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lab summary on preparing a bacterial smear, simple staining, microscopy, cellular morphology. 300 words minimum.
lab summary on preparing a bacterial smear, simple staining, microscopy, cellular morphology. 300 words minimum.
11 Preparing a bacterial smear Good bacterial smears are essential for good Gram stains, a critical method in characterizing and identifying bacteria. In summary, you clean the slide, label the slide, add a drop of water, mix cells into the water, allow the smear to air dry, and heat fix the dry smear. You should only use cells grown on agar media to perform stains. Broth-grown-cells can be used for staining, but I do not suggest this if it is avoidable, especially for Gram stains. Debris from the broth media will interfere with image clarity, and perhaps staining characteristics. This being said, some procedures require the use of cells grown in broth, and certain fastidious (picky) organisms can only be grown in broth, but that is another story. 1. Start with a clean microscope slide. Even if your slide is new from the box it is coated with oil and should be washed. Wet your thumb and forefinger. Dip these into a detergent powder such as Alconox. Wet the slide and rub vigorously with the detergent, being careful not to cut your fingers on the edge of the slide. Rinse the slide thoroughly and dry it with a paper towel. The slide should now be “squeaky clean.” Remove excess lint from the slide that came from the paper towel as it can be problematic later on. If the slide is heavily stained or has many scratches discard it and use another. 2. Place a small piece of tape on one end of the slide. The tape must be no wider than the width of the slide and preferably no longer than ½ inch. Tape that overlaps to the bottom of the slide will “jack up” that end of the slide when it is placed on the microscope stage. The tape is used to label the contents and position of each smear on the slide, and to indicate the top side of the slide. It is possible to place 4 smears on a single slide, but 3 smears allows for larger smears. Space the smears evenly on the slide, but not too near the slide edge (can cause problems when trying to rotate objectives on the scope). Place a small spot of water (about the size of a golf ball dimple or mustard seed) on the top of the slide where you intend to place a smear. To avoid water contaminated with particulates, simply allow the water to run for a few seconds before collecting a drop. Use a sterile loop to collect the water. An entire loop full of water is too much. Lightly “flip” the loop to remove much of the volume of the drop or the smear will take a LOOOONG time to dry. A loop of water that cannot be seen if the loop is held on edge is perfect. Use the corner of a paper towel to soak up water if you still have too much. Tape 3. Using aseptic technique, collect a VERY SMALL AMOUNT of cells from an isolated colony (one that is separated from all others) if one is available. SEE IMAGES: Isolated colonies and Mixed culture. Isolated colonies will likely not be present if you are working with an agar slant. It is a natural tendency for new students to get too many cells. You will likely do this yourself until you have repeated the procedure and been reprimanded a few times. Simply touching the loop to the cells should be sufficient. If you can see the cells on the loop you probably have far too many. On the other hand, for “newbie” students with little or no experience on the scope a thick smear may prove (I hate to admit) beneficial. They are easier to focus on, but after a few slides you need to tighten up and make nice thin smears. Why then do I emphasize thin smears? Viewing the characteristic size, shape and arrangement (cellular morphology) of a cell with other cells piled on top does not work well at all. Furthermore, thick smears do not Gram stain properly. Now, gently mix the cells into the small drop of water for ~3 seconds using a circular motion. DO NOT over-mix! Over-mixing will disturb cellular arrangement, which can cause confusion later, especially if you have a Gram positive coccus. We will talk more about this later. If your slide was cleaned properly your smear should spread out easily on the slide (like water on an un-waxed car), but it doesn’t always work this way. Don’t worry if it doesn’t work. The completed smear should be roughly the size of a dime or a little smaller. 4. Air dry the smear COMPLETELY by waving it back and forth, or by laying it on the bench a waving across the top of it with a piece of paper, etc. When you think the smears are dry, wave another minute to make sure. Is it taking a long time to dry? Now you understand why I said to use SMALL drops of water! Now you are ready to heat fix your smear. SEE IMAGES: heat fixation 1 & 2. Hold the slide between your thumb and index finger with the smear side up. Pass the slide through the flame 3 or 4 times such that the entire slide is heated evenly. Your fingers should get warm but not burned. You are not trying to “cook” the cells. If the smear is not completely dry, heat fixation will burst the cells, and you will have to start over. The 3 functions of heat fixation are: 1) to kill the cells (or at least render them non-viable) 2) to insure that the cells stick firmly to the slide 3) to increase the amount of stain that the cells will accept Look at your completed smears. Can you see them on the slide? If you can barely see the smear in a few spots it may not be too thick. Practice making more smears. Keep in mind that you should use small drops of water, that you need few cells and that the smear must be totally dry before heat fixation. As mentioned above, agar slants will most likely be used in lab as a culture source. If so, it is unlikely that isolated colonies will be present. When possible, it is best to use a petri dish in order that isolated colonies may be used. NOTES: * Avoid making smears from broth cultures. It is best to use isolated colonies from a Petri dish. * Always take cells from an isolated colony if possible. This means it is better to make smears using cells from plates rather than from slants. * Thin smears are good smears. Thick smears make the accurate determination of cellular morphology difficult, and they make for inconsistent Gram stains. *Smears MUST be completely dry before heat fixation. * Avoid collecting cells from “disturbed” agar – debris from the media will cause problems in a smear. *“Newbies” should probably make their first smear and stain using a relatively easy organism to see, such as Bacillus. You should not try to tackle tiny cells such as Alcaligenes until you have some experience. Microscopy Keys to success in microscopy: patience practice confidence read and/or ask if you have questions The microscopes in this lab are compound, binocular, brightfield, light microscopes. The term “compound” refers to the fact that light passes through 2 or more lenses between the light source and your eye. In our case, there are 2 lenses: an ocular lens or eyepiece, and an objective lens. The term “binocular” (vs monocular) indicates the presence of two ocular lenses. There are 2 basic types of microscopes: light and electron. Light microscopes depend on the passage of light through or around the specimen and into our eye. Common types of light microscopes are brightfield, darkfield, phase contrast and fluorescence. In brightfield microscopy, we view contrast between 1.the light passing through the subject (the transmitted light) and 2. the surrounding material (water, resin, air, etc.) based upon a difference in refractive index between the two (i.e. the bending or “refraction” of light as it passes from one medium to another, such as from air to water). Parts of the microscope: be familiar with these parts of the microscope; the location and function of each *eyepiece (ocular) objective *stage clip *condenser focusing knob??? *revolving nosepiece *stage adjustment knobs *course adjustment knob *objective lenses *light condenser *fine adjustment knob *mechanical stage *condenser diaphragm *light source Handling and care of the microscope: These scopes are expensive and fragile pieces of equipment. Certain procedures MUST be followed during their use. I will be unhappy if you do not. Follow these 4 rules in this order EVERY time that you use the microscope. 1. Use 2 hands when carrying the microscope; one on the arm and the other under the base. 2. Place the scope firmly on the working surface, at least 6 inches from the edge where it will not be knocked of the table, and not too near the bunsen burner. 3. Displace the objective from center and THEN move the nosepiece away from the stage. From this “safe position” you can now clean lenses, add or remove microscope slides on the stage, etc. This is done to avoid damaging and objective. 4. Before and AFTER using the scope, clean the ocular and objective lenses with LENS PAPER. Kimwipes are NOT lens paper. Take care to avoid scratching the lenses. Cleaning lenses before you start will improve the image, but that is up to you. Cleaning immersion oil off of the oil immersion objective is absolutely necessary after EVERY use! You don’t want to get caught not doing this. 5. The mechanical stage should be cleaned thoroughly with paper towels after use. Do Nots: * Do not remove ocular lenses or any other portion of the microscope. * Do not “scoot” your microscope. To move the scope, pick it up with 2 hands. * Never use the course adjustment knob on any objective other than the low power (10X). * Do not use alcohol or any other solvents in an attempt to clean the lenses. * Never put immersion oil on any objective other than the oil immersion (100x) objective. Microscope terms and properties * The objective “viewing area” is simply the area beneath the objective when it is in clicked into position. It corresponds to the area where the circle of light passes from the lamp up through the slide. Viewing area is NOT synonymous with the term “field of view” below. * “Working distance” is the distance between the objective lens and the subject in focus. As the magnification of the objective lens becomes greater, the working distance becomes smaller. * Find a small asymmetrical object on your slide and view it using the 10X objective. What do you notice about the image? Using the stage adjustment knob move the object to your right. Which direction does the image appear to move? Move it down. Which direction does it appear to move? What can you conclude from your findings? Consider the way that the light transmitted light must enter the lens. * “Resolution” is the ability to distinguish 2 very small and closely spaced objects. Resolution increases with magnification. To see this point, look at 2 closely spaced cells under the 100x objective, then 40x. * If these microscopes were “parfocal”, a focused image would remain in focus when other objectives were rotated into position. Scopes gradually lose this precision after being used for some time. * The apparent magnification of an image, termed “total magnification”, is the product of the magnification of the ocular lens (10X) and the objective lens (10, 40, or 100X). What is the total magnification when using the 10X objective? What about the other objectives? * “Field of view” refers to the area of the light image viewed when looking into the scope (the diameter of the circle of light). Look at your smear through the 100x objective. Find distinguishable shapes at the extreme edges of opposite sides of the field of view. Now find these objects using the 40x objective. Are they still at the extreme edges of the field? What do you think the mathematical relationship between the field of view and magnification of the objectives is? Using the microscope: 1. Move objectives to the “safe position” as described above. 2. Place your slide flat on the mechanical stage and secure properly with the spring-loaded stage clip. 3. Before bothering to look through the ocular lens, position the subject (a dark area is best if you can see it) in the objective viewing area. Confirm that the condenser is positioned directly beneath the slide. 4. Place the 10X objective (a short one) in position. ALWAYS begin with the 10X objective. MAKE SURE that the objective is clicked firmly into position. While looking through the ocular, slowly decrease the distance between objective and subject with the course adjustment knob until focused (~5mm working distance). Examine the subject. Having trouble? If your subject is dim and you can find nothing to focus on, do one of the following. A) Position your 10X objective to give a working distance of ~5mm. While looking through the ocular, turn either stage adjustment knob repetitively in a back-and-forth motion so that the subject is passing through the objective viewing area. This will usually allow you to see faint subjects as they move back-and-forth. Now focused, line up the subject and continue with step #5. B) Line up an edge of the slide or cover slip (if you are using one) in the center of the viewing area. Focus on the edge, then move over to the subject using a stage adjustment knob. Continue with step #5. C) Using a dark marker, place a small dot on the slide. Focus on this dot, then move over to the subject using a stage adjustment knob. Continue with step #5. 5. Rotate the nosepiece so that the 100x objective is adjacent to viewing position, but in the safe position (it is “on deck”). Place a drop or 2 of immersion oil directly onto the smear, or on the cover slip if using one. Quickly rotate the 100x objective into position while the oil puddle is still heaping, and ensure that an oil bridge now conects the slide and 100x objective. Focus with the FINE ADJUSTMENT KNOB. 6. When finished with a slide, rotate to the safe position, move the nosepiece to the maximum distance from the stage, and ONLY THEN do you remove the slide from the stage. 7. Clean the lenses with LENS PAPER. Clean the stage with paper towels if necessary. 8. The light intensity can be adjusted using the condenser diaphragm. When viewing a subject with very little contrast it helps to “stop down” the amount of light passing through the condenser. NOTES: * Oil immersion objectives are expensive. Good scopes with good objectives are absolutely necessary for viewing bacterial cells, and they are hard to come by. The moral of this story is: take care of these scopes. Listen to me and follow my instruction on the use of the scope. See the “Handling and care..” section above. * I will say it again: always clean the oil off of the oil immersion objective after using the scope. * Feel free to keep good slides in your slide box. Build yourself a “slide library.” Simple staining of a bacterial smear Over 90% of the mass of a cell is water. For this reason most cells are near-colorless. When suspended in water cells are very difficult to see. By staining cells we can add contrast between the cell and its surroundings making the cells much easier to see under the brightfield microscope. Staining procedures can be classified in one of 3 groups: simple stains, differential stains and structural stains. In a simple stain procedure, the cells are stained with a colored dye that simply adds contrast which makes the cell easier to see on the background. Simple stains generally give no further information that could be used to differentiate between cell types. In the next lab session you will learn the Gram stain procedure, perhaps the best example of a differential stain. Differential stains involve the use of a combination of dyes that stain cell types differently as a result of anatomical differences between those cells. Structural stains may involve the use of one or more stains in combination that serve to distinguish certain structural parts of a cell, for example the endospore, flagella, and capsule stain procedures. A note about the stains: they stain everything, including clothes and skin, so be careful. Gloves can be worn to prevent staining fingers, otherwise the stain will wear off skin in a few days. Simple stain procedure: Use aseptic technique and the method for preparing a smear learned in the previous lab session to prepare smears. After cooling the heat-fixed slide, lay the slide on the staining tray with the smear side up. Choose the stain that you will use for your simple stain. Crystal violet is commonly used because it is dark, adding significant contrast. Drip 2 to 3 drops of crystal violet onto the smear to cover it. You do not need to cover the entire slide, only the smears themselves. Allow the stain to sit for 30 to 60 seconds, and then thoroughly rinse off the excess with a gentle stream of water. Blot (do not wipe) the slide in the book of bibulous paper 2 or 3 times, or until dry. Examine the smear: Does your smear look like a dark glob of stain? If so, it is too thick. The smear should be faintly purple and translucent. Notice the consistent and characteristic shape of the cells with each species viewed. This is what we call cellular morphology. Cellular morphology will be discussed further in later labs. You may be able to see endospores of Bacillus species. They appear as clear, unstained, round/ovoid spots either inside intact cells or outside those cells that have ruptured.