A direct smear made from the gumline may also be used as a Gram stain control. Expect numerous Gram-positive bacteria (especially cocci) and some Gram-negative cells, including your own epithelial cells. In this slide, Gram-positive cocci predomi- nate, but a few Gram-negative cells are visible, including Gram- negative rods (circled) and a Gram-negative diplococcus (arrow) on the sur face of the epithelial cell.
3-87 GRAMSTAIN OFSTAPHYLOCOCCUS(+) ANDPROTEUS(–) (X1320)✦Staphylococcushas a staphylococcal arrangement, whereas Proteusis a bacillus.
3-89 POSITIVECONTROLSTOCHECKYOURTECHNIQUE✦Stain-
ing known Gram-positive and Gram-negative organisms on either side of your unknown organism act as positive controls for your technique. Tr y to make the emulsions as close to one another as possible. Spreading them out across the slide makes it dif fi- cult to stain and decolorize them equally.
Receptor protein
Peptidoglycan
Cytoplasmic membrane Outer membrane Periplasm
Porin Lipoprotein LPS
O antigen Lipid A LPS
3-88 BACTERIALCELLWALLS✦AThe Gram-negative wall is composed of less peptidoglycan (as little as a single layer) and more lipid (due to the outer membrane) than the Gram-positive wall B.
Cell wall
Surface protein
Cytoplasmic membrane
Teichoic acid Lipoteichoic Acid
Peptidoglycan
B A
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 field (Figure 3-93) or stain precipitate may be mistakenly identified as bacteria (Figure 3-94).
Age of the culture also affects Gram stain consistency.
Older Gram-positive cultures may lose their ability to re- sist decolorization and give an artifactual Gram-negative result. The genus Bacillusis notorious for this. Staphylo- coccuscan also be a culprit. Cultures 24 hours old or less are best for this procedure.
3-91 UNDER-DECOLORIZEDGRAMSTAIN(X1000)✦This is
a direct smear from the gumline. Notice the purple patches of stain on the epithelial cells. Also notice the variable quality of this stain—the epithelial cell to the left of center is stained better than the others.
3-92 OVER-DECOLORIZEDGRAMSTAIN(X1000)✦This also
is a direct smear from the gumline. Notice the vir tual absence of any purple cells, a cer tain indication of over-decolorization.
3-93 CRYSTALVIOLETCRYSTALS(GRAMSTAIN, X1000)✦If
the staining solution is not adequately filtered or is old, cr ystal violet cr ystals may appear. Although they are pleasing to the eye, they obstruct your view of the specimen. Cr ystals from two dif ferent Gram stains are shown here: Aa gumline direct smear; BMicrococcus roseusgrown in culture.
A
B
3-94 STAINPRECIPITATE(GRAMSTAIN, X1000)✦If the slide is not rinsed thoroughly or the stain is allowed to dr y on the slide, spots of stain precipitate may form and may be confused with bacterial cells. Their variability in size is a clue that they are not bacteria.
✦ Application
The Gram stain, used to distinguish between Gram- positive and Gram-negative cells, is the most important and widely used microbiological differential stain. In ad- dition to Gram reaction, this stain allows determination of cell morphology, size, and arrangement. It typically is the first differential test run on a specimen brought into the laboratory for identification. In some cases, a rapid, presumptive identification of the organism or elimination of a particular organism is possible.
✦ In This Exercise
The Gram stain is the single most important differential stain in bacteriology. Therefore, you will have to practice it and practice it some more to become proficient in its execution. The organisms should be used in the combi- nations given so you will have one Gram-positive and one Gram-negative on each slide.
✦ Materials
Per Student Group
✦compound microscope with oil objective lens and ocular micrometer
✦clean glass microscope slides
✦sterile toothpick
✦Gram stain solutions (commercial kits are available)
⽧Gram crystal violet
⽧Gram iodine
⽧95% ethanol (or ethanol/acetone solution)
⽧Gram safranin
✦squirt bottle with water
✦bibulous paper or paper towels
✦disposable gloves
✦staining tray
✦staining screen
✦slide holder
✦recommended organisms (overnight cultures grown on agar slants or turbid broth cultures):
⽧Staphylococcus epidermidis
⽧Escherichia coli
⽧Moraxella catarrhalis(BSL-2)
⽧Corynebacterium xerosis
Procedure
1 Follow the procedure illustrated in Figure 3-81 to prepare and heat-fix smears of Staphylococcus
epidermidisand Escherichia coliimmediately 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 thick- ness, as thick smears risk being under-decolorized.
2 Repeat step 1 for Moraxella catarrhalisand Corynebacterium xerosison a second slide.
3 Because Gram stains require much practice, we rec- ommend that you prepare several slides of each combination and let them air-dry simultaneously.
Then they’ll be ready when you need them.
4 Use the sterile toothpick to obtain a sample from your teeth at the gumline. (Do not draw blood!
What you want is easily removed from your gingival pockets.) Transfer the sample to a drop of water on a clean glass slide, air-dry, and heat-fix.
5 Follow the basic staining procedure illustrated in Figure 3-95. We recommend staining the pure cul- tures first. After your technique is consistent, stain the oral sample. Be sure to wear gloves.
6 Observe using the oil immersion lens. Record your observations of cell morphology and arrangement, dimensions, and Gram reactions in the chart pro- vided on the Data Sheet.
7 Dispose of the specimen slides in a jar of disinfectant or a sharps container after use.
References
Chapin, Kimberle C., and Patrick R. Murray. 2003. Pages 258–260 in Manual of Clinical Microbiology, 8th ed., edited by Patrick R. Murray, Ellen Jo Baron, James H. Jorgensen, Michael A. Pfaller, and Robert H.
Yolken. American Society for Microbiology, Washington, DC.
Forbes, Betty A., Daniel F. Sahm, and Alice. S. Weissfeld. 2002. Chapter 9 in Bailey & Scott’s Diagnostic Microbiology, 11th ed. Mosby-Year Book, St. Louis.
Koneman, Elmer W., Stephen D. Allen, William M. Janda, Paul C.
Schreckenberger, and Washington C. Winn, Jr. 1997. Chapter 14 in Color Atlas and Textbook of Diagnostic Microbiology, 5th Ed. J. B. Lippincott Co., Philadelphia.
Murray, R. G. E., Raymond N. Doetsch, and C. F. Robinow. 1994. Pages 31 and 32 in Methods for General and Molecular Bacteriology, edited by Philipp Gerhardt, R. G. E. Murray, Willis A. Wood, and Noel R. Krieg.
American Society for Microbiology, Washington, DC.
Norris, J. R., and Helen Swain. 1971. Chapter II in Methods in Micro - biology, Vol 5A, edited by J. R. Norris and D. W. Ribbons. Academic Press, Ltd., London.
Power, David A., and Peggy J. McCuen. 1988. Page 261 in Manual of BBL™ Products and Laboratory Procedures, 6th ed. Becton Dickinson Microbiology Systems, Cockeysville, MD.
1. Begin with a heat-fixed emulsion.
(See Figure 3-81.)
2. Cover the smear with crystal violet stain for 1 minute.
Use a staining tray to catch excess stain. Be sure to wear gloves.
8. Gently blot dry in a tablet of bibulous paper or paper towels.
(Alternatively, a page from the tablet can be removed and used for blotting.) Do not rub. When dry, observe under oil immersion.
3. Grasp the slide with a slide holder.
Gently rinse the slide with distilled water.
Alternatively, you can tap the edge of the slide to remove the excess stain and eliminate the wash step.
Stain Disposal
4. Cover the smear with Gram’s Iodine stain for 1 minute.
Use a staining tray to catch excess stain.
Stain Disposal
5. Grasp the slide with a slide holder.
Gently rinse the slide with distilled water.
6. Decolorize with 95% ethanol or ethanol/acetone by allowing it to trickle down the slide until the run-off is clear.
Gently rinse the slide with distilled water.
Stain Disposal
7. Counterstain with safranin stain for 1 minute.
Rinse with distilled water.
3-95 PROCEDURALDIAGRAM: GRAMSTAIN✦Pay careful attention to the staining times. If your preparations do not give “correct”
results, the most likely source of error is in the decolorization step. Adjust its timing accordingly on subsequent stains.
✦ Theory
The presence of mycolic acids in the cell walls of acid- fast organisms is the cytological basis for the acid-fast differential stain. Mycolic acid is a waxy substance that gives acid-fast cells a higher affinity for the primary stain and resistance to decolorization by an acid alcohol solution. A variety of acid-fast staining procedures are employed, two of which are the Ziehl-Neelsen (ZN) method and the Kinyoun (K) method. These differ pri- marily in that the ZN method uses heat as part of the staining process, whereas the K method is a “cold” stain.
In both protocols the bacterial smear may be prepared in a drop of serum to help the “slippery” acid-fast cells adhere to the slide. The two methods provide comparable results.
The waxy wall of acid-fast cells repels typical aqueous stains. (As a result, most acid-fast positive organisms are only weakly Gram-positive.) In the ZN method (Figure 3-96), the phenolic compound carbolfuchsin is used as the primary stain because it is lipid-soluble and pene- trates the waxy cell wall. Staining by carbolfuchsin is further enhanced by steam-heating the preparation to melt the wax and allow the stain to move into the cell.
Acid alcohol is used to decolorize nonacid-fast cells;
acid-fast cells resist this decolorization. A counterstain, such as methylene blue, then is applied. Acid-fast cells are reddish-purple; non acid-fast cells are blue (Figure 3-97).
The Kinyoun method (Figure 3-98) uses a slightly more lipid-soluble and concentrated carbolfuchsin as the primary stain. These properties allow the stain to pene- trate the acid-fast walls without the use of heat but make this method slightly less sensitive than the ZN method.
Decolorization with acid alcohol is followed by a con- trasting counterstain, such as brilliant green (Figure 3-99) or methylene blue.
✦ Application
The acid-fast stain is a differential stain used to detect cells capable of retaining a primary stain when treated with an acid alcohol. It is an important differential stain used to identify bacteria in the genus Mycobacterium, some of which are pathogens (e.g., M. lepraeand M. tuberculosis, causative agents of leprosy and tuber - culosis, respectively). Mem bers of the actinomycete genus Nocardia(N. brasiliensisand N. asteroidesare
3-8 Acid-Fast Stains
Acid-Fast Acid-Fast Negative Cells prior to staining
are transparent.
After staining with carbolfuchsin, cells are reddish-purple. Steam heat enhances the entry of carbolfuchsin into cells.
Decolorization with acid alcohol removes stain from acid-fast negative cells.
Methylene blue is used to counterstain acid-fast negative cells.
3-96 ZIEHL-NEELSENACID-FASTSTAIN✦Acid-fast cells stain reddish-purple; acid-fast negative cells stain blue or the color of the counterstain if a dif ferent one is used.