- High quality full colour photography - Essential for accurate diagnosis
- Fully revised pathogenicity sections taking into account the major discoveries/incidences of the last 3-5 years
- Reclassification of viruses, including changes to nomenclature
- Appendices of Infectious Diseases - Fast access to vital information
- Unique and practical inclusion of virology, bacteriology and mycology in one text
- Greatly expanded chapter on viruses
- More on PRIONS (including BSE)
- Reclassification of viruses - many changes to nomenclature
- Fully revised pathogenicity sections
- Revised/complete coverage of E coli 0157
- Revised Systems section
- Complete update of Infectious Diseases coverage in the appendices
Bacterial pathogens
Microscopy, culture and identification
Microscopy
A good-quality microscope with a built-in light source (bright-field microscopy) as well as low-power, high-dry and oil-immersion objectives is required. A darkfield condenser is a useful addition necessary for the visualization of unstained preparations such as those of spirochaetes. Microscopes for microbiology require a higher degree of resolution than those used for haematology or histopathology. Figure 2.1 indicates the size of bacteria relative to that of an erythrocyte and to viruses. Bacteria are measured in micrometres (µm) which are 10−6?m while viruses are measured in nanometres (nm) which are 10−9?m. Using an oil-immersion objective lens on a light microsope, a magnification of 1000 × can be achieved to visualize bacteria. On account of their small size viruses are visualized by using an electron microscope, which utilizes a beam of electrons to achieve magnifications of the order of 100,000 ×.
Figure 2.1 Comparison of the relative sizes of a red blood cell, bacteria and viruses.
Stained Smears from Pathological Specimens
Stained smears made from lesions can yield a considerable amount of information inexpensively and quickly. Table 2.1 summarizes the information that can be gained from the various diagnostic staining techniques.
Table 2.1
Diagnostic uses of stained smears
Preparing Bacterial Smears
Microscope slides are not always clean enough to use directly from the supplier. Rubbing with a clean, soft cloth and passing quickly through the Bunsen flame may be sufficient to remove a greasy film. If not, a mildly abrasive liquid cleaner can be used followed by rinsing the slide thoroughly and wiping it dry with a clean cloth.
A blunt scalpel and forceps should be kept in a container of 70% ethyl alcohol. The instruments are flamed and cooled before use. Afterwards they should be placed into a container of disinfectant. When making a smear from tissue lesions, the specimen is held firmly with the forceps and the scalpel is used to scrape deep into the material. A small amount of the scrapings is placed on the cleaned microscope slide. Another clean slide is used with a scissor action to prepare a thin smear. With liquid or semiliquid specimens, a little of the sample is placed on the slide with a sterile swab. The contents of the swab are smeared over the surface of the slide, with the aim of having thick and thin areas of specimen present. The smears are allowed to dry thoroughly before proceeding further.
Fixing the Smears
The reasons for fixing the smears include killing the vegetative bacteria, rendering them permeable to the stain and ensuring that the material is firmly fixed to the slide. Fixed and stained smears should be handled carefully as not all bacteria, especially endospores, may have been killed. After use, the stained smears should be autoclaved or soaked in a reliable disinfectant (24–48 hours) before discarding.
For routine staining the smears are fixed by passing the slide, smear side up, quickly through the Bunsen flame two or three times, taking care not to overheat the smear. This can be tested on the back of the hand; the slide should feel warm but not hot enough to burn.
Dried smears to be stained by the Giemsa stain are first fixed in absolute methyl alcohol for three minutes and then dried.
Staining the Smears: Staining Techniques
The fixed smears are placed on a staining rack over a sink. The staining solutions are flooded over the entire smear and left on the slide for the appropriate time. Between each staining reagent the smear is washed under a gently running tap, excess water tipped off and the next reagent added. Finally the stained smear is washed and air-dried. The preparation method of each of the staining solutions is given in Appendix 1.
Gram stain
Crystal violet 60 seconds
Gram's iodine (mordant) 60 seconds
Gram's decolourizer 15 seconds
Counter-stain (dilute carbol fuchsin or safronin) 60 seconds
Gram-positive bacteria retain the crystal violet-iodine complex and stain purple-blue. Gram-negative bacteria are decolourized and are stained red by the counter-stain. Gram-stained smears are illustrated (Figs 2.2–2.6, inclusive). There can be slight differences in the composition of the reagents for the Gram stain. If using a commercial kit set for Gram staining always follow the manufacturer's directions.
Figure 2.2 Gram-stained porcine faecal smear showing Gram-positive (blue) and Gram-negative (red) bacteria. Note range of morphological forms. (×1000)
Figure 2.3 Gram-stained smear of mastitic milk (bovine) showing Gram-positive streptococci in chains and inflammatory cells. (×1000)
Figure 2.4 Gram-stained smear from an abscess with the Gram-positive, pleomorphic rods of Trueperella (Arcanobacterium) pyogenes predominating. (×1000)
Figure 2.5 Gram-stained smear from the mucosa of the small intestine (lamb recently dead from enterotoxaemia): large Gram-positive rods of Clostridium perfringens. (×1000)
Figure 2.6 Gram-stained smear of urine (dog with cystitis): Gram-negative rods of Escherichia coli. (×1000)
Dilute carbol fuchsin (DCF):
A simple staining technique
Dilute carbol fuchsin four minutes
Wash and air-dry.
The stain is used for some Gram-negative bacteria such as Campylobacter fetus, Brachyspira hyodysenteriae or Fusobacterium necrophorum (Fig. 2.7) where a greater depth of stain aids microscopic visualization.
Figure 2.7 DCF-stained smear from a bovine abscess: red filaments of Fusobacterium necrophorum showing typical irregular staining. (×1000)
Modified Ziehl–Neelsen (MZN)
Dilute carbol fuchsin 15 minutes
Acetic acid (0.5%) 15 seconds
Methylene blue two minutes
Wash and air-dry.
MZN-positive bacteria such as Nocarida asteroides (Fig. 2.8), Brucella spp. (Fig. 2.9) and Chlamydophila abortus stain a bright red with the background and other bacteria staining blue.
Figure 2.8 MZN-stained smear of a thoracic aspirate from a dog with a pleural effusion: MZN-positive branching filaments of Nocardia asteroides. (×1000)
Figure 2.9 MZN-stained smear from a bovine placenta (an abortion case): red MZN-positive coccobacilli of Brucella abortus in clumps. (×1000)
Ziehl-Neelsen (ZN) or acid-fast stain
Strong carbol fuchsin 10 minutes with heat
Acid-alcohol decolourizer 15 minutes with several changes
Methylene blue 20 seconds
Wash and air-dry.
ZN-positive or acid-fast bacteria, such as the pathogenic Mycobacterium sp., stain bright red with the background and other bacteria counter-stained blue (Fig. 2.10). Heating the strong carbol fuchsin can be carried out in one of two ways:
Figure 2.10 ZN-stained smear from a tuberculous lesion in a hen: red ZN-positive thin rods of Mycobacterium avium. (×1000)
• Strong carbol fuchsin is flooded onto the fixed smear with the slide on the rack over a sink. A cotton wool swab, on a metal rod, is dipped in alcohol and set alight. This is used to gently heat the smear and carbol fuchsin from below. The stain is allowed to steam for the 10 minutes but not to boil. The sink should be rinsed with water before starting the heating process in case any inflammable reagents are present.
• Heat the strong carbol fuchsin in a boiling tube to just below boiling point. Wear protective goggles when carrying out this procedure and direct the tube away from you. Add the hot stain to the smear on the staining rack over the sink. Keep topping-up the smear with hot stain for the full 10 minutes.
Alternative method for the Ziehl–Neelsen stain
The advantage of the method using the brilliant green counter-stain is that it is almost impossible to over-do the counter-staining.
Strong carbol fuchsin three minutes
Wash in distilled water
Acid-alcohol decolourizer three minutes exactly
Several washes in distilled water
Alkaline brilliant green three minutes
Wash in distilled water and air-dry.
The...
| Erscheint lt. Verlag | 1.11.2013 |
|---|---|
| Sprache | englisch |
| Themenwelt | Medizin / Pharmazie |
| Veterinärmedizin ► Klinische Fächer ► Mikrobiologie / Immunologie | |
| Veterinärmedizin ► Klinische Fächer ► Parasitologie | |
| ISBN-10 | 0-7020-5588-3 / 0702055883 |
| ISBN-13 | 978-0-7020-5588-1 / 9780702055881 |
| Haben Sie eine Frage zum Produkt? |
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