micro lab ch2&3.pdf

Diversity and Ubiquity of Microorganisms Microorganisms are found everywhere that other forms of life exist. In Exercise 2-1, you will transfer microorganisms from seemingly uninhabited sources and grow them on agar plates....

Diversity and Ubiquity of Microorganisms Microorganisms are found everywhere that other forms of life exist. In Exercise 2-1, you will transfer microorganisms from seemingly uninhabited sources and grow them on agar plates. Each source is likely to have multiple species growing on or in it and represents a mixed culture. When you inoculate the plates you will be transferring untold numbers of unknown cells to them. Any cells that are able to grow on the plate's medium will divide and produce Ell visible colonies of identical cells. In Exercise 2-2 you will learn to identify some of the various growth characteristics produced by the "invisible" cohabitants from Exercise 2-1, as well as examining plates of known species. An ability to recognize differences in colony morphology is often the first clue to a microbiologist that two organisms are different species. If a colony is not contacting other colonies, it is said to be isolated and a portion of it can be transferred to a sterile medium to start a pure culture of the species, which is then frequently used in identifying the species. Pure cultures are usually grown in a broth or on a slanted medium. In Exercises 2-3 and 2-4 you will examine growth characteristics of bacteria on slants and in broth, respectively. + EXERCISE Ubiquity of Microorganisms 2-1 Theory Microorganisms have a long, rich history on Earth and capable of producing a disease state if introduced into a have successfully adapted to a wide range of habitats. The suitable part of the body. Any area, including sites outside literature on microorganisms often describes them as being of the host organism, where a microbe resides and serves "ubiquitous in nature." More specifically, this means that as a potential source of infection is called a reservoir. microorganisms of all sorts can be isolated from soil (of all sorts), water (over a large range of salinities), plants, and animals (including humans). Microbes are even found Application in apparently uninhabitable sites such as hot acid pools This exercise is designed to demonstrate the ubiquitous (Sulfur Caudron in Yellowstone is approximately 85 + °C nature of microorganisms and the ease with which they with a pH close to 1!). can be cultivated. (It should be noted that although we Many microorganisms are free-living-they do not can find living microorganisms virtually everywhere and reside on or in a specific plant or animal host and are confirm their presence by cultivation, molecular techniques not known to cause disease. They are nonpathogenic. developed over the last two or three decades demonstrate Frequently they are saprophytes and perform the impor- that the organisms successfully grown in the lab represent tant ecosystem role of decomposing organic matter. Other a minute fraction of those still uncultivable.) microorganisms reside on or in another species and benefit from the symbiotic association with their host(s)-they have a place to live and reproduce successfully. If they In This Exercise cause damage to their host, that is, they cause disease, Today, you will work in small groups to sample and culture they are pathogens of it. In other instances the microbe several locations in your laboratory. Your instructor may may actually benefit their host, an example of mutualism. have other locations outside of the lab to sample as well. Lastly, some microbes are commensals, where they benefit Remember that even relatively "harmless" bacteria, when but have no significant effect on their host. However, even cultivated on a growth medium, are in sufficient enough many of the commensal or mutualistic strains inhabiting numbers to constitute a health hazard. Treat them with our bodies are opportunistic pathogens. That is, they are care..I Materials Per Student Group D Eight nutrient agar plates D One sterile cotton swab IE Procedure Lab One 1 Number the plates 1 through 8. 2 Open plate number 1 and expose it to the air for 30 minutes or longer. Set it aside and out of the way of the other plates. 3 Use the cotton swab to sample your desk area and then streak plates 2 and 3 in the pattern shown in 2.1 Simple Streak Pattern on an Agar Plate + Roll the Figure 2.1. Press very lightly as you roll the swab swab as you collect the sample from the table and then again as on the table top and then again on the agar to you inoculate the plate. Do not press so hard that you cut the agar. inoculate it. Be sure the contaminated part of the swab contacts the agar so you actually transfer any organisms collected. Lab Two 4 Hold the plate directly in front of your mouth and 1 Using the plate diagrams on the data sheet, page 59, cough several times on the agar surface of plate 4. draw two representative colonies on each of your Be sure you aren't facing anyone when you cough. agar plates. Be sure to label them according to In fact, it is best to cough downward onto the plate incubation time, temperature, and source of to minimize spreading germs throughout the inoculum. It is best to select one colony from each classroom. Send them to the floor instead! plate to draw before doing the second one. That 5 Rub your hands together, and then touch the agar way you will have observed each plate in case time surface of plate 5 lightly with your fingertips. A runs short. Note: Do not remove the plate's lid if light touch is sufficient; touching too firmly will there is fuzzy growth on it or your instructor has crack the agar. Your fingers will feel gooey told you not to. If you are in doubt about "fuzzy afterward, but the agar is sterile so there should growth," ask your instructor. actually be fewer microbes on your fingers than 2 Save these plates in a refrigerator for use in Exercise before you touched it. 2-2. 6 Remove the lid of plate 6 and vigorously scratch your head above it. It's best to bend your head References forward so your hair is actually above the plate. Holt, John G., ed. Bergey's Manual of Determinative Bacterio logy, 9th ed. When inoculating, do so away from plate 1 to Baltimore: Lippincon Williams & Wilkins, 1994. avoid cross-contamination. "Sulphur Caldron." Available online. URL: http://www.yellow- 7 Leave plates 7 and 8 covered; do not open them. stoneparknet.com/geothermal_features/sulphur_caldron.php. Tille, Patricia M. Chap. 7 in Bailey & Scott's Diagnostic Microbiology, 8 Label the base of each plate with the date, type of 13th ed. St. Louis, MO: Mosby, 2014. exposure it has received, and the name or number Varnam, Alan H. and Malcolm G. Evans. Environmental Microbiology. of your group. Washington, DC: ASM Press, 2000. 9 Invert all plates and incubate them for 24 to 48 Winn, Washington C. et al. Koneman's Color Atlas and Textbook of hours at the following temperatures: Diagnostic Microbiology, 6th ed. Baltimore: Lippincott Williams & Wilkins, 2006. Plates 1, 2, and 8: 25°C Plates 3, 4, 5, 6, and 7: 37°C  Date - - - - - - - - - - - - - - - - - - - - - - - - - - - - Lab Section - - - - - - - - - - - - - - - - - - - - - - - - - - I was present and performed this exercise (initials) - - - - - - - - - - - - - Ubiquity of Microorganisms OBSERVATIONS AND INTERPRETATIONS 1 Use the circles below as Petri dishes. Then, for each plate, choose two different colonies and draw each as seen from above and from the side. Simple line drawings are acceptable, but do them with care. Chicken scratches are not very useful! Label the plates according to incubation time, temperature, and source of inoculum. Also include other useful colony information, such as color and relative abundance. l Save the plates for Exercise 2-2. Colony#1 Plate #1 _ _ _ _ _ _ _ _ _ _ _ __ Plate #2 _ _ _ _ _ _ _ _ _ _ _ __ Plate #3 _ _ _ _ _ _ _ _ _ _ _ __ Plate #4 _ _ _ _ _ _ _ _ _ _ _ __ SECTION 2 Microbial Growth 59 1. "----' "T"" ,. r _,..._..._. ) I..  Colony#2 IE Plate #5 _ _ _ _ _ _ _ _ _ _ _ __ Plate #6 _ _ _ _ _ _ _ _ _ _ _ __ Colony#2 Plate #7 _ _ _ _ _ _ _ _ _ _ _ __ Plate #8 _ _ _ _ _ _ _ _ _ _ _ __ 60 MICROBIOLOGY: Laboratory Theory & Application... -:. ·...... ~- Date-------------------------- DATA SHEET Lab Section - - - - - - - - - - - - - - - - - - - - - - - - 2-1 I was present and performed this exercise (initials) - - - - - - - - - - - - (continued) QUESTIONS 1 Consider plates 7 and 8. a. What was the purpose of incubating the unopened plates? Be specific. b. What is an appropriate name for these plates? c. If growth appears on both unopened plates, what are some likely explanations? d. What if growth appears on only one plate? e. How does growth on the unopened plates affect the reliability (your interpretation) of the other plates? SECTION 2 Microbial Growth 61  2 Why do you think the specific types of exposure (air, hair, tabletop, etc.) were chosen for this exercise? IE 3 Consider plates 2 and 3. a. Did you get different-appearing colonies on plates 2 and 3? If so, explain why. b. What is the likely source (reservoir) of organisms that grew best at 37°C, and how do they survive at room temperature without nutrients? 4 Suppose plate 4 (cough) has no growth after incubation. It is highly unlikely the "cougher" has sterile coughs! Suggest reasons why no growth was recovered on the plate. 5 The plates you are using for this lab will be autoclaved to completely sterilize them. The measures taken to disinfect the tabletops (the source of the organisms on plates 2 and 3) are not as extreme. Why? 62 MICROBIOLOGY: Laboratory Theory & Application... -:. ·...... ~-  EXERCISE Introduction to the Light Microscope 3-1 Theory Bright-field microscopy produces an image made from appears below or within the microscope. The amount of light that is transmitted through a specimen (Fig. 3.2A). magnification that each lens produces is marked on the lens 11!'111111 The specimen restricts light transmission and appears (Figs. 3.4A and 3.4B). Total magnification of the specimen 1:.111111 "shadowy" against a bright background (where light enters can be calculated by using the following formula: the microscope unimpeded). Because most biological Total = Magnification by the X Magnification by the specimens are transparent, the contrast between the Magnification Objective Lens Ocular Lens specimen and the background can be improved with the application of stains to the specimen (Exercises 3-5 The practical limit to magnification with a light through 3-11 and 3-13). The "price" of the improved microscope is around 1300 X. Although higher magnifi- contrast is that the staining process usually kills cells. cations are possible, image clarity is more difficult to This is especially true of bacterial-staining protocols. maintain as the magnification increases. Clarity of an Image formation begins with light coming from an image is called resolution (Fig. 3.5). The limit of resolution internal or an external light source (Fig. 3.3 ). It passes (or resolving power) is an actual measurement of how far through the condenser lens, which concentrates the light apart two points must be for the microscope to view them and makes illumination of the specimen more uniform. as being separate. Notice that resolution improves as the Refraction (bending) of light as it passes through the limit of resolution is made smaller. objective lens from the specimen produces a magnified The best limit of resolution achieved by a light mi- real image. This image is magnified again as it passes croscope is about 0.2 µm. (That is, at its absolute best, a through the ocular lens to produce a virtual image that light microscope cannot distinguish between two points N 3.2 Types of Light Microscopy + (A) This is a bright-field micrograph of an amoeba (called a "whole mount") Because of its thickness, the entire organism is not in focus at once. Continually adjusting the fine focus to clearly observe different leve ls of the organism wi ll give a sense of its three-dimensional structure. The nucleus (N) is obvious, as are the numerous cyanobacteria (C), both inside and outside the amoeba! Other granular material also is seen in the cytoplasm but notice its different texture toward the periphery. (B) This is a dark-field micrograph of the same amoeba. Notice the more three-dimensional image and that the peripheral cytoplasm is barely visible. (C) This is a phase contrast image of the same amoeba. Different parts of the interior and its detail are visible than what is seen in the other two micrographs. (D) This is a fluorescence micrograph of Mycobacterium kansasii. The apple green is one of the characteristic colors of fluorescence microscopy.  Condenser IIJ Real image (formed by objective lens) 3.3 Image Production in a Compound Light Microscope + Light from the source is focused on the specimen by the condenser lens. It then enters the objective lens, where it is used to produce a magnified real image. The real image is magnified again by the ocular lens to produce a virtual image that is seen by the eye as being below or within the microscope. (After Chan, et al., 1986) 3.4 Markings of Magnification and Numerical Aperture on Microscope Components + (A) Three plan apochromatic objective lenses on the nosepiece of a light microscope. Plan means the lens produces a flat field of view. Apochromatic lenses are made in such a way that chromatic aberration is reduced to a minimum. From left to right, the lenses magnify 10 x, 20 x, and 40 x, and have numerical apertures of 0.40, 0.70, and 0.85. The 20 x lens has other markings on it The mechanical tube length is the distance from the nosepiece to the ocular and has become standardized at 160 mm. However, this 20 x lens has been corrected so the light rays are made parallel, effectively creating an infinitely long mechanical tube length (co). This allows insertion of accessories into the light path w ithout decreasing image quality. The thick- ness of cover glass to be used is also given (0 17 ± 0.01 mm). Also notice the standard colored rings for each objective: yellow for 10 x, green for 20 x (or 16 x ), and light blue for 40 x (or 50 x ). (B) This is an oil-immersion lens. It is the only lens constructed in such a way as not to be damaged by oil and, as such, is the only one w ith w hich oil is to be used. Oil lenses are indicated by black-and-white rings. This particular lens is also constructed for phase contrast microscopy. It indicates it is to be used w ith the #3 setting on the phase condenser. (C) A 10 x ocular lens. (D) A condenser (removed from the microscope) w ith a numerical aperture of 1.25. The lever at the right is used to open and close the iris diaphragm and adjust the amount of light entering the specimen. closer together than 0.2 µm.) For a specific microscope, are the same as the units for wavelength, which typically the actual limit of resolution can be calculated using the are in nanometers (nm). following formula: Numerical aperture is the measure of a lens's ability to A "capture" light coming from the specimen and use it to D = make the image. As with magnification, it is marked on NAcondenser + NAobjective the lens (Figs. 3.4A, 3.4B, and 3.4D). Using immersion oil where D is the minimum distance at which two points between the specimen and the oil-immersion lens increases can be resolved, A is the wavelength of light used, and its numerical aperture and, in turn, makes its limit of NAcondenser and NA biecrive are the numerical apertures of 0 resolution smaller. (If necessary, oil also may be placed the condenser lens and objective lenses, respectively. between the condenser lens and the slide.) The result is Because numerical aperture has no units, the units for D better resolution.  Application Light microscopy (used in conjunction with cytological stains) is used to identify microbes from patient specimens or the environment. It also may be used to visually examine a specimen for the presence of more than one type of bac- teria, or for the presence of other cell types that indicate tissue inflammation or contamination by a patient's cells. In This Exercise Today you will become familiar with the operation and Ill limitations of your light microscope. You also will examine two practice slides to learn about microscope functioning..I Materials 3.5 Resolution and Limit of Resolution + The headlights of D Compound light microscope most automobiles are around 1.5 m apart. As you look at the cars in D Lens paper the foreground of the photo, it is easy to see both headlights as separate objects. The automobiles in the distance appear smaller D Non-sterile cotton swabs (but really aren't) as does the apparent distance betw een the head- D Lens-cleaning solution or 95% ethanol lights. When the apparent distance between automobile headlights D Letter "e" slide reaches about 0.1 mm, they blur into one because that is the limit of resolution of the human eye. D Colored-threads slide The light microscope may be modified to improve its lnstrudions for Using the Microscope ability to produce images with contrast without staining, Proper use of the microscope is essential for your success in which often distorts or kills the specimen. In dark-field microbiology. Fortunately, with practice and by following microscopy (Fig. 3.2B), a special condenser is used so only a few simple guidelines, you can achieve satisfactory results the light reflected off the specimen enters the objective. quickly. Because student labs may be supplied with a vari- The appearance is of a brightly lit specimen against a dark ety of microscopes, your instructor may supplement the background, and often with better resolution than that of following procedures and guidelines with instructions the bright-field microscope. specific to your equipment. Refer to Figure 3.1 as you Phase contrast microscopy (Fig. 3.2C) uses special read the following (if working independently), or follow optical components to exploit subtle differences in the along on your microscope as your instructor guides you. refractive indices of water and cytoplasmic components (Note: This is a thorough treatment of microscope use, to produce contrast. Light waves that are in phase (that and not all parts may be immediately relevant to your is, their peaks and valleys exactly coincide) reinforce one laboratory. Refer back to this exercise as necessary.) another, and their total intensity (because of the summed amplitudes) increases. Light waves that are out of phase Transport by exactly one-half wavelength cancel each other and 1 Carry your microscope to your workstation using result in no intensity-that is, darkness. Wavelengths both hands-one hand grasping the microscope's that are out of phase by any amount will produce some arm and the other supporting the microscope be- degree of cancellation and result in brightness that is less neath its base. than maximum but more than darkness. Thus, contrast 2 Gently place the microscope on the table. is provided by differences in light intensity that result from Cleaning differences in refractive indices in parts of the specimen that put light waves more or less out of phase. As a result, 1 Lens paper is used for gently cleaning the condenser the specimen appears as various levels of "darks" against and objective lenses. Light wiping is usually enough. a bright background. If that still doesn't clean the lens, call your instructor. Fluorescence microscopy (Fig. 3.2D) uses a fluorescent 2 To clean an ocular, moisten a cotton swab with dye that emits fluorescence when illuminated with ultra- cleaning solution and gently wipe in a spiral motion violet radiation. In some cases, specimens possess naturally starting at the center of the lens and working out- fluorescing chemicals and no dye is needed. ward. Follow with a dry swab in the same pattern.  Basic Operation 9 Scan the specimen to locate a promising region to 1 Raise the substage condenser to a couple of milli- examine in more detail. (Note: If your microscope meters below its maximum position nearly even has a field diaphragm, now would be a good time to with the stage (be sure not to raise it too high if you set it up for Kohler illumination, which is described have already placed a slide on the stage) and open on page 147.) the iris diaphragm. 10 If you are observing a nonbacterial specimen, progress 2 Plug in the microscope and turn on the lamp. Adjust through the objectives until you see the degree of the light intensity slowly to its maximum. structural detail necessary for your purposes. You will have to adjust the fine focus (but not the coarse focus IIJ 3 Using the nosepiece ring, move the scanning objective (usually 4 X) or low-power objective (10 X) into position. Do not rotate the nosepiece by the objectives because the lenses are parfocal-all that should be necessary after changing lenses is a slight adjustment) because this can damage the objective lenses and and illumination for each objective. Before advancing cause them to unscrew from the nosepiece. to the next objective, be sure to position a desirable portion of the specimen in the center of the field or 4 Place a slide on the stage in the mechanical slide you will risk "losing" it at the higher magnification. holder and center the specimen over the opening in the stage. 11 If you are working with a bacterial smear, you will have to use the oil-immersion lens. 5 If using a binocular microscope, adjust the distance between the two oculars as you examine the specimen 12 Follow these instructions to use the oil-immersion to match your own interpupillary distance. Position lens. your eyes above the oculars so the images from the Work through the low (10 X ), then high-dry (40 X) two oculars fuse into one. objectives, adjusting the fine focus and illumination for each. Before advancing to the next objective, be 6 Adjust the iris diaphragm to produce optimum illu- sure to position a desirable portion of the specimen mination, contrast, and image. In the simplest sense, in the center of the field or you risk "losing" it at this means opening the iris diaphragm as you increase the higher magnification. magnification because a smaller portion of the light When the specimen is in focus under high dry, ro- beam is entering the lens. (More specifically, use the maximum light intensity combined with the smallest tate the nosepiece to a position midway between aperture in the iris diaphragm that produces optimum the high-dry and oil-immersion lenses. Then place illumination. Remember: This is bright-field micros- a drop of immersion oil on the specimen. Be careful copy, so don't close down the iris diaphragm too not to get any oil on the microscope or its lenses, much unless necessary to see detail, as in unstained and be sure to clean it up if you do. Rotate the oil specimens.) lens so its tip is submerged in the oil drop, pass through it, and then return the oil lens into the oil. 7 Use the coarse-focus adjustment knob to bring the This minimizes the occurrence of air bubbles. image into focus. (Note: For most microscopes, the Note: Do not move the stage down to add oil to distance from the nosepiece opening to the focal plane the slide or the specimen will no longer be in of each lens has been standardized at 45 mm. This focus. On a properly adjusted microscope, the makes the lenses parfocal and gives the user an idea of oil and the high-dry lenses have the same focal where to begin focusing.) Bring the image into sharp- plane. Therefore, when a specimen is in focus on est focus using the fine-focus adjustment knob. Then high dry, the oil lens, although longer, will also be observe the specimen with your eyes relaxed and in focus and won't touch the slide when rotated slightly above the oculars to allow the images to fuse into position.) into one. If you are using a monocular microscope, Focus and adjust the illumination to maximize keep both eyes open anyway to reduce eye fatigue. the image quality. 8 If you are using a binocular microscope, adjust the oculars' focus to compensate for differences in visual 13 When you are finished, lower the stage (or raise the objective) and remove the slide. Dispose of the freshly acuity of your two eyes. Close the eye with the ad- prepared slides in a jar of disinfectant or a sharps justable ocular and bring the image into focus with container; return permanent slides to storage. the coarse- and fine-focus knobs. Then, using only the eye with the adjustable ocular, focus the image using the ocular's focus ring.  Kohler Illumination Kohler illumination was developed in 1893 by August and/or center the condenser using the centering Kohler while he was a graduate student in invertebrate screws on it (see step 6). zoology. It produces uniform and high-quality illumination 5 Adjust the height of the condenser until the edges of the specimen, but it requires components not found on of the field diaphragm come into focus (Fig. 3.6D). all microscopes. If your microscope has a field diaphragm Should you see red and blue light fringes as you raise (Fig. 3.6A), you will need to adjust it for Kohler illumina- and lower the condenser, settle on the intermediate tion to get the best image your microscope can produce. point between them. 1 Place a prepared slide, preferably a thin section of 6 Once the field diaphragm is in focus, you will need 11!'111111 a specimen, but college microbiology labs may not to center the condenser. Use the centering screws 1:.111111 have these readily available. For today's lab, you may (Figs. 3.6£ and 3.6F) located on the condenser to use the letter "e" slide or another slide provided by move it until the opening is nearly centered. Then, your instructor. We have used a blood smear slide for open the field diaphragm until it almost fills the illustration. After this lab, you will mostly be exam- field of view and do a final adjustment with the ining bacterial smears and you can use them. The centering screws. specimen and field condenser may not be in focus. 7 Open the field diaphragm until it just disappears 2 Make sure the lamp is on. Open the field diaphragm from the field of view (Fig. 3.6G). to its maximum. Do the same with the iris diaphragm. 8 Adjust the light with the iris diaphragm so the 3 Bring the specimen into focus at low (10 X) power specimen is optimally illuminated (Fig. 3.6H). (Fig. 3.6B). Adjust the light with the iris diaphragm 9 You will need to make these adjustments for each if it is too bright. objective lens. However, when examining bacterial 4 Completely close the field diaphragm so you can smears you will be using the lower-power objectives see its edges (Fig. 3.6C). Depending on how out of only to focus and center the specimen; only the oil- adjustment your microscope is, the edges of the immersion lens will be used for serious examination. field diaphragm may only be a dark, blurry shadow, This means that once you set up the oil lens for as in the lower left of Figure 3.6C or you may not Kohler illumination, you can leave it there. High- be able to see them all. If you can't see the edges, quality illumination is not needed for the lower you may need to raise the condenser (see step 5) powers. Kohler Illumination Kohler illumination allows focusing of the light source on the specimen, which produces more uniform illumination. All photos in this sequence were shot with the same microscope light intensity. The only adjustments made are those written in the captions as part of the process. 3.6A The Field Diaphragm The field diaphragm is located 3.68 Bring the Specimen into Focus Notice the uneven in the microscope's base. It can be adjusted by rotating the ring illumination of this field. It is darker on the left than on the right, around the lamp (arrow). which means it needs adjusting. For perspective, the circled white blood cells are the ones seen in Figures 3.6D and 3.6F. IIJ 3.6( Close the Field Diaphragm + Closing the field dia- 3.6D Adjust the Condenser Height until the Edges of the phragm has cut down the amount of light entering the objective Field Diaphragm are in Focus + What was barely visible in lens, making the image dark. The actual light intensity setting on Figure 3.6C now is illuminated at the same level as in Figure 3.6B the microscope is the same in this micrograph as in all the others and is still in focus. (except Fig. 3.6H). 3.6E Use the Condenser Centering Screws (Arrows) to 3.6F Center the Field Diaphragm + Notice that it is the Center the Light in the Field of View + This may take some lighted area that has moved, not the slide itself. Compare this practice, because they apply and release pressure on the con- lighted area with Figure 3.6B. denser to move it in different directions. 3.6G Open the Field Diaphragm until It Just Goes Out of 3.6H Adjust the Light With the Iris Diaphragm + For View+ Readjust the centering if it is a little off. bright-field microscopy, the background should be white, but not so bright as to lose detail. Note the more uniform illumination compared to Figure 3.6B. Storage 8 Examine the colored-threads slide without the When you are finished for the day: microscope (Fig. 3.7). See if you can tell where in 1 Move the scanning objective into position. the stack of three threads each color resides. That is, is the red thread on the top, bottom, or middle? Do 2 Center and lower the mechanical stage. the same for the yellow and blue threads. Record 3 Lower the light intensity to its minimum, and then your observations on the data sheet. turn off the light. 9 Now, place the slide on the microscope and deter- 4 Wrap the electrical cord according to your particular mine the order of the threads using the low- and lab rules. high-power objectives. Record your observations 11!'111111 5 Clean any oil off the lenses, stage, etc. Be sure to on the data sheet. 1:.111111 use only cotton swabs or lens paper for cleaning any of the optical surfaces of the microscope (see "Cleaning," page 145). 6 Return the microscope to its appropriate storage place. Procedure 1 Get out your microscope and record the magnifica- tions and numerical aperture values in the chart on the data sheet, page 151. 2 Clean your microscope lenses as outlined in "Instructions for Using the Microscope." 3 Plug in the microscope and position the scanning objective over the stage. Make condenser and lamp adjustments appropriate for scanning power. 4 Pick up the letter "e" slide and examine it without the microscope. Record the orientation of the letter when 3.7 Challenge of the Threads + Even w ith the microscope, the slide label is on the left. Sketch the orientation of determining the order of threads from top to bottom is a challenge. the letter "e" on your data sheet. This will require patience and use of the fine focus I Making it wo rse, not all the slides will be the same. Good luck I 5 Place the slide on the stage in the same position as you examined it with your naked eyes. Now, center the "e" in the field and examine it with the scanning References objective. After focusing, sketch the orientation of Abramowitz, Mortimer. Microscope Basics and Beyond. Olympus Amer- the letter "e" image as viewed with the microscope ica Inc. Melville, NY: Scientific Equipment Group, 2003. on your data sheet. (If your microscope has a field Ash, Lawrence R. and Thomas C. Orihel. Pages 187-190 in Parasites: A condenser, you should adjust it for Kohler illumina- Guide to Laboratory Procedures and Identification. Chicago: Ameri- can Society for Clinical Pathology (ASCP) Press, 1991. tion after focusing.) Bradbury, Sa vile and Brian Bracegirdle. Chap. 1 in Introduction to Light 6 Now, move the stage to the right and record the Microscopy. Oxford, UK: BIOS Scientific Publishers Limited, 1998. direction the image moves. 1 Forbes, Betty A., Daniel F. Sahm, and Alice S. Weissfield. Pages 119-121 in Bailey & Scott's Diagnostic Microbiology, 11th ed. St. Louis, MO: 7 Position the "e" in the center of the field again. Move Mosby, 2002. the stage toward you and record on your data sheet the direction the image moves. Then remove the slide from the microscope. 1 If your microscope doesn't have a mechanical stage, move the slide with your hands in the appropriate direction.  Date - - - - - - - - - - - - - - - - - - - - - - - - - - - Lab Section - - - - - - - - - - - - - - - - - - - - - - - - - I was present and performed this exercise (initials) - - - - - - - - - - - - - Introduction to the Light Microscope DATA AND CALCULATIONS 1 Record the relevant values off your microscope and perform the calculations of total magnification for each lens. Magnification of Magnification of Total Numerical Calibration of Objective Lens Ocular Lens Magnification Aperture Ocular Micrometer Scanning Low power High dry Oil immersion Condensor lens NA NA NA NA l Sketch your observations of the letter "e" slide in the table below. Be sure the slide is right side up with the label at the left. When the stage moves When the stage moves Appearance of the "e" Appearance of the "e" to the right, the image toward you, the image with the Naked Eye Under the Microscope moves to the... moves... ] Record your observations of the colored-threads slide below. Check it under low and high power and see if your answer changes. Top: Low power (usually the 10 x objective) Middle: Bottom: Top: High power (usually the 40 x objective) Middle: Bottom: SECTION 3 Microscopy and Staining 151 '...,....., - s. t ·.. "--,.,,' ,_,,. :-: --~:_.1 -.  QUESTIONS 1 Why aren't the magnifications of both ocular lenses of a binocular microscope used to calculate total magnification? IIJ 2 What is the total magnification for each lens setting on a microscope with 15 X oculars and 4 X, 10 X, 45 X, and 97 X objectives lenses? 3 Assuming that all other variables remain constant, explain why light of shorter wavelengths will produce a clearer image than light of longer wavelengths. 4 Why is wavelength the main limiting factor on limit of resolution in light microscopy? 5 On a given microscope, the numerical apertures of the condenser and low-power objective lenses are 1.25 and 0.25, respectively. You are supplied with a filter that selects a wavelength of 520 nm. a. What is the limit of resolution on this microscope? b. Will you be able to distinguish two points that are 300 nm apart as being separate, or will they blur into one? 152 MICROBIOLOGY: Laboratory Theory & Application 1,,,- _.._ ' :I".... :..1:.,. > ! ''\..,1  Date - - - - - - - - - - - - - - - - - - - - - - - - - - - DATA SHEET Lab Section - - - - - - - - - - - - - - - - - - - - - - - - - 3-1 I was present and performed this exercise (initials) - - - - - - - - - - - - - (continued) 6 On the same microscope as in question 5, the high-dry objective lens has a numerical aperture of 0.85. a. What is the limit of resolution on this microscope? b. Will you be able to distinguish two points that are 300 nm apart as being separate, or will they blur into one? 7 Calculate the limit of resolution for the oil lens of your microscope. Assume an average wavelength of 500 nm. 8 Examine Figure 3.3 and explain the results you observed with the letter "e" slide. 9 With which objective was it easier to determine the sequence of colored threads? 1Q Why should closing the iris diaphragm improve your ability to determine thread order? SECTION 3 Microscopy and Staining 153 '...,....., - s. t ·.. "--,.,,' ,_,,. :-: --~:_.1 -.  11 What does the colored-threads slide demonstrate about specimens you will be observing later in the class? IIJ 154 MICROBIOLOGY: Laboratory Theory & Application 1,,,- _.._ ' :I".... :..1:.,. > ! ''\..,1  EXERCISE Calibration of the Ocular Micrometer 3-2 Theory An ocular micrometer is a type of ruler installed in th e microscope eyepiece, composed of uniform but unspeci- fied graduations (Fig. 3.8). As such, it must be calibrated before any viewed specimens can be measured. The device used to calibrate ocular micrometers is called a stage micrometer. As illustrated in Figure 3.9, a stage micrometer 2001,1m is a type of microscope slide containing a ruler with 10 µm and 100 µm graduations. Other measuring instruments + + 50 m may be used in place of a stage micrometer, as shown in Figure 3.10. When the stage micrometer is placed on the stage, it is magnified by the objective being used; therefore, the size of the graduations (relative to the ocular micrometer di- visions) increases as magnification increases. Consequently, the value of ocular micrometer divisions decreases as 3.10 Hemacytometer + Any instrument w ith markings of magnification increases. For this reason, calibration must known distance apart may be used as a stage micrometer. The he- macytometer is a grid w ith lines 50 µm apart (red arrows). A larger be done for each magnification. grid is formed by lines 200 µm apart (blue arrows). Use any horizontal As shown in Figure 3.11, the stage micrometer is line as the micrometer, w ith the smallest divisions 50 µm apart. placed on the stage and brought into focus such that it is superimposed by the ocular micrometer. Then the first 0 10 20 30 40 50 (left) line of the ocular micrometer is aligned with one of 0 10 20 30 40 50 1,11 , 1111111111111 1111 1111111111111111111 1111111111 IIIIII 3.11 What They Look Like in Use + When properly aligned, I I I I I I I I I I I I I I I I I I I I I I I I I 111111111111111 the ocular micrometer scale is superimposed over the stage mi- crometer scale. Notice that they line up at their left ends. These 3.8 Ocular Micrometer + This illustration representing an two micrometers have intentionally not been drawn to the correct ocular micrometer shows a scale w ith uniform increments of proportions relative to each other because they are intended only unknown size. It has to be calibrated for each objective lens on to show how to use them. When you work w ith your microscope the microscope. and stage micrometer, expect different numbers to align. o I mm oo 1mm [] 111111111111111111111 I I I I I I I I I I I I I I I I I I I I 3.9 Stage Micrometer + (A) The stage micrometer is a microscope slide w ith a microscopic ruler engraved into it (not visible in the dark center of the slide) The markings on this micrometer indicate that the major increments are 0.1 mm (100 µm) apart. There is also a section of the scale that is marked off in 0.01 mm (10 µm) increments. (B) This drawing represents what the stage micrometer on the slide in A looks like. The micrometer is 2,200 µm long. The major divisions are 100 µm apart. The 200 µmat the left are divided into 10 µm increments.  the marks on the stage micrometer. (The line chosen on larger when changing from low power to oil immersion. the stage micrometer depends on the power of the lens But, because the magnification of the ocular micrometer being calibrated. Lower powers use the large graduations; does not change, an ocular division now covers only higher powers use the smaller graduations on the left. one-tenth the distance. Thus, the size of an ocular unit Figure 3.11 illustrates proper alignment with the scanning using the oil-immersion lens can be calculated by dividing objective.) the calibration for low power by 10. Notice in Figure 3.11 that line 25 of the ocular mi- Ocular micrometer values can be calculated for any crometer and the eighth major line of the stage micrometer lens using values from any other lens, and they provide a are perfectly aligned. (Note: This is a hypothetical situation. good check of measured values. As calculated from Figure IIJ Your stage and ocular micrometers won't align in the same way.) This indicates that 25 ocular micrometer divisions 3.11, 32 µm/OU was the calibration for the scanning objective of a hypothetical microscope. For practice, cal- (also called ocular units, or OU) span a distance of 800 µm culate the low, high-dry and oil-immersion calibrations. because the stage micrometer lines are 100 µm apart. Write the values in Table 3-2. Notice also that line 47 of the ocular micrometer is aligned You are given the calibration of the ocular micrometer with the fifteenth major stage micrometer line. This means of the scanning objective for the hypothetical microscope that 47 ocular units span 1,500 µm. These values have been as calculated in the text (Table 3-1). You are also given the entered for you in Table 3-1. total magnifications produced when using the other ob- To determine the value of an ocular unit on a given jectives. Use this information to calculate the remaining magnification, divide the distance (from the stage microm- calibrations as described in the text. eter) by the corresponding number of ocular units. TABLE 3-2 Using one Calibration to Calculate the Others- 800 µm 32 µm A Sample Problem 25 ocular units OU Total Calibration Power Magnification (11m/OU) 1,500 µm µm µm 3 9 32 Scanning 40X 32 47 ocular units = 1. OU = OU Low Power 100x As shown in Table 3-1, it is customary to record more High-Dry Power 400X than one measurement. Each measurement is calculated separately. If the calculated ocular unit values differ, use Oil Immersion 1000x their arithmetic mean as the calibration for that objective lens. Once you have determined the ocular unit values for each objective lens on your microscope, use the ocular TABLE 3-1 Sample Data from Figure 3.11 micrometer as a ruler to measure specimens. For instance, Stage Ocular if you determine that under the scanning objective a cell Micrometer Micrometer Calibration is 5 ocular units long, the cell's actual length would be 32 µm determined as follows (using the sample values from 800 µm 250U Table 3-2): OU Cell Dimension = Ocular Units X Calibration 31.9 µm = 32 µm 1,500 µm 47 OU Cell Dimension = 5 Ocular Units X 32 µrn/OU OU OU Cell Dimension = 160 µm Be sure to include the proper units in your answer! As mentioned previously, each magnification must be calibrated. Because of its short working distance, calibrat- ing the oil-immersion lens may be difficult to accomplish Application using the stage micrometer. It also may be difficult because The ability to measure microbes is useful in their identi- the distance between stage micrometer lines is too large. fication and characterization. If this is the case, its value can be calculated using the calibration value of one of the other lenses. Refer to Table 3-2 for the total magnifications for each objective In This Exercise lens on a typical microscope. Notice that the magnification This lab exercise involves calibrating the ocular micrometer of the oil-immersion lens is 10 times greater than the on your microscope. Actual measurement of specimens will low-power lens. This means that objects viewed on the be done in subsequent lab exercises as assigned by your stage (stage micrometer or specimens) appear 10 times instructor../ Materials 5 Change to low power and repeat the process. Per Student 6 Change to high-dry power and repeat the process. o Compound microscope equipped with an ocular 7 Change to the oil-immersion lens and repeat the micrometer process. Be sure to look at the oil lens from the side o Stage micrometer as you rotate it into position. If it looks like it is going to contact the stage micrometer, stop! Return the high-dry lens into position and complete the calibration from the value of another lens. You will Procedure also need to calculate the oil lens' calibration if the 11!'111111 Following is the general procedure for calibrating the stage micrometer lines are too far apart for direct 1:.111111 ocular micrometer on your microscope. Your instructor measurement. will notify you of any specific details unique to your laboratory. 8 Compute average calibrations for each objective lens and record these on the data sheet. 1 Check your microscope and determine which ocular has the micrometer in it. 9 As long as you keep this microscope throughout the term, you may use the calibrations you recorded 2 Move the scanning objective into position. without recalibrating the microscope. 3 Place the stage micrometer on the stage and posi- tion it so its image is superimposed by the ocular micrometer and the left-hand marks line up. References Abramoff, Peter and Robert G. Thompson. Pages 5-6 in Laboratory 4 Examine the two micrometers and, as previously Outlines in Biology-III. San Francisco: W. H. Freeman, 1982. described, record two or three points where they Ash, Lawrence R. and Thomas C. Orihel. Pages 187-190 in Parasites: line up exactly. Record these values on the data A Guide to Laboratory Procedures and Identification. Chicago: sheet, page 159, and calculate the value of each American Society for Clinical Pathology (ASCP) Press, 1991. ocular unit.  Date - - - - - - - - - - - - - - - - - - - - - - - - - - - Lab Section - - - - - - - - - - - - - - - - - - - - - - - - - I was present and performed this exercise (initials) - - - - - - - - - - - - - Calibration of the Ocular Micrometer DATA AND CALCULATIONS 1 Record two or three values where the ocular micrometer and the stage micrometer line up for the scanning, low, high-dry, and oil-immersion objective lenses. Then calculate the calibration for each. Be sure to include proper units in your calibrations. Scanning Objective Lens Stage Micrometer Ocular Micrometer (µm) (OU) Calibration Low-Power Objective Lens Stage Micrometer Ocular Micrometer (µm) (OU) Calibration SECTION 3 Microscopy and Staining 159 '...,....., - s. t ·.. "--,.,,' ,_,,. :-: --~:_.1 -.  High-Dry Objective Lens Stage Micrometer Ocular Micrometer (µm) (OU) Calibration IIJ Oil-Immersion Objective Lens Stage Micrometer Ocular Micrometer (µm) (OU) Calibration 2 Calculate the average value for each calibration and record them in the table below. If necessary, use one of the values to calculate the calibration of the oil lens. Be sure to include proper units in your answer. Average Calibrations for My Microscope Objective Lens t... Scanning Lower power High-dry power Oil immersion 160 MICROBIOLOGY: Laboratory Theory & Application 1,,,- _.._ ' :I".... :..1:.,. > ! ''\..,1  EXERCISE Simple Stains 3-5 Theory ) Stains are solutions consisting of a solvent (usually water ~o or ethanol) and a colored molecule (often a benzene deriva- tive), the chromogen. The portion of the chromogen that gives it its color is the chromophore. A chromogen may (. J Ill have multiple chromophores, with each adding intensity to the color. The auxochrome is the charged portion of a chromogen and allows it to act as a dye through ionic or covalent bonds between the chromogen and the cell. Basic stains 1 (where the auxochrome becomes positively charged as a result of picking up a hydrogen ion or losing a hydrox- ide ion) are attracted to the negative charges on the surface of most bacterial cells. Thus, the cell becomes colored (Fig. ) r (" 3.82). Common basic stains include methylene blue, crystal violet, and safranin. Examples of basic stains may be seen ) in Figures 3.69, 3.80, and 3.83. F Basic stains are applied to bacterial smears that have (' been heat-fixed. Heat-fixing kills the bacteria, makes them adhere to the slide, and coagulates cytoplasmic proteins to 3.83 Safranin Dye in a Simple Stain + This is a simple stain make them more visible. It also distorts the cells to some using safranin, a basic stain. Notice that the stain is associated with extent. the cells and not the background. The organism is Rhodospirillum rubrum grown in broth culture. Application Because cytoplasm is transparent, cells usually are stained In This Exercise with a colored dye to make them more visible under the Today you will learn how to prepare a bacterial emulsion microscope. Then cell morphology, size, and arrangement (smear) and perform simple stains. Several different can be determined. In a medical laboratory, these are usu- organisms will be supplied so you can begin to see the ally determined with a Gram stain (Exercise 3-7), but you variety of cell morphologies and arrangements in the will be using simple stains as an introduction to these. bacterial world. We suggest that you perform all the stains on one or two organisms (to get practice) and 1 Notice that the term basic means " alkaline," not " elementary"; however, look at your lab partners' stains to see the variety of cell coincidentally, basic stains can be used for simple staining procedures. types. Be sure that you view all the available organisms../ Materials Per Student Group D Clean glass microscope slides Apply basic stain (Positive Chromogen e +) D Methylene blue stain D Safranin stain D Crystal violet stain Negatively charged cell Cell is stained D Disposable gloves D Chemical eye protection 3.82 Chemistry of Basic Stains + Basic stains have a posi- D Squirt bottle with water tively charged chromogen ( +), which forms an ionic bond with the negatively charged bacterial cell, thus colorizing the cell. D Staining tray  D Staining screen o Bibulous paper (or paper towels) D Slide holder D Compound microscope with oil-immersion lens and 1. Place a small drop of water (not too much) on a clean slide using an inoculating loop. If you are ocular micrometer staining from a broth culture, begin with Step 2. D Immersion oil o Lens paper D Recommended organisms: IIJ Bacillus cereus Micrococcus luteus 2. Aseptically add bacteria to the water. Note: If you find that you are transferring too much organism with a loop, use an inoculating needle. If you are transferring a BSL-2 organism, use a sterile Neisseria sicca wooden stick or a disposable loop and dispose of it properly when Rhodospirillum rubrum finished. Up to three emulsions can comfortably be made on a single slide. Mix in the bacteria and spread the drop out. Avoid Staphylococcus epidermidis spattering the emulsion as you mix. Flame your loop when done. Vibrio spp. 3. Allow the smear to air dry. If prepared correctly, the smear should be slightly cloudy. Procedure 1 A bacterial emulsion (smear) is made prior to most staining procedures. Follow the procedural diagram in Figure 3.84 to prepare smears of two different organisms on a slide. Be sure not to mix them. Make a second and third slide with the remaining four organisms at the same time so they can be air drying simultaneously. Emulsions should be about 4. Using a slide holder, pass the smear through the upper part of the size of a dime. a flame two or three times.This heat-fixes the preparation. Avoid overheating the slide because aerosols may be produced. 2 Heat-fix each slide as described in Figure 3.84. If you are using a bacterial incinerator, hold the slide (with a slide holder) near the opening for a few seconds (Fig. 3.85). 5. Allow the slide to cool, then continue with the staining protocol. 3 Following the basic staining procedure illustrated in 3.84 Procedural Diagram: Making a Bacterial Smear the procedural diagram in Figure 3.86, stain one of (Emulsion) + You will apply this technique as the first step in per- your slides with each stain using the following times: forming most stain procedures (the negative and capsule stains are the exceptions). It is an important skill to learn, because preparation crystal violet: stain for 30 to 60 seconds of uniform bacterial smears will make it easier to obtain consistent safranin: stain for up to 1 minute staining results. Caution: Avoid producing aerosols. Do not spatter the smear as you mix it, do not blow on or wave the slide to speed methylene blue: stain for 30 to 60 seconds up air-drying, and do not overheat when heat-fixing. If working with Be sure to wear gloves when staining. Record your a BSL-2 organism, use a wooden stick or disposable loop to transfer actual staining times in the table provided in the the organism to the slide and heat-fix by holding the slide near the data sheet, page 189, so you can adjust for over- or opening of a bacterial incinerator (Fig. 1.14). under-staining. 4 Using the oil-immersion lens, observe each slide. Record your observations of cell morphology, ar- rangement, and size in the chart provided on the data sheet. 5 Dispose of the slides and used stain according to your laboratory's policy. 3.85 Heat-fixing a BSL-2 Organism + In order to reduce aerosol producti on, heat-fixing a slide can be done by hol ding it near the opening of a microincinerator or over the grill for 20-30 seconds. This is an absolute requirement if staining a BSL-2 organism. 1. Begin with a heat-fixed emulsion (see Figure 3-84). 2. Wearing gloves, place the slide on a rack over a staining More than one organism can be put on a slide. In this tray. Cover the smear(s) with the stain. Make sure any exercise, we recommend putting three organisms on excess stain falls into the staining tray. each slide. Stain disposal 3. Grasp the slide with a slide holder and hold it on an angle. 4. Gently blot dry in a tablet of bibulous paper or paper towels. Gently rinse the slide with distilled water into the staining tray. (Alternatively, a page from the tablet can be removed and used Dispose of stain in the tray at the end of lab according to your for blotting.) Do not rub. When dry, observe under oil immersion. lab's practices. 3.86 Procedural Diagram: Simple Stain + Staining times differ for each stain, but cell density of your smear also affects staining time. Strive for con sistency in making your smears. You also only need to cover the emulsion(s) on the slide, not the slide's w hole surface. Caution: Be sure to flame your loop after cell tran sfer and properly di spose of the slide w hen you are finished observing it.  References Chapin, Kimberle. Chap. 4 in Manual of Clinical Microbiology, 6th ed. Norris, J. R. and Helen Swain. Chap. II in Methods in Microbiology, Vol. Patrick R. Murray, Ellen Jo Baron, Michael A. Pfaller, Fred C. Tenover, SA. J. R. Norris and D. W. Ribbons, eds. London, UK: Academic Press, and Robert H. Yolken, eds. Washington, DC: American Sociery for Ltd., 1971. Microbiology, 1995. Power, David A. and Peggy J. McCuen. Page 4 in Manual of BBL"' Chapin, Kimberle C. and Patrick R. Murray. Pages 257-259 in Manual Products and Laboratory Procedures, 6th ed. Cockeysville, MD: of Clinical Microbiology, 8th ed. Patrick R. Murray, Ellen Jo Baron, Becton Dickinson Microbiology Systems, 1988. James H. Jorgensen, Michael A. rfaller, and Robert H. Yolken, eds. Tille, Patricia M. Pages 70- 71 in Bailey & Scott's Diagnostic Microbiology, Washington, DC: American Sociery for Microbiology, 2003. 13th ed. St. Louis, MO: Mosby, 2014. Murray, R. G. E., Raymond N. Doetsch, and C. F. Robinow. Page 27 in IIJ Methods for General and Molecular Bacteriology. Philipp Gerhardt, R. G. E. Murray, Willis A. Wood, and Noel R. Krieg, eds. Washington, DC: American Sociery for Microbiology, 1994.  Date - - - - - - - - - - - - - - - - - - - - - - - - - - - Lab Section - - - - - - - - - - - - - - - - - - - - - - - - - I was present and performed this exercise (initials) - - - - - - - - - - - - - Simple Stains OBSERVATIONS AND INTERPRETATIONS 1 Record your observations in the table below. Stain and Cellular Morphology and Arrangement Organism Duration (include a sketch) Cell Dimensions SECTION 3 Microscopy and Staining 189 '...,....., - s. "--,.,,' ,/ _,,~f I. '... "  QUESTIONS 1 What are some consequences of leaving a stain on a bacterial smear too long (over-staining)? 2 What are some consequences of not leaving a stain on a smear long enough (under-staining)? IIJ 3 Choose a coccus and a bacillus from the organisms you observed and calculate their surface-to-volume ratios. Consider the coccus to be a perfect sphere and the bacillus to be a cylinder. Use the equations supplied. Cell Morphology Surface Area Volume Coccus SA= 4'1Tr 2 SA= _!_'1Tr 2 3 Bacillus SA = 2'1Trh + 2'1Tr 2 V = '1Tr 2 h r = radius, h = height, 7T = 3.14 Surface-to-Volume Ratio of Sample Cells Cell Surface Area Volume Surface-to-Volume Organism Morphology (µm2) (µm3) Ratio 4 Consider a coccus and a rod of equal volume. a. Which is more likely to survive in a dry environment? Explain your answer. b. Which would be better adapted to a moist environment? Explain your answer. 190 MICROBIOLOGY: Laboratory Theory & Application 1,,,- _.._ ' :I".... :..1:.,. > ! ''\..,1  EXERCISE Negative Stains 3-6 Theory.-.-.-.-.-.-.-.-.-.-.-.-Ill The negative staining technique uses a dye solution in Apply acidic stain which the chromogen is acidic and carries a negative (Negative Chromogen. - ) charge. (An acidic chromogen gives up a hydrogen ion, - which leaves it with a negative charge.) The negative charge on the bacterial surface repels the negatively charged chromogen, so the cell remains unstained against Negatively charged cell.-.-.-.-.- Background is stained a colored background (Fig. 3.87). A specimen stained with the acidic stain nigrosin is shown in Figure 3.88. 3.87 Chemistry of Acidic Stains + Acidic stains have a negatively charged chromogen ( - ) that is repelled by negatively charged cells. Thus, the background is colored and the cell remains transparent Application The negative staining technique is used to determine morphology and cellular arrangement in bacteria that are too delicate to withstand heat-fixing. A primary example is the spirochete Treponema, which is distorted by the heat- fixing of other staining techniques. Also, where determining the accurate size is crucial, a negative stain can be used because it produces minimal cell shrinkage. In This Exercise Today you will perform negative stains on three different organisms. You also will have the opportunity to compare the sizes of Bacillus cereus, Micrococcus luteus, and Rhodospirillum rubrum measured with the negative stain to their sizes as determined using a simple stain../ Materials 3.88 Nigrosin Negative Stain + Notice that the Bacillus Per Student Group megaterium cells are unstained against a dark background. The circular objects are bubbles. D Nigrosin stain or eosin stain D Clean glass microscope slides D Disposable gloves D Chemical eye protection Procedure D Compound microscope with oil-immersion lens 1 Follow the procedural diagram in Figure 3.89 to and ocular micrometer prepare a negative stain of each organism. Do NOT D Immersion oil heat-fix your slides after air-drying them. o Lens paper 2 Dispose of the spreader slide in a disinfectant jar or D Bibulous paper or paper towel sharps container immediately after use. D Recommended organisms: 3 Observe using the oil-immersion lens. Record your Bacillus cereus observations in the chart on the data sheet, page 193. Micrococcus luteus 4 Dispose of the specimen slide in a disinfectant jar Rhodospirillum rubrum or sharps container after use.  1. Begin with a drop of acidic stain 2. Working on the table top covered with a paper at one end of a clean slide. Be towel , aseptically add organisms and emulsify sure to wear gloves. with the loop. Don't over-inoculate and avoid spattering the mixture. Sterilize the loop after emulsifying. Note: If you find you are transferring IIJ too much organism with a loop, use an inoculating needle. If you are transferring a BSL-2 organism, use a sterile wooden stick or disposable loop/ needle and dispose of it properly when finished. 3. Take a second clean slide, place 4. When the drop flows across it on the surface of the first slide, the width of the spreader slide... and draw it back into the drop. 5.... push the spreader slide to the other end. 6. Air-dry and observe under the microscope. Dispose of the spreader slide in a jar Do NOT heat-fix. of disinfectant or sharps container. 3.89 Procedural Diagram: Negative Stain + Be sure to sterili ze your loop after transfer and to appropriately dispose of the sprea der slide immediately after use. Do not set it on the table. Once air-dried, the slide is rea dy for view ing. No heat-fixing is required in thi s procedure. References Claus, G. William. Chap. 5 in Understanding Microbes- A Laboratory Textbook for Microbiology. New York: W. H. Freeman and Co., 1989. M urray, R. G. E., Raymond N. Doetsch, and C. F. Robinow. Page 27 in Meth ods for General and Mo lecular Bacteriology. Philipp Gerhardt, R. G. E. Murray, Willis A. Wood, and Noel R. Krieg, eds. Washington, DC: American Society for Microbiology, 1994.  Date-------------------------- Lab Section - - - - - - - - - - - - - - - - - - - - - - - - I was present and performed this exercise (initials) - - - - - - - - - - - - Negative Stains OBSERVATIONS AND INTERPRETATIONS 1 Record your observations in the table below. Cellular Morphology and Arrangement Cell Dimensions Cell Dimensions (include a detailed sketch from Negative from Simple Organism of a few representative cells) Stain Stain QUESTIONS 1 Why doesn't a negative stain colorize the cells in the smear? SECTION 3 Microscopy and Staining 193 '...,....., - s. t ·.. "--,.,,' ,_,,. :-: --~:_.1 -.  2 Eosin is a red stain and methylene blue is blue. What should be the result of staining a bacterial smear with a mixture of eosin and methylene blue? IIJ 3 Compare the sizes of B. cereus, M. luteus, and R. rubrum cells as measured using a basic stain (Exercise 3-5) and an acidic stain. What might account for any difference? 194 MICROBIOLOGY: Laboratory Theory & Application 1,,,- _.._ ' :I".... :..1:.,. > ! ''\..,1  Differential and Structural Stains Differential stains allow a microbiologist to detect differences between organisms or differ- ences between parts of the same organism. In practice, these are used much more frequently than simple and negative stains because they not only allow determination of cell size, mor- phology, and arrangement (as with simple or negative stains) but provide information about other features as well. The Gram stain is the most commonly used differential stain in bacteriology. Other differential stains are used for organisms not distinguishable by the Gram stain and for those that have other important cellular attributes, such as acid-fastness, a capsule, spores, or flagella. With the exception of the acid-fast stain, these other stains sometimes are referred to as structural stains. Ill EXERCISE Gram Stain 3-7 Theory The Gram stain is a differential stain in which a decolor- mixture of varying proportions) whereas Gram-positive ization step occurs between the applications of two basic cells are not. Gram-negative cells can thus be colorized stains. The Gram stain has many variations, but they all by the red counterstain safranin, but Gram-positive cells work in basically the same way (Fig. 3.90). The primary are already violet and cannot. Upon successful completion stain is crystal violet. Iodine is added as a mordant to of a Gram stain, Gram-positive cells appear purple and enhance crystal violet staining by forming a crystal violet- Gram-negative cells appear reddish-pink (Fig. 3.91). iodine complex. Decolorization follows and is the most Electron microscopy and other evidence indicate critical step in the procedure. Gram-negative cells are that the ability to resist decolorization or not is based on decolorized by the solution (generally an alcohol/acetone the different wall constructions of Gram-positive and Gram- Gram- negative positive cells cells Cells are transparent prior to staining. Crystal violet stains both Gram- positive and Gram-negative cells. Iodine is used as a mordant. Decolorization with alcohol or acetone removes crystal violet from Gram-negative cells. Safranin is used to counterstain Gram-negative cells. 3.91 Micrograph ofTwo Species Illustrating Gram Stain 3.90 Gram Stain Overview + After application of the primary Results + The violet staphylococcal cells are Gram positive; the stain (crystal violet), decolorization, and counterstaining with safranin, pink rods are Gram negative Depending on your Gram-stain kit, the Gram-positive cells stain violet and Gram-negative cells stain pink/ safranin may be anywhere from a light pink (as in this micrograph) red. Notice that crystal violet and safranin are both basic stains, and to a more intense reddish color. In either case, it should be easily that the decolorization step is w hat makes the Gram-stain differential. distinguishable from the crystal violet color.  Gram-negative cells. Gram-negative cell walls have a Neither of these situations changes the actual Gram higher lipid content (because of the outer membrane) reaction for the organism being stained. Rather, these and a thinner peptidoglycan layer than Gram-positive are false results because of poor technique. cell walls (Fig. 3.92). The alcohol/acetone in the decolor- A second source of poor Gram stains is inconsistency izer extracts the lipid, making the Gram-negative wall in preparation of the emulsion. Remember, a good emul- more porous and incapable of retaining the crystal v