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LABORATORY 4

Acid-Fast Respiratory Pathogens: Mycobacterium and Nocardia Corynebacterium diphtheriae

1. Evaluate the quality of a collected sputum sample.
2. Demonstrate the cultural and biochemical characteristics used for identification of mycobacteria.
3. Demonstrate the acid-fast staining procedure.
4. Describe the protocol used by the University Hospital's Diagnostic Microbiology Laboratory to identify mycobacteria from clinical specimens.
5. Descibe the morphological characteristics of the Nocardia.
6. Describe the cultural and biochemical characteristics of Corynebacterium diphtheriae.
7. Perform a methylene blue stain of C. diphtheriae.
8. Describe the Elek test for toxigenicity of C. diphtheriae.

1. Sputum Cultures

– Observe a Gram stain of a sputum smear to determine criteria used to reject or accept specimen for clinical tests.

2. Mycobacterium spp.
   
   – Perform the acid-fast stain on M. tuberculosis in groups of 2-4.
3. Nocardia spp.
   
   – Observe a Gram stain and acid-fast stain of N. asteroides in groups of 2-4.
4. Corynebacterium diphtheriae
   
   – Observe cultures of C. diphtheriae on tellurite medium.
   
   – Perform a methylene blue stain on C. diphtheriae in groups of 2-4.
   
   – Observe "Chinese characters" and metachromatic granules.

1. Sputum Cultures

Bacterial pneumonia, pulmonary tuberculosis, and chronic bronchitis constitute a clinically important group of diseases. Since the choice of appropriate treatment frequently depends upon a bacteriological diagnosis, the prompt and accurate examination of a properly collected sputum specimen by microscopic examination of a smear, culture, and antibiotic susceptibility testing becomes imperative.

The organisms most frequently associated with acute bacterial infections of the lower respiratory tract include Streptococcus pneumoniae; Klebsiella pneumoniae and, in certain patients, other Gram-negative enteric bacteria; Haemophilus influenzae; Staphylococcus aureus; Pseudomonas, and Mycoplasma pneumoniae. With the exception of Mycobacterium tuberculosis and Gram-negative enterics other than Klebsiella, it generally is possible to recover these important pulmonary pathogens from the upper respiratory tract of apparently healthy people. The recovery of these organisms in small numbers, therefore, is not always sufficient evidence of their etiological role in a particular infection. In acute bacterial pneumonia, however, the pathogen is generally present in large numbers and in pure culture. With immunocompromised patients a common pathogen encountered in sputum specimens is the yeast Candida albicans which stains positive by the Gram procedure.

An early presumptive diagnosis of bacterial pneumonia can often be made by microscopic examination of a smear of sputum properly stained by the Gram method. The presence of numerous organisms of a particular morphologic type indicates the etiologic agent of the pneumonia, e.g., Gram-positive diplococci (S. pneumoniae), Gram-positive cocci in clusters (S. aureus), large Gram-negative rods (K. pneumoniae, Pseudomonas, etc.) or small Gram-negative rods (H. influenzae).

The Gram stain will also reveal if the sputum specimen has been properly collected or if it is mainly saliva containing normal oral flora. The quality of sputum samples can be evaluated by counting the relative numbers of squamous epithelial cells and segmented neutrophils per low-power field in a stained smear. The presence of squamous epithelial cells indicating a preponderance of oropharyngeal secretions is indicative of an improperly collected specimen. In contrast, bacterial pneumonia will produce large numbers of segmented neutrophils in the sputum. If the number of epithelial cells (10-25 per field) is greater than the number of neutrophils and there is a mixed flora of bacteria with no single type predominating, the specimen should not be processed by the laboratory. Recollection of the specimen should be requested. If the number of neutrophils is greater than 5-10 per oil immersion field, the epithelial cells are fewer than neutrophils, or bacteria are of pure or nearly pure type, the specimen should be processed by the laboratory. If intracellular organisms are seen, the specimen should be processed regardless of other characteristics. If a patient is identified as being immunocompromised (renal transplant, liver transplant, certain leukemia patients) neutrophils may not be present. This type of sputum specimen should be processed as requested.




 

Student Work A-1a. Microscopic Observation of a Sputum smear.

Scan a Gram-stained sputum smear (in slide box at the center of each bench) and determine whether the collected sputum is acceptable for clinical testing using the criteria discussed above. If you are unsure of whether you would accept or reject the sample, please talk to an instructor. It is important that you understand the reasons why clinical labs may accept or reject various sputum collections. After you are done, please blot the immersion oil off the slide gently using lens paper and return the slide to the box.

 

2. Mycobacterium

Introduction:

The genus Mycobacterium includes many innocuous saprophytes in soil and water but also several medically important species. For nearly a century M. tuberculosis and M. leprae have been known to be the causes of tuberculosis (TB) and leprosy, respectively. These are chronic diseases, lasting many years and requiring prolonged therapy. However, since the early 1950s, disease states resembling pulmonary tuberculosis have been identified as due to various mycobacteria other than M. tuberculosis (MOTT) or to a branching bacterium, Nocardia. The correct bacteriologic diagnosis is important, since therapy against these organisms is very different from that for tuberculosis.

1. Acid-fastness— Mycobacteria characteristically have a much higher content of lipid (including several unique ones such as mycolic acid) than other bacteria. This property causes the Gram stain not to be useful for mycobacteria, because neutral dyes do not readily penetrate and fix to them. To overcome this difficulty alternative staining procedures have been devised. These procedures would stain all bacteria and be of no value were it not for the fact that an "acid-alcohol" rinse decolorizes all bacteria readily except Mycobacterium and some Nocardia. Hence these organisms are termed "acid-fast." This property alone is sufficient to distinguish Mycobacterium and Nocardia from most other bacteria.
2. Hydrophobic surface— This feature, also due to the high lipid content of the mycobacterial wall, leads to growth in irregular massive clumps, which have a greater tendency toward parallel orientation of the bacteria (cord formation) in pathogenic than in avirulent strains of M. tuberculosis. As a result, growth in ordinary liquid media appears as a surface pellicle rather than as a dispersed suspension.
3. Slow growth—Most mycobacteria can grow on a very simple media, but even on a rich solid medium colonies of virulent M. tuberculosis require several weeks to become visible, whereas most other bacteria will grow up in a day or so. In rich liquid media virulent M. tuberculosis have a doubling time of 12 hours or so compared with 20-50 minutes for E. coli. M. leprae has not been successfully grown on laboratory media, but in vivo its doubling time is 10-30 days, making it the "champion slow grower" in the wide world of microbes.

Primary isolations from patients are carried out on various complex solid media, such as Lowenstein, Petragnani, Peizer, Dorset, etc. The special characteristics of these media include a high concentration of eggs and a dye that inhibits the faster growing contaminants.

I. Runyon Group I.

--Photochromogen (photo = light; chromogen = color producing)

IV. Runyon Group IV.

-- Rapid growers --growth is visible within a few days like M. tuberculosis

A. Classical saprophytes (non-pathogenic for humans) include  

M. phlei: timothy grass bacillus

M. butyricus: butter bacillus

 

Demonstration B-2a. Pigment production and growth rate of mycobacteria:

Do Not Open These Cultures

Three acid-fast organisms:

were inoculated on Lowenstein's slants (an egg medium); M. tuberculosis also was inoculated on the clear medium 7H10 (a plate sealed with tape).

These cultures were incubated at 37^o C for the length of time designated on each (7, 14, or 21 days).

Record:

1. the amount of growth (0 to 4+) for each incubation interval;
2. the color of the growth (white, buff, yellow, orange, etc.);
3. the texture of the growth and size of any colonies; and
4. the rate of growth -- e.g., < 7 days, < 14 days, > 14 days, > 21 days.

Note: Be aware that the above laboratory strains used in this demonstration have been carried on artificial media for some time and, therefore, may grow faster than freshly isolated ones.

Also, do not mistake for growth any clumps of organisms which may have been transferred in the original inoculum. There is usually a fading of the green color of the medium or a yellowish tinge in the immediate vicinity of the organisms if growth has occurred.

At your bench should be a Bactrol^TM Acid Fast slide (one per 2-4 students). The positive circle contains a smear containing acid-fact bacteria and the negative circle contains a smear without acid-fast bacteria.

Heat fix the slide and perform an acid-fast stain as follows.

Procedure for Acid fast stain (Kinyoun Carbolfuchsin Method): To be performed in groups of 2-4 students.

1. Place your fixed slide on a staining rack and flood the smear with the carbolfuchsin stain for 5-10 minutes.
2. Wash the slide under running water.
3. Decolorize to a faint pink with acid alcohol while continuously agitating the slide until no more stain comes off in the washings. Add more acid alcohol and leave the slide flat for approximately 30 seconds. Pour off and add more acid alcohol for approximately 30 seconds. Thoroughness in decolorization is essential in order to prevent the possibility of a false positive reading.
4. Wash the slide with water.
5. Counterstain with brilliant green for 1 minute; then wash with water, blot dry, and examine under oil. Acid-fast bacteria are red; other organisms and most of the background are blue. Before examining your stained smear, observe the demonstration slide of acid-fast bacilli that is available at your bench.
6. Show your acid-fast stained smear to your group instructor. Sketch two or three representative bacilli.

Although the following procedure will not be carried out in this laboratory exercise, you should be familiar with it in a general sense. The elimination of contaminating organisms, which generally grow much faster, is important in preparing clinical specimens for the culture of tubercle bacilli. A treatment must be used that is not destructive to tubercle bacilli in the specimen, but does destroy most of the other organisms. The procedure used for sputa also reduces the viscosity by liquefying the mucus, thus permitting the tubercle bacilli to be concentrated by centrifugation. The method used in the Diagnostic Microbiology Laboratory depends on brief digestion with the mucolytic agent, N-acetyl L-cysteine, and decontamination with a weak (2%) NaOH solution.

Gastric washings are obtained when sputum specimens are unobtainable or consistently negative. The washings are collected in Na₂CO₃ to neutralize the gastric acidity. The sediment from the washings is treated as above for sputum and cultured. Sediment from morning specimens of urine is treated as above and cultured in cases of suspected renal tuberculosis.

Smears are of no value for either gastric washings or urine specimens. Saprophytic, acid-fast bacilli may be present in stomach contents after ingestion of food or even tap water (see M. scrofulaceum) and can be found in urine (M. smegmatis).

Results may be reported as follows:

 Far more important than the number of bacilli in the preparation is the mere fact that they are present. If clumps of bacilli appear, the number of individual bacilli is estimated. In practice it is desirable also to culture the specimen both for definitive identification and for testing chemotherapeutic sensitivity. The microscopic findings suggest whether the specimen should be concentrated or diluted in order to get isolated colonies that may be more easily identified. A method for digesting and concentrating specimens has been described above. In general, it is used for material which appears negative on smear or which is heavily contaminated with other bacteria.

The Diagnostic Microbiology Laboratory inoculates conventional media (e.g., a Lowenstein-Jensen slant) from treated patient specimens, and, in addition, uses the Bactec system for radiometric detection of mycobacteria. A portion of the processed patient specimen is inoculated into a Bactec vial containing liquid medium and ¹⁴C-labeled oleic acid. The ability to metabolize oleic acid with evolution of CO₂ is diagnostic for mycobacteria. In the Bactec system, ¹⁴CO₂ is detected. This permits the physician to have a presumptive-positive detection of mycobacteria in 4-7 days (depending on the severity of the infection and on whether the patient has been receiving antibiotic therapy). A ++++ smear permits presumptive detection of mycobacteria in as few as 4 days; a + smear requires a week or more. Once mycobacteria have been detected in the Bactec system, the culture in the Bactec vial is stained by the Kinyoun acid-fast method and examined microscopically. Here, morphology helps in the identification: if roping (cord formation) is seen, M. tuberculosis is suspected.

The mycobacteria in a Bactec vial that gives a positive detection reaction are then pelleted by centrifugation and speciated by polymerase chain reaction (PCR). The Diagnostic Microbiology Laboratory uses five nucleic acid probes that allow sorting of mycobacteria into M. tuberculosis, M. kansasii, M. avium-intracellulare, and M. gordonae. If all probes are negative, biochemical tests are carried out to determine the mycobacterial species that grew in the Bactec bottle.

In some diagnostic labs (e.g., State laboratories), HPLC is being used instead of nucleic acid probes. This method speciates mycobacteria by their different compositions of cell wall lipids.

Pulmonary nocardiosis and pulmonary tuberculosis are sometimes indistinguishable clinically or by X-ray. Nocardia may account for some of the reported cases of streptomycin-resistant tuberculosis, since it is resistant to streptomycin; however, it is responsive to sulfonamides. Nocardia grow readily on the same media used to isolate M. tuberculosis.

1. Gram-positive
2. Some strains are weakly acid-fast
3. Forms branching filaments in liquid cultures (best seen in "wet mount" slide preparations)
4. Grows more rapidly than M. tuberculosis on media used for mycobacteria
5. Aerobic, with a predilection for lung.

Nocardia spp. are opportunists—they are regularly isolated from soil and, therefore, may be found normally on the skin or in the upper respiratory tract. Two medically important species are:

1. Nocardia asteroides—produces:

a) pulmonary infections especially in persons with lymphomas or other neoplasms, and

b) chronic subcutaneous abscesses (mycetomas) with draining sinuses which discharge "sulfur" granules (colonies embedded in a matrix of calcium phosphate and named for their yellow color);

2. N. brasiliensis— found predominantly in Mexico; causes mainly mycetomas.

Note that the genera Mycobacterium and Nocardia belong to the order Actinomycetales. This order also includes the branching, non-acid-fast bacterium, Actinomyces naeslundii, and the genus Streptomyces, which produces some medically important antibiotics. The term Actinomycete refers to any genus of Actinomycetales except Mycobacterium.

5. Corynebacterium diphtheriae

Corynebacterium diphtheriae is a Gram-positive, nonmotile rod that is facultatively anaerobic but grows best aerobically. It infects the upper respiratory tract and skin, causing the disease called diphtheria. Its major established virulence property is diphtheria toxin. This toxin is an exotoxin that accounts for most of the pathologic effects of diphtheria: it causes both local and systemic symptoms. Diphtheria toxin is produced only by strains of C. diphtheriae that are lysogenized by a temperate bacteriophage, corynephage b, carrying the structural gene for toxin. There are many serotypes of C. diphtheriae, but all make an identical toxin. Humans are the main reservoir (the bacteria are carried in the respiratory tract). Infection is spread by droplet transmission or contact with nasal secretions

In diphtheria, the bacteria spread from the nasopharynx to the larynx and trachea and cause severe disease. In addition to serious systemic effects of the toxin (such as cardiac arrest or respiratory paralysis), diphtheria patients can suffer asphyxiation due to a pseudomembrane that forms at the site of the infection. The airway can be obstructed by this and by the local swelling. Although diphtheria is primarily a disease of the upper respiratory tract, primary or secondary lesions can occur in other parts of the body. Skin is the most common of these (due to infection of an abrasion) and results in chronic, spreading ulcers covered by a grayish membrane. This is more of a problem in the tropics than in the temperate zone (however, physicians here will see immigrants with skin diphtheria, and this infection is increasingly seen among street people in the U.S.). People also can be infected by a nontoxigenic strain of C. diphtheriae, with a resulting mild "surface" diphtheria due to effects of undefined virulence factors.

"Diphtheroids" are members of our normal flora that are morphologically similar to diphtheria bacilli -- e.g., C. pseudodiphtherium and C. xerosis. These are relatively nonpathogenic; they are opportunistic pathogens.

In throat cultures plated onto blood agar, corynebacteria can be overgrown by throat flora; however, a BAP will reveal any group A streptococci that may be part of a dual infection. McLeod's tellurite medium is good for permitting selective growth of corynebacteria. Tellurite salt (K₂TeO₃; colorless) is reduced by the bacteria, producing a precipitate of metallic tellurium, giving gray-black colors. Tellurite salts also reduce the number of contaminants: this is why tellurite medium is a selective medium.

Observe the gray-black colonies of C. diphtheriae growing on the tellurite plate at your bench.

Typically, in cases of suspected diphtheria, the physician will provide the diagnostic lab with nose and throat swabs, which are used to streak a tellurite plate, a BAP, a CAP, and a Loeffler slant. The last one is a suboptimal medium that accentuates some diagnostically useful morphological characteristics of C. diphtheriae, such as pleomorphism and the presence of metachromatic granules of polyphosphate called Babes-Ernst granules. C. diphtheriae stains poorly with the Gram stain, and other stains, such as methylene blue, are useful. Methylene blue will reveal any Babes-Ernst granules present. C. diphtheriae is a pleomorphic rod, often club-shaped. Some of the bacteria fail to separate after division, giving rise to V- or L-shaped pairs of bacilli in smears. These tend to form arrangements reminiscent of "Chinese characters".

Procedure: Use cells from the Loeffler slant.

Prepare and heat-fix a smear of C. diphtheriae as you would for a Gram stain.

Add 2-3 drops of methylene blue stain to the slide for at least one minute.

Rinse with water and blot dry.

Examine your stained smear in the microscope and sketch several representative groups of cells. Do you see "Chinese character"-like groupings or metachromatic granules?

To complete the identification of suspected C. diphtheriae, confirmatory fermentation tests are then made: the Diagnostic Microbiology Laboratory uses the API Coryne Strip to make a set of such tests that are read in 24 hours.

Because the incidence of diphtheria in the United states is so low, suspected isolates of C. diphtheriae are sent to the Center for Disease Control (CDC) via the State Health Department where they are tested for toxigenicity by an antigen-antibody immunodiffusion assay (Elek test) and by polymerase chain reaction (PCR) to detect the toxin gene.

The Elek test is subject to error (false negative results) due to small differences in media preparation and technical skills of personnel performing the test. For this reason, it is almost never performed outside a reference laboratory. A schematic diagram of an Elek plate is shown on the next page.

The diagram of an Elek plate above illustrates the differences in immunoprecipitation patterns between non-toxin-producing and toxin-producing strains of C. diphtheriae. A filter paper strip impregnated with diphtheria antitoxin is buried just beneath the surface of a special agar plate before the agar hardens. Strains to be tested and known positive and negative toxigenic strains are streaked on the agar’s surface in a line across the plate and at a right angle to the antitoxin paper strip. After incubation at 37⁰C for 24 hours, plates are examined for the presence of fine white precipitin lines at a 45⁰ angle to the streaks (starting at the intersection of antitoxin-impregnated filter paper and the culture streak). The presence of precipitin lines indicates that the strain produced toxin that reacted with the homologous antitoxin. Line 1 is a nontoxigenic strain. Line 2 is a toxigenic strain.

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