Searching for microbes Part II. Microscopical diagnostics II Gram staining + capsulla Author of the slideshow: Ondřej Zahradníček To practical education of VLLM0421c Contacs to myself: 777 031 969 zahradnicek@fnusa.cz ICQ 242-234-100 Contents of presentation A Tale (based on real story, but still more fiction than truth J) • There was a man from Denmark, named Christian Gram. He stained bacteria and was angry. Sometimes he stained a pacient‘s sample, but beside bacteria, epitheliae were also stained. „Awful epitheliae, they hide my bacteria!“, said he. • So he started a research. He wanted to find something that would stain him bacteria, but not epitheliae… To be continued Tale continues • He found, that when the sample is stained by gentiane or crystaline violet, and then binding of the dye to the cell wall is supported by Lugol Solution, bacteria do not decolorize even by alcohol. Epitheliae, on the other hand, decolorize. „Great!“ • But soon he saw, that with epitheliae, part of bacteria decolorize, too. „Stupid staining“, said he, drunk remaining decolorizing alcohol, and throught his work into the corner of the room. And end of the tale… • Some twenty years later, one young researcher found in a corner of a laboratory the dusty work of Mr. Christian Gram. • As he read it, he thougt – it is not bad, it only needs a bit more to be added. • And so, to the end of the processus, he added counterstain of safranin (or Gabbet = carbolfuchsin). Not only bacteria, but also epitheliae stained red, but he said – why not? To know, if epitheliae are present, may be quite usefull! • And so Gram staining of today was developped. Survey of methods in medical microbiology Survey of methods • Direct methods: We search for a microbe, its part or its product (e. g. a bacterial toxin) – Direct detection in specimen – we use the whole specimen (blood, urine, CSF etc.) – Strain identification – isolate determination • Indirect methods: We search for antibodies. An antibody is neither a part nor a product of a microbe – it is a macroorganism product, after being challenged by a microbe Survey of direct methods Microbiological laboratory Microscopy: review What we can see in a microscope • When we work with a strain, we can see one type of microbial cells • When we work with a specimen, we can see – microbes – sometimes no microbes, sometimes more than ten various species of organisms – cells of host organism – usually epitheliae, WBCs, sometimes RBCs and other cells – other structures, e. g. fibrin fibers, cellullar detritus etc. Types of microscopy • Electron microscopy – in viruses, rather research than routine diagnostics • Optical microscopy – Wet mount – large and/or motile organisms – Wet mount – dark field (mostly spirochets) – Fixated and stained preparations, e. g. • Gram staining – most important bacteriological stain • Ziehl-Neelsen staining – e. g. for TB bacilli • Giemsa staining – to some protozoa • Fluorescent staining – for better visualisation What we allready know • Microbes have various size. Yeasts are larger than bacteria, and bacteria than viruses • Bacteria have various shape (cocci, coccobacilli, bacilli of various shapes, spirochets) • Bacteria have various arrangement (clusters, chains, couples); cocci in chains should not be named „streptococci“, because they could be for example enterococci. • Some bacteria form endospores, that do not stain Comparison of size: yeast of genus Candida and bacterium Staphylococcus Gram stain: principle Bacterial cell wall • There are bacteria, that are mechanically strong, their cell wall is thick and simple. They are called gram-positive bacteria. • There are other bacteria, that are rather chemically strong, their cell wall is thiner, thin, but more complex. They are called gram-negative bacteria. • Besides these and those, there are also so named Gram non-staining bacteria. Gram-positive cell wall Gram-negative cell-wall Gram staining – principle 1 •Gram -positive bacteria have a thick peptidoglycan layer in the cell wall. – So, gentiane/crystallin violet binds more firmly to them, and… – …after confirmation of this bound by Lugol iodine solution… – …even alcohol is not able to decolorize them. •Gram-negative bacteria are decolorized by alcohol and then stained pink by safranin. Gram staining – principle 2 Other structures than bacteria: how do they stain? • Yeasts mostly stain violet like G+ bacteria. They have a cell wall of their own type, but in Gram staining it works similarly like that of Gram positive bacteria • Human cells stain mostly red, although nuclei may be partially blue Do not forget, that preparations of specimens might contain various fibers, cell detritus etc., and all preparations may contain staining artifacts, too. Sometimes they are very confusing and may be mistaken for bacteria by a non-experienced observer! Gram stained preparation Gram stain: procedure Part One: fixated preparation • We make a small saline drop • We sterilize your loop and wait until it stops to be too hot • We take some mass of microbes by your loop (stains A to E) • We mix in the drop • We sterilize your loop again and place back • We let the drop dry, or dry AROUND your burner • We fixate the slide by passing it THOUGH the flame of the burner • We move the slide to the sink for staining Part Two: proper Gram staining • Gentian/crystaline violet (20 –) 30 sec. • (rinse by tap water – not necessary) • Lugol (20 –) 30 sec. • (rinse by tap water –not ncessary) • Alkohol 15 (– 20) sec. • rinse by tap water!!! imporant! • Safranin 60 – 120 sec. • rinse by tap water • dry by filtration paper • microscopy as in Task One Mixture of gram-positive and gram-negative bacteria Capsule. Burri staining Capsule and biofilm • Capsule surrounds an individual bacterium or a couple of bacteria. It is not an integral part of a bacterial cell, rather a layer of molecules (mostly polysaccharides) that protect the cell. Usually negative staining is used (capsule is an unstained place on a stained background) • Biofilm is a complex layer, composed of bacteria, their capsullae and other material. Biofilm is much stronger than individual bacteria, living in so named planctonic form. Burri capsular staining In Burri staining, bacteria were stained red and the bacground by black ink. Capsule is the unstained place between the red bacterium and the black ink. The End Lugol iodine = mixed I[2] + KI Jean Guillaume Auguste Lugol (18 August 1786 – 16 September 1851) was a French physician. He was born in Montauban. He studied medicine in Paris and graduated MD in 1812. In 1819 he was appointed acting physician at the Hôpital Saint-Louis a post he held until he retired. Lugol was interested in tuberculosis and presented a paper to the Royal Academy of Science in Paris in which he advocated the use of fresh air, exercise, cold bathing and drugs. He also published four books on scrofulous diseases and their treatment (1829, 1830, 1831, 1834). He suggested that his iodine solution could be used to treat tuberculosis. This assertion attracted much attention at the time. Although not efficacious in treating tuberculosis, Lugol's iodine was successfully used to treat thyrotoxicosis by Plummer. http://en.wikipedia.org/wiki/Jean_Guillaume_Auguste_Lugol Prof. Christian Gram Lipopolysacharide of G– cell wall • It contains lipid A. This lipid is also called endotoxin. It is released when the cell is broken. It is an important factor of virulence. • In also contains the polysaccharide part. It is a virulence factor; it also contains body antigens (called also O-antigens). These antigens are often important in diagnostics (especially in enterobacteria like Escherichia coli or Salmonella)