Spore stain

It is advisable to use old cultures grown on nutrient agar, as spore production is a survival mechanism found in ageing cultures. A smear should be made and fixed in the normal manner. The slide should be placed on the rim of a beaker half full of boiling water, with the bacterial film uppermost, and then the following procedure used. 

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Check spore production after 5 days by spore stain. If the percentage of cells sporing is less than 70 to 80%, continue incubation. When a 70 to 80% spore yield is achieved, wash off the growth from the flats with 20 ml of sterile normal saline and dispense into sterile centrifuge tubes. 

Subculture about 3 to 5 ml of the broth onto each of hve 45-ml solid RCM agar slopes (in 100-ml medical flats) and incubate anaerobically at 32°C for until 70 to 80% of the population is sporing (approximately 2 weeks). Spore production should be checked every few days by spore stain. 

Figure 21.1 Photomicrograph of Bacillus anthracis Uom an agar culture demonstrating spores Fuchsin-methylene blue spore stain. Anthrax CDC...

It is not necessary to carry out an acid fast stain (Chapter 4) on Gram-positive rods giving a positive spore stain, but this test should be carried out on Grampositive rods which cannot be demonstrated to contain spores. This stain is intended to differentiate the genus Mycobacterium and some Nocardia spp. on the basis of the lipid material present in the outer layers of the cell. 

Schaeffer-Fulton Spore Staining A differential stain used to make endospores easier to visualize. 

The same generally impervious properties make spores difficult to stain by simple stains. However, if a slide preparation of spores is warmed with a stain the spores are dyed so effectively that dilute acid will not wash out the colour. This is the basis of the acid-fast stain for spores. 

Due to a waxy component in the cell wall these organisms are difficult to stain with ordinary stain solutions, the hydrophobic nature of the wall being stain repellent however, if the bacterial smear on the slide is warmed with the stain, the cells are dyed so strongly that they are not decolorized by washing with dilute acid, hence the term acid-fast. Many bacterial spores exhibit the phenomenon of acid fastness. 

Tlie morphology of some bacteria, especially those that form spores, is distinctive enough under the light microscope to have value for identification. This means that differential staining techniques, such as the Gram stain or acid-fast stain, and fluorescence microscopy may help to determine the iden-... 

Some bacteria measure as large as 80 p in length others as small as 0.2 ix. However, the majority of the commonly encountered bacteria, including the disease producers, measure about 0.5 n in diameter for the spherical cells and 0.5 by 2 to 3 p for the rod forms. Bacteria producing spores are generally larger than the nonspore-producing species. The sizes of some common species in dried and stained smears are as follows Escherichia coli, 0.5 by 1 to 3... 

Figure 4.6. A view of soil microorganisms obtained after removing and staining a slide buried in soil for several days. Actinomycetes (A), bacteria (B), spore (S), and fungi (F) can be seen clearly.

Iodine in a 1 20,000 solution is bactericidal in 1 minute and kills spores in 15 minutes. Tincture of iodine USP contains 2% iodine and 2.4% sodium iodide in alcohol. It is the most active antiseptic for intact skin. It is not commonly used because of serious hypersensitivity reactions that may occur and because of its staining of clothing and dressings. 

Figure 9 shows a germinating spore on a treated leaf surface, an appressorium positioned above a stoma and two epidermal cells which are attacked by the fungus. Their characteristic yellow stain indicates necrotic cells characteristic of a hypersensitive reaction. 

Green, L.C., LeBlanc, P.J. and Didier, E.S. (2000) Discrimination between viable and dead encephalitozoon cuniculi (microsporidian) spores by dual staining with Sytox Green and Calcofluor White M2R. Journal of Clinical Microbiology 38, 3811-3814. 

Some psilocybian mushrooms reveal a striking blue color characteristic in fresh specimens this can aid in identification along with other traits, such as spore color and size, appearance of die gills, etc. When these mushrooms are scratched or bruised by handling, they stain blue or, if the surface color is yellowish, greenish blue. Some of these mushrooms exhibit this stain naturally, perhaps because of the heat of the sun or the pressure of raindrops. 

Owing to its applications in homeland security, sterilization validation, and astrobiology, bacterial spore detection has become a hot field. However, direct detection of bacterial spores can be challenging for the same reasons that make endospores difficult to irradicate. The tough spore coat is impermeable to staining techniques, so most microscopy and flow cytometry methods are not useful. Endospores are also highly resistant to lysis, meaning that common DNA extraction protocols are difficult to perform. The lack of measurable metabolism renders microcalorimetry and cellular respiration techniques ineffective. 

It should also be mentioned that the majority of microbial airborne particles are dead or noncultivable , which means that they do not grow under the laboratory conditions chosen. This is, for example, true for microorganisms hke Stachybotrys chartarum. To fully survey the indoor microbial situation and to avoid overlooking fungal species which may be indicators of damage, additional samples have to be taken using methods hke direct microscopy which are not dependent on hving, cultivable cultures. Here, for example, microscopic slides with an adhesive surface are inserted in special impactor samplers. The airborne particles collected are stained in the laboratory. EspeciaUy spores with a characteristic shape hke those of S. chartarum are easily detected under the microscope. 

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