Micro Lesson 3 - DEA 1135 Introduction to Microbiology...

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Unformatted text preview: DEA 1135 Introduction to Microbiology Lesson 3 - microscopy: techniques used to study microorganisms microorganisms the microscope the The microscope is the essential tool of the microbiologist. Because of the inability of the unaided human eye to perceive objects smaller than 0.1mm, a microscope is essential to see microbial cells and to determine their morphological (outline and shape) characteristics. used to see objects smaller than 0.1 mm based widely on Leeuwenhoek’s design 10x ocular 10x, 45x, 95x or 100x objective Leeuwenhoek’s microscope microscope The earliest microscope based on Leeuwenhoek’s design was extremely simple. It consisted of a single piece of ground glass fixed between two perforated metal plates and it could magnify objects about 400 times. compound microscope microscope •Today’s compound microscope employs a two­ lens system for magnification. • Light enters in and passes through the specimen and is magnified through a series of lenses. parts of a compound light microscope microscope •The eyepiece is called the ocular and usually has a power of 10X, which means it magnifies the object 10 times, and the other lens through which the object is viewed is called the objective. The objective can be several different powers but it usually cannot be any higher than 100X. A typical compound light microscope will typical have a 10X ocular and 3 objective lenses objective which can be rotated into position, a 10X low power objective, one a 45X high power objective, and a 95X or 100X oil immersion lens. To calculate the total magnifying power of To the microscope, multiply the ocular power by the objective power. For example: If the 10X objective is in place For for viewing, multiply the 10X ocular by the 10X objective and you get 100X magnification power. In order to view bacteria at high In magnification, the light must be concentrated and made stronger or the image will appear too dark, so the 95X or 100X oil immersion lenses must be used. A small drop of immersion oil, usually small cedar oil, is placed on the slide over the bacterial smear and the objective is slowly lowered into the oil and focus. The oil helps to concentrate the light passing concentrate through it so that the image can be viewed. through Newer compound microscopes have a spring in the oil immersion objective spring to protect the slide. If the objective is lowered too far and actually touches the slide, the spring keeps the slide’s cover plate from fracturing. Older microscopes didn’t have this feature and the slide and the bacterial smears were at risk of damage if the user wasn’t careful. compound microscope parts compound In addition to the optical parts, the compound microscope has several mechanical parts and an illuminating system. The mechanical parts include: 1. the body tube 2. a revolving nosepiece that houses the objectives 3. the stage 4. clips for the slides 5. coarse and fine adjustment knobs 6. an arm 7. a base. The coarse adjustment knob moves the body tube The over a greater vertical distance, bringing the specimen into approximate focus. The fine adjustment knob raises and lowers the The body tube slowly for finer focusing or resolution. Resolution is the precision and clarity of the image Resolution precision clarity under observation. The illuminating system of the microscope The illuminating consists of the mirror or a built-in lamp, a diaphragm and a condenser lens. If there is a mirror present, it directs the light from the external source through the diaphragm and onto the specimen. specimen. The diaphram regulates the amount of light diaphram entering the objective. The greater the magnification, the greater the amount of light needed. needed. resolution resolution •Magnification alone is of little value unless the enlarged image possesses increased detail and clarity. •The image clarity depends on the microscope’s resolving power or resolution, which is the ability of the lens to distinguish two adjacent points or objects at a particular distance apart. The resolving power depends on the wavelength of The wavelength the light source and the numerical aperture of the microscope. The shorter the wavelength of light and the greater The the numerical aperture, the better the resolving power of the microscope. Blue light has a shorter wavelength than a red Blue red light, so greater resolution can be achieved with a blue light source or using a blue filter over a normally white light source. normally The numerical aperture is a property of the lens The that describes the amount of light that can enter it. A larger numerical aperture allows the objective to larger be placed closer to the specimen for greater magnification. This is usually achieved using an oil immersion lens at 100X and a drop of cedar oil placed on the specimen slide. Remember the take-home point, Remember the greater the magnification, the greater greater the amount of light needed light for resolution. The cedar oil allows more light to be concentrated into the objective, but the condenser lens usually needs to be opened more to allow more light to pass through the condenser and the specimen. Fixation and staining Fixation Magnification only gets you part­way there as far as seeing the specimen. Even magnified under the compound microscope, most organisms appear almost structure less and transparent. To distinguish the fine details of the specimen, a series of chemical procedures called fixation and staining is performed. Fixation ensures that the organisms do not move around on the slide and the process of fixation can be as simple as spraying the specimen with hair spray to hold it firmly on the slide. Once the specimen is fixed to the slide, it is dyed with various stains that are specific for certain types of bacteria and their structures. This allows the specimen to be viewed as best as possible. Gram stain Gram A stain is a solution of dye that can be used to color objects to improve their visibility. Stains can be prepared that are very selective and will attach only to specific kinds of cells or parts of cells. In order for an object to be seen, it must contrast with its background. One simple staining method is to flood the specimen with methylene blue or crystal violet stain. This will allow the basic anatomical differences of the cells to be seen. A differential staining method uses multiple differential colors of stains to show differences in characteristics of different types of cells. characteristics The most widely used differential staining method is called a Gram stain. Gram This process divides bacteria into two major groups: Gram-positive and Gram-negative. Other staining methods help to visualize specific Other cellular structures like endospores, or if a bacteria causes a specific disease like tuberculosis or leprosy. tuberculosis bright field microscope microscope There are other types of microscopes that can be used. The basic type of microscope that we’ve already discussed is called a bright­field microscope. When the light is concentrated through the specimen, the resulting image appears as a shaded object in a bright field background. bright field microscope slide bright A dark­field microscope uses a dark­field ring to block direct light from the source of illumination so that the only light reaching the objective is light reflected from the source. This makes the specimen appear light on a dark background and allows the user to distinguish anatomical characteristics of the microorganisms. dark field microscope microscope dark field microscope slide dark phase contrast microscope microscope A phase­contrast microscope allows specimens to be alive, giving very definite contrast which can eliminate the need for fixation and staining. It’s based on the fact that different components of cells bend light in different ways and it can produce an image based on the differences in refracted wavelengths of light. (It can be found in dental offices to educate and motivate patients in their oral hygiene goals). Phase contrast microscope in dentistry dentistry Some dental offices/clinics will utilize this Some microscope by taking a sample of the patients plaque and creating a slide for the patient to view. This is often a motivating factor for patients to see first hand the type of living microorganisms they are harvesting in their mouth. phase contrast microscope slide phase •The electron microscope is a method of magnifying specimens by using beams of electrons rather than waves of light. • Since the wavelength of an electron beam is about 10,000 times smaller than the wavelength of a beam of ambient light, the electron microscope can magnify objects that are much smaller. electron microscope electron It was developed in 1940 and uses a It magnetically focused beam of electrons that travel through the specimen and produce an image on a fluorescent screen. produce The image can be photographed, developed and enlarged up to 250,000X or more. Because of the small size of a virus, this is the only method we have for viewing the smallest nonliving particles of smallest microbiology. microbiology. Scanning electron microscope photomicrograph of a root surface at 3000 x magnification. This is a sample photomicrograph from my thesis from Old Dominion University. This is a root surface with no instrumentation – used as the control. Note the irregular root surface and concretions from the plaque and calculus. Photomicrograph following scaling with a curet (again at 3000x magnification). Note the striations and deep grooves in the root surface. Photomicrograph following scaling with an ultrasonic thin precision insert. (3000x magnification) Note the smooth and clean root surface. electron microscope slide electron culturing culturing The ability to examine and study microorganisms depends largely on being able to grow or culture them in a controlled environment. Microorganisms require much of the same elements humans do for nutrients, carbon, hydrogen, oxygen, nitrogen, sulfur, phosphorus, potassium, magnesium, calcium, iron, sodium. The growth, or culture, of a given bacterium requires a culture medium that provides all the essential nutrients, the proper concentration of salts and ions, and the proper pH for optimal bacterial growth to occur. A pure culture is one that is free of any pure other type of microorganism other than the original microorganism. Isolation of pure cultures involves Isolation separating samples of microorganisms into individual cells that are then allowed to reproduce to form clones of the single clones microorganism. In order to get the microorganisms to grow, In we need a culture medium which can support their nutritional needs. MacConkey agar lactose fermentation MacConkey There are basically 2 types of culture media: synthesized and natural, and both can be used in a liquid or a solid form. The liquid media is available in test tubes or it can be solidified by the addition of agar and poured into petri dishes or test tubes. Special ingredients can be added to the media to support growth of certain types of bacteria, like RBCs from sheep, powdered hemoglobin, or chemicals that will allow selective growth of only one type of bacteria, like the bacteria that causes gonorrhea. This slide shows colonies of bacteria growing on solid media in a petri dish. The bacteria utilize the lactose in the media as they grow so the media turns clear. This helps us to know what type of microorganism is growing on the media. Some types of media allow differentiation of Some organisms, such as differentiating between bacteria that ferment lactose and those that don’t. The culturing media will contain the carbohydrate lactose, which is a source of nutrition for many bacteria, and a pH neutral dye that turns red when the acidity level drops below 6.8. below When the bacteria grow, if they can ferment or use lactose, the culturing media and the bacteria turn red because when the bacteria grow, they make acids as a metabolic bygrow, product. Any bacteria that can’t ferment lactose remain clear or colorless. So this type of culturing media is called a differential media. differential streaking streaking The most common method of isolating pure cultures is called streaking. Here a wire loop or cotton swab is first placed in contact with the source of microorganisms to be examined and then rubbed over one edge of the agar surface in a petri dish. A petri dish is a small flat container petri with vertical sides and a cover. It permits the liquid agar to harden into a readily available flat surface. The loop or swab deposits those The bacteria picked up from the source onto the surface of the agar. onto Next, a sterile wire inoculating inoculating loop is moved through the deposited bacteria and then streaked over about one-fourth of the untouched agar surface. This step deposits bacteria from the area inoculated by the swab along the lines of the streak. streak. The loop is sterilized and moved through the second streaking, followed by streaking over a fresh onestreaking fourth of the plate. This fourth procedure is repeated one more time. Each streak increasingly dilutes the dilutes population of bacteria until single cells are deposited along the streak lines. After incubation for several days, individual colonies will develop where single cells were deposited. Bacteria from each colony can then be transferred to a separate sterile medium to produce a pure culture. To ensure that the culture is pure, a second streak plate may be made from a single colony. This procedure is repeated until all the colonies appear identical and culture is assumed to be pure. streaking streaking biologic tests biologic •Once a pure culture is obtained, a series of tests can lead to identification of the isolated bacterium. • A trained microbiologist is able to select those colonies from the isolation media that are most likely to represent disease­ producing bacteria. The morphology and staining The characteristics can readily be determined by microscopic examination and a colony from a pure culture is then inoculated onto a variety of differential and selective media. variety Besides specifically identifying the microbe that is causing the disease, it is important to know which antibiotic will inhibit its growth and could be used for therapy. Antibiotic therapy is often performed performed • Because organism Because identification and antibiotic susceptibilities are often critical to proper patient care and the time required to obtain these data is an important factor in clinical clinical microbiology. • Using traditional procedures, Using it may require from several hours to several days to complete identification on most bacteria. • This factor becomes This significant in the care of a critically ill patient. • Today modern technology is applied in Today various forms to shorten the time required to obtain the needed information for optimal treatment of the patient. • Once the organism has been identified, the Once doctor will know what type of antibiotic to antibiotic administer to effectively treat the infection. •These are small disks that contain various antibiotics that are placed on a culture. The areas where the drug kills the microorganisms is very clear. This helps us to determine if a microorganism is susceptible to a particular drug. •Biochemical tests are not only useful for determining the type of a bacterium, but the tests are also useful in trying to prevent the disease the bacterium can cause. • In a later lecture, we’ll talk about the body’s ability to produce antibodies to certain diseases which prevent a person from getting a disease a second time, but research has given us the tools to prevent a person from getting some diseases a first time. • penicillin disks penicillin • Most of these vaccines that we get today were at Most one time tested on animals to verify the vaccine would work. Animal testing gives us the chance to develop a drug, test it and have it approved by the government before subjecting humans to it. • Recently, I saw a TV documentary about how Recently, sheep are used to produce antibodies to snake venom. These sheep are injected with minute amounts of the snake venom and over a period of time, they produce antibodies to the poisonous effects of the venom. • The blood is collected from the sheep, the The antibodies are isolated and then made into an injection for humans. Even though the injection may be fairly expensive, around $1000 for the shot, given the choice of life or death, it’s a small price to pay for the miracle a herd of sheep can provide. to The End The Reading Assignment: Chapter 2, Microscopy Chapter pp. 25-40 in your text pp. ‘Microbiology for the Health Sciences’ ...
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This note was uploaded on 08/19/2011 for the course DEA 1135 taught by Professor Guilford during the Spring '05 term at Gulf Coast Community College.

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