15 Microorganisms
Introduction
• The increased mobility of the world’s population through tourism, immigration, and international commerce has distorted natural geographic boundaries to infection, exposing weaknesses in host defenses, and in knowledge. Some agents, such as Ebola, have been around for many years but the first human outbreaks were not recorded until 1976. Previous outbreaks would flare up and then burn themselves out, undetected and confined, before deforestation and the like altered this state.
• Immunodeficiency states occurring either as part of a natural disease, such as acquired immune deficiency syndrome (AIDS), or as an iatrogenic disease. As treatment becomes more aggressive, depression of the host’s immunity often occurs, enabling organisms of low virulence to become life-threatening, and allows latent infections, accrued throughout life, to reactivate and spread unchecked.
• Emerging, re-emerging, and antibiotic-resistant organisms such as the tubercle bacillus and staphylococcus are a constant concern.
• Adaptive mutation occurring in microorganisms, which allows them to jump species barriers and exploit new physical environments, thus evading host defenses, and resisting agents of treatment.
• Bioterrorism has become an increasing concern. The world’s public health systems and primary healthcare providers must be prepared to address varied biological agents, including pathogens that are rarely seen in developed countries. High-priority agents include organisms that pose a risk to national security because they:
The following are listed by the Centers for Disease Control and Prevention (CDC) in the United States as high-risk biological agents:
Size
The term ‘microorganism’ has been interpreted liberally in this chapter. Space limitation precludes a comprehensive approach to the subject, and the reader is referred to additional texts such as that of von Lichtenberg (1991) for greater depth. The organisms in Table 15.1 are discussed, and techniques for their demonstration are described.
Organisms | Size |
---|---|
Viruses | 20–300 nm |
Mycoplasmas | 125–350 nm |
Chlamydia | 200–1000 nm |
Rickettsia | 300–1200 nm |
Bacteria | 1–14 µm |
Fungi | 2–200 µm |
Protozoa | 1–50 µm |
Metazoans | 3–10 mm |
General principles of detection and identification
The Gram stain
Gram method for bacteria in smears (Gram 1884)
Method
1. Fix dry film by passing it three times through a flame or placing on a heat block.
2. Stain for 15 seconds in 1% crystal violet or methyl violet, and then pour off excess.
3. Flood for 30 seconds with Lugol’s iodine, pour off excess.
4. Flood with acetone for not more than 2–5 seconds, wash with water immediately.
5. Alternatively decolorize with alcohol until no more stain comes out. Wash with water.
6. Counterstain for 20 seconds with dilute carbol fuchsin, or freshly filtered neutral red for 1–2 minutes.
7. Wash with water and carefully blot section until it is dry.
Modified Brown-Brenn method for Gram-positive and Gram-negative bacteria in paraffin sections (Churukian & Schenk 1982)
Solutions
Crystal violet solution (commercially available)
Crystal violet, 10% alcoholic | 2 ml |
Distilled water | 18 ml |
Ammonium oxalate, 1% | 80 ml |
Modified Gram’s iodine commercially available, or
Iodine | 2 g |
Potassium iodide | 4 g |
Distilled water | 400 ml |
0.5% basic fuchsin solution (stock) commercially available, or
Basic fuchsin or pararosaniline | 0.5 g |
Distilled water | 100 ml |
Basic fuchsin solution (working)
Basic fuchsin solution (stock) | 10 ml |
Distilled water | 40 ml |
Picric acid-acetone
Picric acid | 0.1 g |
Acetone | 100 ml |
Staining method
1. Deparaffinize and rehydrate through graded alcohols to distilled water.
2. Stain with filtered crystal violet solution, 1 minute.
3. Rinse well in distilled water.
5. Rinse in distilled water, blot slide but NOT the tissue section.
6. Decolorize by dipping in alcohol-acetone solution until the blue color stops running. (One to two dips only!)
7. Counterstain in working basic fuchsin for 1 minute. Be sure to agitate the slides well in the basic fuchsin before starting the timer.
8. Rinse in distilled water and blot slide but not section.
10. Dip in picric acid-acetone until the sections have a yellowish-pink color.
11. Dip several times in acetone-xylene solution. At this point, check the control for proper differentiation. (Go back to picric acid-acetone if you need more differentiation.)
Gram-Twort stain (Twort 1924; Ollett 1947)
Solutions
Crystal violet solution (see previous method)
Gram’s iodine (see previous solution)
1% neutral red in ethanol | 9 ml |
0.2% fast green in ethanol | 1 ml |
Distilled water | 30 ml |
Method
1. Deparaffinize and rehydrate through graded alcohols to distilled water.
2. Stain in crystal violet solution, 3 minutes.
3. Rinse in gently running tap water.
4. Treat with Gram’s iodine, 3 minutes.
5. Rinse in tap water, blot dry, and complete drying in a warm place.
6. Differentiate in preheated acetic alcohol until no more color washes out (2% acetic acid in absolute alcohol, preheated to 56°C). This may take 15–20 minutes; the section should be light brown or straw colored.
7. Rinse briefly in distilled water.
8. Stain in Twort’s, 5 minutes.
10. Rinse in acetic alcohol until no more red runs out of the section; this takes only a few seconds.
Techniques for mycobacteria
Ziehl-Neelsen (ZN) stain for Mycobacterium bacilli (Kinyoun 1915)
Solutions
Carbol fuchsin commercially available, or
Basic fuchsin | 0.5 g |
Absolute alcohol | 5 ml |
5% aqueous phenol | 100 ml |
Mix well and filter before use.
Acid alcohol | |
Hydrochloric acid | 10 ml |
70% alcohol | 1000 ml |
Method
1. Deparaffinize and rehydrate through graded alcohols to distilled water.
2. Carbol fuchsin solution, 30 minutes.
4. Differentiate in acid alcohol until solutions are pale pink. (This usually only takes 2–5 dips.)
5. Wash in tap water for 8 minutes, then dip in distilled water.
6. Counterstain in working methylene blue solution until sections are pale blue.
7. Rinse in tap water, then dip in distilled water.
Results
Mycobacteria, hair shafts, Russell bodies, Splendore-Hoeppli immunoglobulins around actinomyces, and some fungal organisms | red |
Background | pale blue |
Notes
a. The blue counterstain may be patchy if extensive caseation is present. Care should be taken to avoid over-counterstaining as scant organisms can easily be obscured.
b. Decalcification using strong acids can destroy acid-fastness; formic acid is recommended.
c. Victoria blue can be substituted for carbol fuchsin and picric acid for the counterstain if color blindness causes a recognition problem.
Fluorescent method for Mycobacterium bacilli (Kuper & May 1960)
Solution
Auramine O | 1.5 g |
Rhodamine B | 0.75 g |
Glycerol | 75 ml |
Phenol crystals (liquefied at 50°C) | 10 ml |
Distilled water | 50 ml |
Method
1. Deparaffinize (1 part groundnut oil and 2 parts xylene for M. leprae).
2. Pour on preheated (60°C), filtered staining solution, 10 minutes.
4. Differentiate in 0.5% hydrochloric acid in alcohol for M. tuberculosis, or 0.5% aqueous hydrochloric acid for M. leprae.
5. Wash in tap water, 2 minutes.
6. Eliminate background fluorescence in 0.5% potassium permanganate, 2 minutes.
7. Wash in tap water and blot dry.
8. Dehydrate (not for M. leprae), clear, and mount in a fluorescence-free mountant.
Results
Mycobacteria | golden yellow (using blue light fluorescence below 530 nm) |
Background | dark green |
Modified Fite method for M. leprae and Nocardia
Solutions
5% sulfuric acid in 25% alcohol
25% ethanol | 95 ml |
Sulfuric acid, concentrated | 5 ml |
Methylene blue, working
Stock methylene blue | 5 ml |
Tap water | 45 ml |
Xylene-peanut oil | 1 part oil: 2 parts xylene |
Method
1. Deparaffinize in two changes of xylene-peanut oil, 6 minutes each.
2. Drain slides vertically on paper towel and wash in warm, running tap water for 3 minutes. (The residual oil preserves the sections and helps accentuate the acid fastness of the bacilli.)
3. Stain in carbol fuchsin at room temperature for 25 minutes. (Solution may be poured back into bottle and reused).
4. Wash in warm, running tap water for 3 minutes.
5. Drain excess water from slides vertically on paper towel.
6. Decolorize with 5% sulfuric acid in 25% alcohol, two changes of 90 seconds each. (Sections should be pale pink.)
7. Wash in warm, running tap water for 5 minutes.
8. Counterstain in working methylene blue, one quick dip. (Sections should be pale blue.)
9. Wash in warm, running tap water for 5 minutes.
10. Blot sections and dry in 50–55°C oven for 5 minutes.
11. Once dry, one quick dip in xylene.
Results See Figure 15.1
Acid-fast bacilli including M. leprae | bright red |
Nuclei and other tissue elements | pale blue |
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