Microscopy 7
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7 MICROSCOPY Authors: Reviewer: Jeppe L. Nielsen Jiři Wanner Robert J. Seviour Per H. Nielsen 7.1 INTRODUCTION One widely applied method for studying the microbiology dihydrochloride, Neisser, Gram, Nile Blue) for detecting of activated sludge is microscopy. It provides an insight cell viability and intracellular accumulation of storage into the hidden world of microbes, which cannot be seen compounds including poly-hydroxy-alkanoates (PHA) otherwise by the naked eye. In this chapter we describe and poly-phosphate granules (poly-P) and in situ protocols for the microscopic examination of activated identification of targeted microbial populations using sludge samples. These include staining techniques to give Fluorescence in situ Hybridization (FISH). Combinations an understanding the huge taxonomic and functional of these techniques provide powerful tools for elucidating diversity found among the microbes in activated sludge. metabolic features of cells at a single cell level. Combining Direct observations and staining can enable the differences FISH with staining techniques is often problematic, and so between bacterial, fungal and protozoan populations to be careful planning is required to ensure that the distinguished. Studying these microorganisms effectively interpretation of this information is unequivocal. This requires the correct use of the microscope to reveal chapter provides the basis for carrying out standard differences in their shape and size and to diagnose cellular protocols. structures. Standard light microscopes with appropriate performance for routine purposes are available from 7.2 THE LIGHT MICROSCOPE several manufacturers, but using more sophisticated microscopic techniques can enhance the level of The purpose of the microscope is to provide sufficient information generated. magnification to distinguish between the objects examined. The most commonly used microscope is a In this chapter the basic principles of the light and bright field microscope that projects a focussed beam of fluorescence microscope are explained and methodologies light onto the image on a glass slide. Nowadays, almost all for relevant staining techniques and data interpretation are these microscopes are (i) binocular (Figure 7.1), meaning provided. The aim of the experimental protocols outlined that both eyes are used to view the object, making here is to serve as a user-friendly guide for cell persistent use less tiring, and (ii) compound, where more morphological examination characterization, staining than one lens system is used to achieve the required sample techniques (e.g. DAPI: 4',6-diamidino-2-phenylindole resolution. © 2016 Jeppe L. Nielsen et al. Experimental Methods In Wastewater Treatment. Edited by M.C.M. van Loosdrecht, P.H. Nielsen, C.M. Lopez-Vazquez and D. Brdjanovic. ISBN: 9781780404745 (Hardback), ISBN: 9781780404752 (eBook). Published by IWA Publishing, London, UK. 264 EXPERIMENTAL METHODS IN WASTEWATER TREATMENT Camera Lamp housing Camera adapter (light source for incident Eyepiece or reflected illumination) Binoculair photo tube Objective + Reflector filter holders for epifluorecense Condenser Filter receptable (e.g. polarizer) Objective α Light collector 2 α Rotating nosepiece aperture angle focus control Oblective(s) Specimen Stage Lamp housing plane Condenser diaphragm (light source for transmitted illumination) Condenser focus Condenser α Field diaphragm aperture angle Filters Course/fine focus control Condenser (b) Figure 7.1 The compound microscope. The eyepiece (ocular) lenses and the objective lenses provide the resolving and magnifying power of the Figure 7.2 The light path in microscopy illustrating the concept of the microscope, while the condenser lens system, by focussing numerical aperture and condensers. the light source onto the specimen (Figure 7.2), maximises the resolution of the systems by increasing the numerical aperture or light-capturing ability of the objective lenses. At the highest magnifications used (100×), because it Its position is critical for optimization of the performance, has a low light-capturing ability (high NA), it is necessary and therefore it should supply a cone of light capable of to increase the refractive index (RI) of the medium filling the objective lens aperture. The resolution reflects between the specimen and the objective lens. For light the ability of the microscope to discriminate between two microscopes, the highest resolution is obtained when the closely positioned entities. If they are closer than the condenser aperture angle matches the objective. Lenses resolution distance then they appear blurred in the in air as a medium cannot have NA that exceed 0.65, microscope image. Actual size, image size and whereas in water and certain immersions such as oils the magnification are related as follows: objective lens NA can be increased theoretically to ca. 1.515, and the resolution of the microscope is then approx. Image size = Actual size · Magnification Eq. 7.1 0.2 µm, suitable for viewing/studying most bacteria, but The distance between two distinct objects is referred not viruses. to as the resolution d and its relationship to numerical Reflections and loss of illumination intensity can be aperture (NA) and wavelength (λ): eliminated by using immersion oil with a RI that matches 1.22 · λ 1.22 · λ the RI of the lens glass. The NA of an objective is also d = or d = Eq. 7.2 N · sin α NAobjective+NAcondenser partially dependent upon the amount of correction for any optical aberration. Highly corrected objectives have Where, much larger NA for the respective magnification (Table λ is the light wavelength; 7.1). α equals one-half of the objective's opening angle and; N is the refractive index of the immersion medium used below the objective lens. MICROSCOPY 265 Table 7.1 Examples of objectives and their numerical aperture (NA) and 7.2.2 Low power objective optical correction. At the first setup of the microscope for optimal Magnification Plan Achromat Plan Fluorite Plan Apochromat performance, one should always start using a low (NA) (NA) (NA) magnification, e.g. 10× objective or lower. The eyepieces 2× 0.06 0.08 0.10 should be adjusted to a position suitable for viewing with 4× 0.10 0.13 0.20 both eyes. Then the next steps should be followed: 10× 0.25 0.30 0.45 • Lower the stage and insert the prepared slide so that 20× 0.40 0.50 0.75 the specimen is centred in the cone of light after 40× 0.65 0.75 0.95 passing through the condenser. 40× (oil) n/a 1.30 1.40 • Lower the lens until it is located a few millimetres 63× 0.75 0.85 0.95 from the slide surface. Look down the ocular while 63× (oil) n/a 1.30 1.40 carefully turning the coarse focus knob until the 100× (oil) 1.25 1.30 1.40 image becomes visible, and then by adjustment with the fine focus knob bring the specimen image into sharp focus. 7.2.1 Standard applications of light • Adjust the iris diaphragm to optimise the contrast to where the image is sharp. With the eyepiece taken microscopy 2 3 out, check that approx. 3 to 4 of the back lens is light-filled. If not, adjust the condenser position until The microscope must be set up and used properly to the iris diaphragm is sharply focussed and, if generate high quality images. The routine for setting up necessary, readjust the iris diaphragm until it the microscope as described below is recommended. superimposes on the circle of light. The best More experienced users may omit some of the steps in resolution is when the condenser is raised to near its the procedure as they may have gained familiarity with maximum height. the microscope and the samples under examination • Reinsert the eyepiece and examine the preparation already. Microscopes are precision instruments, sensitive using the fine focus adjustment and adjustable and expensive to repair or replace. Thus, they require mechanical stage. careful handling and maintenance. The following advice should be observed: • Avoid large temperature variations, store the 7.2.3 High power objective equipment in a cool and dark place, and protect it from dust. Always use a dust cover when it is not in use. In a good quality microscope the objectives should be • Objective lenses must be cleaned using a lens tissue. parfocal, meaning that all the objectives have very similar Do not dry wipe any lens as this may lead to focus settings. • scratching. Begin by blowing off dust or any loose Rotate the nosepiece to the high power objective and material with a pressurized optical duster. then the fine adjustment to sharpen the image. • • Be careful not to spread residual immersion oil onto Adjust the iris diaphragm to increase the illumination other oculars or clean objectives. Use a commercial and optimise contrast and then re-examine the cleaner or solvent (e.g. 70 % ethyl alcohol - EtOH) specimen. to remove any oil or grease. Flooding or placing solvents directly onto the lens is discouraged, and a 7.2.4 Immersion objective lens tissue must always be used. • Keep the microscope clean at all times. Check the required medium designated for the high • Objectives immersed in oil must be cleaned magnification objective (usually written on the objective immediately after use by wiping with a lens tissue. If or indicated by a black (oil) or blue (water) ring). Add a not, the oil may dry and thus need removal with a small drop of immersion oil or water (< 2 cm in diameter) suitable lens cleaning solution. Do not use any on the slide while rotating the objective lens into place. solvents (e.g. alcohol) as this might lead to lens The space between the slide and the lens should be filled damage. with the immersion medium. Some microscopes are only equipped with oil or water objectives. These should be used by adding a small drop 266 EXPERIMENTAL METHODS IN WASTEWATER TREATMENT of the required medium onto the slide and slowly lowering • Use the correct thickness for the cover slip.