than si Differential and Structural Stains Differential stains allow a microbiologist to detect differences between organisms or differ- fences between parts of the same organism. In practice, these are used much more frequently ple 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 for organisms not ‘the most commonly used differential stain in bacteriology. Other differential stains are used. \Quishable 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. © EXERCISE 3-7 = Theory ‘The Gram stain is a differential stain in which a decolor- ization step occurs between the applications of two basic stains. The Gram stain has many variations, but they all ‘work in basicaly the same way (Fig. 3.90). The primary stain is crystal violet. lodine is added as a mordant to ‘enhance crystal violet staining by forming a crystal violet iodine complex. Decolorization follows and is the most critical step in the procedure. Gram-negative cells are decolorized by the solution (generally an alcohol/acetone Gram Gram- negative positive els alls Cals are wansparent prior to staining. ‘Crystal violet stains both Gram- positive and Gram-negative cals, Todine ie used as a morsant Decolorization with aleohol or acetone removes crystal violet ‘rom Gram-negative cel. Satranin is used to counterstain Gram-negative cots 3.90 Gram Stain Overview $ Aiter application ofthe primary Stain (crystal violet), decolorization, and counterstaining with satrain, Gram-positive oels stan violet and Grarr-negatve cals stan pink! red, Notice that crystal violet and safranin are both basic stains, and thatthe decolozation stepis what makes the Gram-stan differentia, rmiscture of varying proportions) whereas Gram-positive cells are not. Gram-negative cells can thus be colorized by the red counterstain safranin, but Gram-positive cells are already violet and cannot. Upon successful completion of a Gram stain, Gram-positive cells appear purple and Gram-negative cells appear reddish-pink (Fig. 3.91). Electron microscopy and other evidence indicate that the ability to resist decolorization or not is based on the different wall constructions of Gram-positive and 3.911 micrograph of Two Species tlustrating Gram Stain Results ¢ The violet staphylococcal cells are Gram positive; the pink rods are Gram negative. Depending on your Gramestain Kit, the sefranin may be anywhere from a light pink [asin this micrograph) to. more intense redalish color. In either case, it shouldbe easiy distinguishable from the crystal volt color. SECTION 3. Microscopy and StainingGram-negative cells. Gram-negative cell walls have a higher lipid content (because of the outer membrane} and a thinner peptidoglycan layer than Gram-positive cell walls (Fig. 3.92). The alcohol/acetone in the decolor- izer extracts the lipid, making the Gram-negative wall ‘mote porous and incapable of retaining the crystal violet~ iodine complex, thereby decolorizing it. The thicker pep- tidoglycan and greater degree of cross-linking (because of teichoic acids) trap the crystal violet-iodine complex mote effectively, making the Gram-positive wall less susceptible to decolorization. Although some organisms give Gram-variable results, ‘most variable results are a consequence of poor technique. ‘The decolorization step is the most crucial and most likely source of Gram-stain inconsistency. It is possible to over- decolorize by leaving the decolorizer on too long and get reddish Gram-positive cells. It also is possible to under- decolorize and produce purple Gram-negative cells. a ‘O antigen Lipid A Porin Ls. Receptor Lipoprotein LPS Outer membrane Peripiasm —| ytopiasmic. membrane [E] —Teichoicacid Surface —_—Lipoteichoie cia Cytoplasmic. memarane 3.92 Bacterial Cell Walls The Gramnegative wal (A) is composed of less peotidoglcan la ite asa single layer) and more lini {due to the outer membrane) then the Grarpositve wal (8) ‘Thought is shown as a solid layer, the peptidoglycan is quite po- ‘ous, The thicker Gram-positive peptidoglycan is ikea three-dimen- sional chaindink fnce, and when the crystal violet~odine Complexes form within it, they ae dificult to remove by decoloriza- tion, The greater amount of iid end thinner peptidoglycan ofthe Gram-negative wll makes exizaction ofthe crystal voet-odine ‘complex by the decolorizer much easier. MICROBIOLOGY: Laboratory Theory & Application Neither of these situations changes the actual Gram reaction for the organism being stained, Rather, these are false results because of poor technique. A second source of poor Gram stains is inconsistency in preparation of the emulsion. Remember, a good emul- sion is about dime size and dries to a faint haze on the slide. AA third source of inconsistent Gram stains may be the organisms themselves. Some Gram positives, especially Bacillus and Staphylococcus species, lose their ability to retain the crystal violet-iodine complex in as little as 24 hours of incubation. Figure 3.93 illustrates a Bacillus culture stained at 24 hours of growth. Always plan on doing your Gram stains on cultures no older than 24 hours for best results. Until correct results are obtained consistently, itis recommended that control smears of Gram-positive and Gram-negative organisms be stained along with the or ganism in question (Fig. 3.94), As an alternative control, a direct smear made from the gumline may be Gram stained (Fig. 3.95) with the expectation that both Gram- positive and Gram-negative organisms will be seen. Overdecolorized and under-decolorized gumline direct smears are shown for comparison (Figs. 3.96 and 3.97). Positive controls also should be run when using new reagent batches. Interpretation of Gram stains can be complicated by nonbacterial elements. For instance, stain crystals from an old or improperly made stain solution can disrupt the ficld (Fig, 3.98) or stain precipitate may be mistakenly identified as bacteria (Fig, 3.99). o. X a XN 2 3.93 24-Hour Bacillus Mustrating Loss of Gram-Positive Reaction with Age = This Bacilus was isolated from aerobic estuarine mucland stained within 24 hours of making the pure culture. Notice somme cells ae pink and have lost their abit to resist deco ‘tization, This result does not change the fact that Bacis hes & Gram positive wall, though,cof your unknown organism act as postive contios for your tec “yy to make the emulsions as close to one another as possible. Spreading ther out across the side makes it ficult o stain and decolorize them equally tis aso beneficial to put the Gram-negative control atthe end where you willbe applying the decolaries. This wll make it easier to see when its cunning clear. Putting it tthe other end means i will ave runoff from the Gram-positive control ‘and the unknwn obscuring i L_ 3.97 over-Decolorized Gram Stain > This also isa direct ‘smear from the gumline, Notice the virtual absence of any purple calls, a certain inication of over-decolorization. 3,95 direct Smear Positive Control (Gram Stain) = A direct ssmaer made fom the gumine may also be used as a Grarrstan co trol Expect numerous Gram-positive bacteria (especialy coccl and some Gram-negative cels, including your own epithelial col. inthis ide, Gram-positive cocci predominate, but afew Gram negative cols ‘0 visible, including Gram-negative rods (clad and a Grar-negatve Procedure 1 Follow the procedure illustrated in Figure 3.84 to prepare and heat-ix smears of Staphylococcus epi- dermidis and Escherichia coli immediately next to ‘one another on the same clean glass slide. (If you make the emulsions at opposite ends of the slide, you may find it difficult to stain and decolorize each equally.) Strive to prepare smears of uniform thickness, because thick smears risk being underdecolorized. 2 Repeat step 1 for Neisseria sicca and Corynebacte~ rium xerosis on a second slide, 3. Because Gram stains require much practice, we recommend that you prepare several sides of each combination and let them air-dry simultaneously. Then they will be ready when you need them.‘Wash your hands, and then use the sterile toothpick to obtain a sample from your teeth at the gumline. (Do not draw blood! What you want is easly re- moved from your gingival pockets.) Transfer the sample to a drop of water on a clean glass slide, air-dry, and heat-ix. Follow the basic staining procedure illustrated in Figure 3.101, We recommend staining the pure cul- tures first. After your technique is consistent, stain the oral sample. Be sure to wear gloves. Also, pay special attention to the decolorization “tips” in steps 6 and 7 of Figure 3.101 and in its caption, Observe using the oil-immersion lens. Record your observations of cell morphology and arrangement, dimensions, and Gram reactions in the chart provided oon the data sheet, page 201. Dispose ofthe specimen slides ina jar of disinfectant or a sharps container after use, References ‘alas Ronald M. and James WE Syder Pages 278-279 in Manual of Clinical “Microbiology, 10th ed James Versalovic, Kazen C. Carell, Guido Funke, James H, Jorgensen, Mare Louise Landry, and David W. ‘Wamock, ed, Washington, DC: ASM Fess, 2011 ‘Chapin, Kimborle Cand Patrik R. Mureay Pages 258-260 in Mama of ‘Clinical Microbiology, 8th ed. Patrick R. Murray, Elen Jo Baron, James H, Jorgensen, Michael A. Piller, and Robert H. Yolken, eds. ‘Wathingion, DC: American Society for Microbiology, 2003 Koneman, Elner W, Stephen D. Allen, Wiliam M. Janda, Pal C. ‘Schreckenberges and Wathingron C. Winn, J Chap. 14 in Color Atlas ‘and Textbook of Diggostic Microbiology, Sth ed. Philadelphia: JB. Lippincott Co. 1997. ‘Murray, R. G. Ey Raymond N, Doetsch, and C.F Robinow: Pages 31 and 32 in Methods or General and Molecular Bacteriology. Philipp Geharat, GE, Muteay Willis A, Wood, and Noel R. Kreg, eds. Washington, DC: American Society for Microbiology, 1994. [Nomi J:R and Helen Swain. Chap. Il in Methods in Microbiology, Vo. 'SA.I.R Noris and D. W, Ribbons, eds London, UK: Academic res, Led, 1971, Powe, David A. and Peggy J. McCuen. Page 261 in Manual of BBL! ‘Product and Labontory Procedures, th ed. Cockeysvile, MD: Becton Dickinson Microbiology Systems, 1988 “ile, Patcicia M. Pages 70-73 in Bailey & Scot's Diagnostic Microbiology, 3th ed, Se Louis, MO: Mosby, In, am afflat of Elsevier, Ine. 2014, SECTION 3 Microscopy and Staining3.101 Procedural Diagram: results, the most likely source of eror is in the decolorzation step. When the runoff is clear (generally within 10 seconds, shorter ifthe acetone concentration is high, IMMEDIATELY rinse with dH,0. The decolorizer continues decolonizing as you put its botle down andl pick _up the water bot, so anticipate the and point of decolonization to compensate for this, 41. Bogin with up to threo heat-fhxod 2. Cover the smoa(s (pot the whole side) 3. Grasp the sie with a side holder and ‘emulsions on on with crystal violet stain for one minuto, holdit on an angi. Genty rinse te side Wearing gloves, Be sure the siining tray catches all, with distlled water Into the staing tray. ‘vera staring vay ‘excess stain Alternatively, s0e stops 4 and 5, Staining Staining Tay, Tray 4. Cover the smear(s) with Gramiodine stain 5, Grasp the slide with aside holder 6. Holding the slide on an angle, decolorize {for one minute. Be sure the staning tay ‘and ho ton an angle. Gently ringe with Gram decolorizr by alvin it to catches all excess stain, the slide with cstiled water into ths treke down the side unt the runt is staining tray Clear. Catch the runoff inthe staring tray. ‘rinse the sie wth luted water nto the for one minute. Be sure the staining hold ton an angle. Gently rinse the slide ‘aining tray (Note: Iwill stil be decolorzing ay catches al excess stain, ith dstiled water into the staining tray while you're crabbing the water bottel Pan ‘hea for that ransiion). qa=Z v4 "Note: Decoloizers citer depending on the kts manufactures The mare acetone inthe decolrizay, the fastr twit decolorze is Staining Tay 8.Counterstan the srea(s) with safrarin 9, Grasp the side witha slide holder and 10. Gently blot dry ina tabtet of bbulous paper or paper towels. (Alternatively, a page rom the tablet can be removed and Used for blotting.) Do not rub, When ory, ‘observe under ol immersion, ram Stain Pay careful attention tothe staining times, I your preparations do not give “correct” ‘MICROBIOLOGY: Laboratory Theory & ApplicationDate Lab Section | was present and performed this exercise (nial) Gram Stain OBSERVATIONS AND INTERPRETATIONS |] Record your observations in the table below. Use separate lines for citferent organisms found in the Gram stain of your gumline. Include a drawing of your own epithelial cali. Pare he bart cana fe (include a detailed sketch Lee Pek of a few representative cells) oo (+/-) SECTION 3 Microscopy and Staining 201QUESTIONS ‘1. Predict the effect on Gram-positive and Gram-negative cells of the following “mistakes” made when performing 4 Gram stain. Consider each mistake independently. a. Failure to add the iodine, b, Failure to apply the decolorizer. «. Failure to apply the safranin. d. Reversal of erystal violet and safranin stains. 2 Both crystal violet and safranin are basic stains and may be used to do simple stains on Gram-positive and Gram-negative cells. This being the case, explain how they end up staining Gram-positive and Gram-negative cells differently in the Gram stain. 3, If you saw large, eukaryotic cells in the preparation made from your gumline, they were most likely your own epithelial cells. Are you Gram-positive or Gram-negative? (You can make a good guess about this even if you didn’t see your cells.) 4, One of your lab partners has followed the recommended procedure of running Gram-positive and Gram-negative control organisms on her Gram stain of an unknown species. Her choices of controls were Escherichia coli «and Bacillus subtilis. She tries several times and each time concludes she is decoloriing too long because both controls have pink cells (one more than the other). What might you suggest she try and why? 202 MICROBIOLOGY: Laboratory Theory & ApplicationEXERCISE 3-9 = Theory Capsules are composed of mucoid polysaccharides or polypeptides that repel most stains because of their neutral charge. The capsule stain technique takes advantage of this characteristic by staining around the cells. Typically, an acide stain such as Congo red or nigrosin, which stains the background, and a basic stain that colorzes the cell proper are used in combination, The capsule remains unstained and appears as a white halo berween the cells and the colored background (Fig. 3.109). This technique begins as a negative stain; cells are spread in a film with an acidic stain and are not heat fixed. Heat-fixing causes the cells to shrink, leaving an artifactual white halo around them that might be interpreted as a capsule In place of heat-fxing, cells may be emulsified in a drop of serum to promote their adhering tothe glass slide, = Application “The capsule stain isa differential stain used to detect cells capable of producing an extracellular capsule. Capsule production increases virulence in some microbes (such as the anthrax bacillus Bacillus anthracis and the pneumo- coceus Streptococcus pneumoniae) by making them less vulnerable to phagocytosis, 3.109 capsule Stain. ¢ The acidic stain colonzes the back- {grOund while the basic stain colorizes the cel leaving the capsules as unstained, white clearings around the cells. Notice the lack of ‘uniform capsule size, and even the absence ofa capsule in some cals. There are several variations ofthe capsuie stain, The capsule stain protocal provided in this exercise wil produce different colors ‘than in this micrograph, but the prncipais the same—capsules wil appear as white earings. "In This Exercise ‘The capsule stain allows you to visualize an extracellular capsule, if present. Be careful to distinguish between a tiny white halo (as a result of cell shrinkage) and a true capsule. Materials 13 Clean glass slides a Sheep serum © Maneval’s stain © Congo red stain Squirt bottle with water » Staining tray Staining sereen © Coplin staining jar (or a beaker} with distilled water © Bibulous paper or paper towel Slide holder © Disposable gloves themical eye protection © Sterile toothpicks © Compound microscope with oilimmersion fens and ocular micrometer co Immersion oil © Lens paper © Recommended organisms (18- to 24-hour skim milk, or tryptic soy agar slant pure cultures} + Aeromonas sobria + Rhizobiuin leguminosarum + (Optional) Klebsiella pneumoniae (BSL-2} (SD Procedure 1 Follow the protocol in the procedural diageam (Fig. 3.110} to make stains of the organisms supplied. Use BSL-2 precautions if K. pneumoniae is stained. Use a separate slide for each specimen and do not heat-fix them. Be sure to wear gloves and eye protection. 2 Using a sterile toothpick, obtain a sample from below the gumline in your mouth. (Do not draw blood!) Mix the sample into a drop of water or serum on a slide, and then perform a capsule stain on it. SECTION 3 Microscopy and Staining3 Observe, using the oil-immersion lens. Record your observations of cell morphology and arrangement, cell dimensions, and presence or absence of a capsule in the table on the data sheet, page 213. 4. Dispose of the specimen slides in a jar of disinfectant or sharps container after use. 41. Working onthe table top covered with a paper towel, place alogptu of sheep 2. Asoptically add organisins and emutsiy References Hughes, Roxana B. and Ann C Smith "Capsule San Frococals” ASM Microbe ary epee 23, 2007 Updated Jay 22,2013 Avalale ‘online, URL: hepfrwaniceobibraryor/ibeary Tabortry esd 3041 -apsle stain poco Murcay, 8. G.E, Raymond N. Dosic, and C.F Robinow: Page 35 ia “Methods for General and Molecular Bacteriology. Pip Geshart, R.G.E Murray Wills A Wood, and Nol R. Kegel Washington, DC: American Soci for Microbiology, 194. Norris J. Rand Helen Swain. Chap in Methods ie Micmbilrgy Vol. SA. 1J.R. Novis and D.W. Ribbons, eds. Landon, UK: Academic Press, Lid 1971, 9. Take a second clean slide, piace ‘Serum at one end of a clean glass sie. ‘Adda drop of Congo red stain, Bo sure ‘ith alaop. Steriize the loop after ‘ermuleying. Note: I you are transferring BSL? organism, use a store wooden Ron the surtace ofthe rst sie, ‘and draw it back Into the drop, to wear gloves. ‘lek or disposable loopineacle and ‘spose oft propetiy when finished 4 Wnen the doo flows across. 6....immediataly push the spreacer side 6. Al-cry and do NOT hatin. ‘the width ofthe spreader sige {o the other end. Dispose ofthe spreader (or slighty befor. ‘ide in ajar of disinfectant or sharps 7. Place the slide an a rack over a staining tray. Cover the spread with Manevals £8. Grasp the slide witha side holder and dip into ajar of 8. Gently tap the edge ofthe slide on the paper towel to romove excess Staln for? minuto, Mako sure the dlstlod wator two or three ater, then sett down to akc staining tay catches all excess stain times to rinse off the Manaval's ‘When dry, observe under oilimmersion. stain. Change the waterin the iar frequently. 3.110 Procedural Diagram: Capsule Stain ® Be sure to stelze your loop attr transfer and to appropriately cispose of the spreader ‘lide immediately after use. Do not setiton the table. Once ai-cie, te slide is ready for counterstaining, No hest-txing is required. Gently ‘insing the Manevat stain and not blotting the sie cry will hep with the quality of your preparations. Properly dispose ofthe side ater viewing. MICROBIOLOGY: Laboratory Theory & ApplicstionDate Lab Section | was present and performed this exercis (initials) Capsule Stain OBSERVATIONS AND INTERPRETATIONS 1 Frocor your observations nthe tbl slow. Use a separate Ine foreach ergenam fom your gun sample that strates a capsule or an unusual cell morphology or arrangement. Measure cell dimensions and the width of the capsule from the cell's ‘surface to the capsule's edge. rma Proc ce (include a detailed sketch fon and Width of Capsule Cee ue) G/-) (any) SECTION 3 Microscopy and Staining 213QUESTIONS 1. copsutes are nentraly charged. This being the case, what isthe purpose of emulsifying the sample in serum in this staining procedure? 2. some oral bacteria produce an extracellular “capsule.” Of what benefits a capsule to these cells? 3 Sketch any cells from your mouth sample that display an unusual morphology or arrangement, 214 MICROBIOLOGY: Laboratory Theory & ApplicationEndospore Stain EXERCISE EL ‘Theory Some bacterial species are able to differentiate into dormant cells called endospores when environmental conditions, such as nutrient depletion or high temperatures, are unsuitable for growth. Endospores are highly resistant to heat and chemicals, which allows them to survive in this state for long periods of time. The total absence of ATP within endospores is an indication of how dormant they are! In addition to nutrient depletion, sporulation has also been shown to be dependent on population density. With increased density, a secreted peptide (called “competence and sporulation factor,” or CSF) reaches a critical concen- tration and results in derepression of sporulation genes, which is a fancy way of saying, “Taking the brakes off” cof them. Endospores’ resistance is due to a combination of factors, including a tough outer covering made of the protcin keratin, its dehydrated state, DNA protective proteins, and other adaptations, When conditions are suitable again, endospores germinate into metabolically active vegetative cells, ‘The keratin in the spore coat also resists staining, so extreme measures must be taken to stain an endospore. In the Schaeffer Fulton method (Fig. 3.111), primary stain of malachite green is forced into the spore by steaming the Spore Spore producer ‘nonproducer CCals and spores prior to staining are transparent. ° ‘After staining with ‘malachite green, ces ‘and spores are green, Heat is used to force ‘the stain inte spores, if present. Decotoriztion with water removes stain 2 trom cals, but not spores. Safran is used t0 counterstain cals. B.1TT the schaetter-Futton Endopore Stain» Uson ‘completion, endospores are green, vegetative and spore mother calls are red. bacterial emulsion, Alternatively, malachite green can be left on the slide for 15 minutes or more to stain the spores. ‘Malachite green is water soluble and has a low affinity for cellular material, so vegetative cells and spore mother cells (which are responsible for producing the endospore} can, be decolorized with water and counterstained with safranin (Big 3.112) Endospores may be located in the middle of the cell (central), at the end of the cell (terminal), or between the end and middle of the cell (subterminal). Endospore location in some species is variable and may be a combination of terminal and subterminal, for instance. 3.112 cutture Age Can Affect Sporulation + Bacteria c2oable ‘of producing endospores do not do so unifermly during their culture's ‘growth, Sporulaton is done in response to nutient depletion, anc ois characteris of older cutures. These two cultures lustrate Shown s Glostciuen {efani stained by a different endospore stain protocol using carbot fuchsin. Notice how the endospores have caused the ends ofthe. cells to dstend (swell ‘ ' + i by - tb o ’ Se 3.115 subterminal Etiptical Endospores » Thisisa stained preparation of Clostridlum botulinum using an alternative procedure, The black, elliptical endospores slightly cistend the cell MICROBIOLOGY: Laboratory Theory & Application ‘© Application The endospore stain is a differential stain used to detect the presence, shape, and location of endospores in bacterial cells, Only a few genera produce spores. Most common are the genera Bacillus (over 100 species) and Clostriditem (over 160 species). In addition, several “new” genera formerly classified as Bacillus species are also endospore producers. These include Brevibacillus, Geobacillus, Paenibacillus, and Virgibacillus, among others. Most Bacillus species are soil, freshwater, or marine saprophytes, but two are pathogens. B. anthracis is the causal agent of anthrax and B. cereus causes two kinds of food poisoning: emetic and diarrheal. Most members of (Clostridium are soil or aquatic saprophytes, or inhabitants of human intestines, but four pathogens are fairly well known: C. tetani (tetanus), C. botulinum (botulism), C. perfringens (gas gangrene), and C. difficile (pseudomem- branous colitis). @In This Exercise In the endospore stain you will examine endospores from different-aged Bacillus cultures. You will not have to stain each species, Divide the work within your lab group, but be sure to look at each other’s slides. Also be sure to compare spore shape and location for each species. Materials 1 Clean glass microscope slides © Malachite green stain © Safranin stain 1 Squirt bottle with water 0 Heating apparatus (steam apparatus or hot plate} 11 Fume hood 10 Bibulous paper or paper towel © Staining tray © Staining sereen © Slide holder 15 Disposable gloves © Lab coat © Chemical eye protection © Compound microscope with oil-immersion lens and. ocular micrometer © Immersion oil © Lens paper © Nonsterile Petri dish for transporting slides© Recommended organisms (per student group: += 48-hour and 5-day sporulating agar slant pure cultures of Bacillus cereus + 48-hour and S-day sporulating agar slant pure cultures of Bacillus coagulans + 48-hour and 5-day sporulating agar slant pure cultures of Bacillus megaterium + 48-hour and 5-day sporulating agar slant pure cultures of Bacillus subtilis (Note: tryptic soy agar can be substituted for sporulating agar) Medium Recipe Spork + Pancreatic digest of gelatin + Pancreatic digest of casein + Yeast extract + Beef extract + Dextrose + Agar + Manganous sulfate * Distilled or deionized water Final pH 6.6 * 0.2 at 25°C ny Agar ew ‘Begin with up to tree heat-fixed emulsions ‘on onesie. (Fig. 284, Use BSL-2 precautions, if appropriate, Wearing gloves ‘rd chemical eye protection, cover te ‘smear witha stip of bibulous paper cut siighty small tan the slide. F you need {otranspor the side to another part ofthe lab for staining, putt ina covered Pet dish, — 4. Place the side on the staining rack and ‘counterstain with cffanin for ore minute. Be ‘Sue excess stain falls into the staining tray. 6.0% 40g 3.08, 15g 10g 15.08 038 1.0L 2.Set tho slide on a steaming apparatus ‘and saturate the bibulous paper with malachite green stain. Heatitto steaming for 50 7 minutes (as shown in Figure 3117. {Be eur to Koop the paper most with stain, bat don’t have so wet tat stain runs of ‘he sige, Perform this step with adequate ventilation, preferably in a fume hood. Procedure 1 For each organism, prepare and heat-fix two smears on the same slide as illustrated in Figure 3.84. One smear should be the 48-hour culture, and the other the S-day culture. Divide the work within your lab group. Minimally, each student should prepare one slide with smeats of the 48-hour and S-day culeures ‘of one species. Note: Use BSL-2 precautions if you have subcultured soil isolates for endospore staining 2 Follow the instructions in the procedural diagram in Figure 3.116. Use a steaming apparatus like the one shown in Figure 3.117. Be sure to have adequate ventilation (a fume hood is highly recommended), eye protection, and gloves. If you must carry the slide to and from the steaming apparatus, put it in a covered Petri dish. 3 Observe using the oil-immersion lens. Record your observations of cell morphology and arrangement, cell dimensions, and endospore presence, position and shape in the table provided on the data sheet, page 219. 4. Dispose of specimen slides in a disinfectant jar or a sharps container, oS v4 = | 3. Grasp the side wih ase holder and propery depose ofthe bibulous paper with {oreope (to prevent stan from getting on your gloves). Then, hold he side onan angle var stain tray and gentiy rinse both sides with tiled wator unl the runffis clea Then, If not already there, return to your lb station ‘carrying your sgein a Pet dish Y [ stanig Ta ‘5. Grasp the sie wit a side holder and hokd iton an angie. Gently ins the side with sted water into te staling tray. a 6, Gently blot ry in tablet of bbulous paper or paper towels Aeratvely page fom the faoet can bo removed and used for bloting) ‘Dornot rub. When dry, observe under of Immersion 3.116 Procedural Diagram: Schaeffer-Fulton Endospore Stain ~ Steam the staining prepertion; do not bal. Be sure to perform this procecure with adequate ventiaton (preferably afurne hood), eye protection, alo coat, and gloves. Employ BSL-2 precautions if you have subcultured a sol isolate for endospore staining, SECTION 3 Microscopy and Staining‘3.117 steaming the Stide During the Endospore Stain © Carefully stoam the side to force the malachite green into t endospotes. Do not bol the side or ltt dry out, Keep it mois wi slain for up to 10 minutes of steeming nat 10 minutes on the ‘apparatus—10 minutes of steaming!), Caution: This should be performed in a wellventiated ares (preferably a furne hood) with hand, clothing, and eye protection ‘MICROBIOLOGY: Laboratory Theory & Application References Claus, G. Willies. Chap. 9 in Understanding Mirobes—A Laboratory Texthook for Microbiology. New Yorks W.HL. Freeman and Co. 1989, Logan, Nall A. and Paul De Vos. Gens I. Bails in Bergey's Manual of Systematic Bacteriology, 2nd ed, Vol The Firmicutes, Paul De Vos, ‘George M. Garety, Dorothy Jones, Noe R. Kreg, Wolfgang Ludwi, Fred A. Rainey, Kae-Heing Schleifer and Wallam B, Whitman, es, [New York: Springer, 2009, Murray, R.G, Ey Raymond N. Dossch, ad C.F Robinow, Page 34 in ‘Methods for General and Molecular Bacteriology. Philipp Gerhardt, R. G.E. Musray, Wills A. Wood, and Nol R. Kreg ds, Wosbington, DC: American Society for Microbiology, 1994,Name Date Lab Section | was present and performed tis exerise (tials) Endospore Stain OBSERVATIONS AND INTERPRETATIONS |] Record your observations in the table below. Cellular Morphology Bes Erreur Pod Pres rey mee ce cei Cell Creseee cues (include culture age) | representative cells and spores) | Dimensions | _ or absent) (if present) SECTION 3 Microscopy and Staining 219QUESTIONS 1 Why does this exercise call for an older (5-day) culture of Bacillus? 2. What does a positive result for the endospore stain indicate about the organism? What does a negative result for the endospore stain indicate about the organism? 3 Whyis it not necessary to include a negative control for this stan procedure? A. tndospores do not stains east Perhaps you have seen them as unstained white objects inside Bacillus species inother staining procedures If they are visible as unstained objects in other stains, of what use is the endo- spore sain? 220 MICROBIOLOGY: Laboratory Theory & Application