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Bacterial Morphology + Gram Stains

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Veterinary Nursing Flashcards on Bacterial Morphology + Gram Stains, created by Princess Banana Hammock on 04/06/2018.
Princess Banana Hammock
Flashcards by Princess Banana Hammock, updated more than 1 year ago
Princess Banana Hammock
Created by Princess Banana Hammock over 4 years ago
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Question Answer
Why do we stain bacteria? Most cells are colourless, consisting mostly of water, so can be hard to differentiate from their surroundings. Using simple, non-specific stains or dyes is a relatively fast, cheap, and straightforward method to add colour and contrast to cells (regardless of the exact type of cell) and therefore helps us to visualise the cells. With improved visualisation, we are able to differentiate between the shapes, sizes, and structures (e.g. cilia, flagella, internal organelles) of different types of bacterial cells. This in turn allows us to classify and identify different bacteria. Differential stains take all of this a step further by having distinctly different effects on different types of microorganism (e.g. a certain colour dye may stain only gram-positive bacteria whilst another colour stains only gram-negative bacteria), allowing for even faster and easier classifications of bacterial samples.
How do dyes and stains work? Dyes and stains contain 'chromophores', which are colour-imparting chemicals. Usually these are a type of salts with a single ion (electricity-conducting atom) of each molecule possessing the colour. (Salts are compounds composed of one positively charged ion to one negatively charged ion, e.g. Na+ and Cl- combine to form the sodium chloride salt, NaCl.)
How does the methylene blue stain work? Methylene blue chloride is a salt which, when added to water, splits into its two component ions. These are the blue-coloured, positively-charged methylene blue ion, and the colourless, negatively charged chloride ion.
What is a basic dye and how does it work? Basic dyes such as crystal violet, safranin, basic fuchsine, and methylene blue each have a positive charge. They therefore react with negatively charged materials. Bacterial cytoplasm can have a slight negative charge when its growth medium is of neutral or very close to neutral pH (7). When this is the case, the positively charged basic dyes are attracted to and bind with the negatively charged bacterial cytoplasm.
What is an acid dye and how does it work? Acid dyes such as nigrosine and Congo red contain negatively charged chromophores (colour-imparting ions). Having a negative charge, these are repelled by the (also slightly negatively charged, in some environments) bacterial surface. Thus they deposit around the organism, staining the background but leaving the microbe transparent, which allows us to easily identify through a microscope where in a specimen these microbes are.
What is the purpose of using oil immersion? This method can be used to improve the resolution (sharpness) of the image seen through the microscope. Without oil, the light passing through the slide can be refracted (redirected/scattered away from) the objective lens. Having the same refractive index as glass, oil prevents this refraction of light from happening, and in doing so aids in the production of a sharper, clearer image. Oil immersion is an essential technique for the identification of bacteria via a microscope. Note, however, that oil immersion should not be used with any objective lens other than the white x100 magnification lens.
How is oil immersion carried out? Find and focus on the specimen as normal, working your way up from the red objective lens (x4 magnification) to the blue objective lens (x40 magnification). Once the specimen is clearly visible and in the centre of the field of view under the blue objective lens, wind the stage back down (this will initially cause it to go out of focus, but that's ok). The objective nosepiece should be turned to an intermediate position between the red (lowest magnification) and white (highest magnification) objective lenses in order to create space. Now one small drop of oil is placed on the slide over the spot of light. The white objective lens (x100 magnification) is brought into position. Watching from the side, wind the coarse focus control clockwise until the lens makes contact with the slide and the drop of oil starts to compress. Looking through the microscope, the focus control is *very* slowly adjusted until a sharp image appears (the lens should never leave the oil during this process).
How does a bacterial cell differ from an animal cell? Genetic material is stored differently. In an animal cell, DNA is bound within a nucleus by a nuclear membrane but in the bacterium, DNA is present only in the form of circular DNA and sometimes of plasmids. Bacteria are prokaryotic cells which is a classification that essentially means that they don't have a defined nucleus. Bacteria also lack a variety of different organelles found within the animal cell including mitochondria, the Golgi apparatus, endoplasmic reticulum and lysosomes. The ribosomes found in the bacterial cell are a smaller type than those found in the eukaryotic animal cell.
What is the size range of bacteria? All bacteria are single celled, with the smallest spanning 0.1μm in diameter, and the largest spanning 6 x 60μm in diameter.
What are some of the most common bacterial cell shapes? Bacilli (rods), Cocci (small round/ovoid shape), and Spirilla (cylindrical).
Why is Gram staining used? Gram staining is a fast and straightforward way of differentiating between the two major classifications of bacteria: gram-negative and gram-positive. Bacterial cells are fixed on a glass slide and stained with crystal violet, then briefly rinsed in water. Iodine solution is then added and forms a complex with crystal violet in the cells. Alcohol or acetone is added to make the crystal violet-iodine complex soluble. Finally, cells are counterstained with safranin, rinsed with water and dried for microscopy. Gram positive bacteria appear purple under the microscope following gram-staining as they retain the crystal violet-iodine complex despite treatment with alcohol or acetone. Gram-negative cells are initially stained purple by the crystal violet, but the next step of alcohol/acetone treatment removes this colourisation. (This is because gram-negative bacteria have a much thinner cell wall than gram-positive bacteria). The addition of safranin then re-stains them pink.
What are the characteristics of gram-positive bacteria? Their main characteristic is a thick cell wall composed of numerous layers of peptidoglycan. Examples of gram-positive bacteria include listeria, streptococcus, and staphylococcus. Certain gram-positive bacteria produce exotoxins which can cause diseases such as tetanus and diphtheria.
What are the characteristics of gram-negative bacteria? The cell wall of gram-negative bacteria contains only a thin layer of peptidoglycan. This is surrounded by a thin outer membrane composed of lipopolysaccharide. Examples of gram-negative bacteria include Escherichia coli, Salmonella typhi, Vibrio cholerae, & Bordetella pertussis.
What are bacterial spores? Spores are bacterial reproductive cells with the potential to grow into new bacteria. They consist of the cell's genetic material and some cytoplasm surrounded by a thick outer coat which can survive environmental conditions which would kill fully-grown bacteria, such as boiling temperatures, only developing into adult bacteria once conditions are more favourable again.
What are the 8 basic elements of bacterial colony morphology? Colony shape, grouping, size, margin, elevation, opacity, chromogenesis, and surface texture.
How can bacteria be fixed onto a slide?
What are the potential shapes of bacterial colonies as seen by the naked eye?
What are the different types of margins a bacterial colony may possess?
What are the different potential types of elevation of a bacterial colony?
How can the size of a bacterial colony be described? Large, medium, small, or punctiform.
What terms can be used to describe the potential different surface textures of bacterial colonies? Smooth and glistening, rough, wrinkled, and/or dry and powdery.
What are some of the most common pigments/colourations displayed by bacterial colonies? White, red, purple, and buff (a shade of pale yellow-brown, like manila paper -- see image!)
Where bacterial colonies group together, what are the three main types of groupings observed? Pairs (diplo-), chains (strepto-), and clusters (staphylo-)
What are the 4 main terms used to describe the opacity of a bacterial colony? Transparent, opaque, translucent (like looking through frosted glass), and iridescent (changing colours due to the reflection of light).
What are the most commonly observed shapes of individual pathogenic bacterial cells? Coccus (spherical), bacillum (basic rod), vibrio (curved rod-shape with flagella; motile), and the spiralling, corkscrew-like bacterial shapes (spirillum and spirochaete).
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