyrazinamidase hydrolyzes pyrazinamide to pyrazinoic acid and ammonia in 4 days; pyrazinoic acid reacts with ferrous ammonium sulfate to produce a red band/pigment.

Which biochemical test is demonstrated by the formation of a black precipitate at the bottom of the tube on the far right?

Tellurite reduction test; potassium tellurite is reduced to black metallic tellurium within $3\text{ to }4\,\text{days}$ (positive for Mycobacterium avium complex and rapid growers).

What biochemical test is shown in this image, which differentiates Mycobacterium scrofulaceum (+) from Mycobacterium gordonae (-) by a pink-to-red color change?

Urease test; hydrolysis of urea produces ammonia and turns the pH indicator pink to red in a positive reaction.

What is the primary diagnostic utility of the p-nitroacetylamino-$\beta$-hydroxypropiophenone (NAP) test?

NAP selectively inhibits the Mycobacterium tuberculosis complex, differentiating it from nontuberculous mycobacteria.

How does the thiophene-2-carboxylic acid hydrazide (TCH or T2H) test distinguish Mycobacterium bovis from Mycobacterium tuberculosis?

Mycobacterium bovis is susceptible (S) to TCH, whereas Mycobacterium tuberculosis is resistant (R).

Which mycobacterial species are capable of growing on egg-based media containing $5\%\,\text{NaCl}$?

Mycobacterium flavescens, Mycobacterium triviale, and most rapidly growing Mycobacterium species.

Who first described Mycobacterium tuberculosis and in what year?

Robert Koch in 1882.

What organism is shown growing on this Lowenstein-Jensen slant, characterized by rough, warty, buff-colored 'cauliflower-like' colonies?

Mycobacterium tuberculosis (Koch's bacillus).

What is meant by the terms 'eugenic' and 'dysgonic' in mycobacterial culture?

'Eugenic' describes luxuriant growth enhanced by glycerol (characteristic of Mycobacterium tuberculosis); 'dysgonic' describes slow, scanty growth (seen in Mycobacterium bovis).

What is Cord Factor chemically, and what morphological phenomenon does it cause?

Trehalose-6,6'-dimycolate; it causes virulent bacilli to align in parallel, forming tight 'serpentine cords' in smears and on cord media.

What are sulfatides, and how are they detected in virulence testing of Mycobacterium tuberculosis?

Sulfolipids associated with virulence that bind neutral red dye, causing neutral red-treated virulent colonies to fix the dye and turn red at $37^\circ\text{C}$.

What is the typical droplet size involved in airborne transmission of primary pulmonary tuberculosis?

$1\text{ to }5\,\mu\text{m}$.

What is Purified Protein Derivative (PPD), and what is the standard dose administered for tuberculin skin testing?

A partially purified preparation of Old Tuberculin prepared by ammonium sulfate fractionation; the standard dose is $0.1\,\text{mL}$ containing $5\,\text{TU}$ (tuberculin units).

Which tuberculin skin testing method involves an intracutaneous injection into the forearm?

The Mantoux test.

Which tuberculin administration technique utilizes a cloth soaked in tuberculin placed over the skin for infants?

The Vollmer patch test.

When should a tuberculin skin test be read, and how are indurations of $>10\,\text{mm}$ versus $>5\text{ to }<10\,\text{mm}$ interpreted?

Read at $48\text{ to }72\,\text{hours}$; an induration of $>10\,\text{mm}$ indicates infection with Mycobacterium tuberculosis, whereas $>5\text{ to }<10\,\text{mm}$ is considered doubtful (possibly due to other mycobacteria).

What is Miliary Tuberculosis, and what are its common anatomical sites of dissemination?

Widespread hematogenous seeding of acid-fast bacilli outside the lungs; common sites include the spleen, liver, lungs, bone marrow, kidney, adrenal gland, and eyes.

What is Pott disease, and what is its most common clinical symptom?

Skeletal tuberculosis (tuberculous spondylitis) involving invasion of the spinal vertebrae; its primary presenting symptom is back pain.

What are the five primary first-line antituberculosis drugs?

Isoniazid (INH), Rifampin, Pyrazinamide (PZA), Ethambutol, and Streptomycin.

What is the standard treatment regimen and duration for newly diagnosed pulmonary tuberculosis?

Isoniazid and Rifampin daily for $9\,\text{months}$, or shortened to $6\,\text{months}$ by adding Pyrazinamide during the first $2\,\text{months}$.

How are Multidrug-Resistant Tuberculosis (MDR-TB) and Extensively Drug-Resistant Tuberculosis (XDR-TB) defined?

MDR-TB is resistant to at least isoniazid and rifampin; XDR-TB is MDR-TB plus resistance to any fluoroquinolone and at least one injectable second-line drug (amikacin, kanamycin, or capreomycin).

How is Mycobacterium bovis naturally acquired by humans, and what vaccine is derived from it?

Acquired through the ingestion of unpasteurized contaminated cow's milk or exposure to infected animals; the Bacillus Calmette-Guérin (BCG) vaccine is an attenuated strain of Mycobacterium bovis.

What geographic region is associated with Mycobacterium africanum, and how is it definitively detected?

Tropical Africa; detected using spoligotyping (spacer oligotyping).

What are the unique growth and colonial characteristics of Mycobacterium canettii?

It forms smooth colonies, grows rapidly in $6\,\text{days}$ on solid media, was isolated from an AIDS patient with mesenteric TB, and is niacin and nitrate positive.

What phenotypic criteria form the basis of the Runyon classification of nontuberculous mycobacteria (NTM)?

Growth rate (slow growers requiring $>7\,\text{days}$ versus rapid growers requiring $<7\,\text{days}$) and colonial pigmentation in response to light and darkness.

How are Runyon Group I (Photochromogens) defined, and which two principal species belong to this group?

They develop yellow/orange pigmentation only upon exposure to light after being grown in the dark and take longer than $7\,\text{days}$ to appear; includes Mycobacterium kansasii and Mycobacterium marinum.

What clinical disease and microscopic appearance are characteristic of Mycobacterium kansasii ('Yellow Bacillus')?

Chronic cavitary pulmonary lesions (second only to MAC in NTM lung disease); microscopically appears as long rods with distinct crossbanding.

Which photochromogenic Mycobacterium species is shown growing on Middlebrook 7H10 agar in this image?

Mycobacterium marinum.

What clinical lesion is produced by Mycobacterium marinum, and what is its optimal growth temperature?

A 'swimming pool granuloma' (tender red or blue-red subcutaneous nodule on elbows, knees, or fingers) acquired from aquariums or water; optimal growth occurs at $28^\circ\text{ to }32^\circ\text{C}$.

How are Runyon Group II (Scotochromogens) defined, and which three principal species belong to this group?

They produce yellow-to-orange pigmentation when grown in either the light or the dark and take longer than $7\,\text{days}$ to appear; includes Mycobacterium scrofulaceum, Mycobacterium szulgai, and Mycobacterium gordonae.

What is the primary clinical presentation of Mycobacterium scrofulaceum in pediatric patients?

Cervical lymphadenitis (scrofula) in children.

How does incubation temperature uniquely alter the colonial pigmentation of Mycobacterium szulgai?

It acts as a scotochromogen (yellow-orange in dark or light) at $37^\circ\text{C}$, but acts as a photochromogen (buff in dark, yellow with light exposure) at $22^\circ\text{C}$.

What is the common nickname of Mycobacterium gordonae, and why is it clinically significant in the laboratory?

'Tap Water Bacillus'; it is a common benign laboratory or clinical contaminant derived from tap water used to rinse patients' mouths before sputum collection.

How are Runyon Group III (Nonphotochromogens) defined, and what is the most significant clinical complex in this group?

They remain nonpigmented (buff, cream, or transparent) regardless of whether they are incubated in light or darkness and require longer than $7\,\text{days}$ to grow; the Mycobacterium avium complex (MAC) is the most significant member.

Which nonphotochromogenic Mycobacterium species is shown growing on Middlebrook 7H10 medium in this image?

Mycobacterium malmoense.

What organism and cellular arrangement are demonstrated in this tissue biopsy showing clusters of acid-fast bacilli inside macrophages?

Mycobacterium leprae arranged in parallel palisades ('cigar packets') inside lepra cells (mononuclear/epithelioid structures).