(EMULSION)✦Preparation of uniform bacterial smears will make consistent staining results easier to obtain. Heat-fixing the smear kills the bacteria, makes them adhere to the slide, and coagulates their protein for better staining. Caution: Avoid pro- ducing aerosols. Do not spatter the smear as you mix it, do not blow on or wave the slide to speed-up air-dr ying, and do not overheat when heat-fixing.
Chapin, Kimberle. 1995. Chapter 4 in Manual of Clinical Microbiology, 6th ed., edited by Patrick R. Murray, Ellen Jo Baron, Michael A. Pfaller, Fred C. Tenover, and Robert H. Yolken. American Society for Microbiology, Washington, DC.
Chapin, Kimberle C., and Patrick R. Murray. 2003. Pages 257–259 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, Inc. St. Louis.
Murray, R. G. E., Raymond N. Doetsch, and C. F. Robinow. 1994. Page 27 in Methods for General and Molecular Bac teriology, edited by Philipp Gerhardt, R. G. E. Murray, Willis A. Wood, and Noel R. Krieg. American Society for Micro biology, 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 4 in Manual of BBL™
Products and Laboratory Procedures, 6th ed. Becton Dickinson Micro - biology Systems, Cockeysville, MD.
3-82 PROCEDURALDIAGRAM: SIMPLESTAIN✦Staining times dif fer for each stain, but cell density of your smear also af fects staining time. Strive for consistency in making your smears.
Caution: Be sure to flame your loop after cell transfer and properly dispose of the slide when you are finished obser ving it.
1. Begin with a heat-fixed emulsion (see Figure 3-81).
More than one organism can be put on a slide.
2. Cover the smear with stain. Use a staining tray to catch excess stain. Be sure to wear gloves.
3. Grasp the slide with a slide holder. Rinse the slide with water.
Dispose of the excess stain according to your lab’s practices.
4. 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.
Stain disposal
✦ Theory
The negative staining technique uses a dye solution in which the chromogen is acidic and carries a negative charge. (An acidic chromogen gives up a hydro gen 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 a colored background (Figure 3-83). A specimen stained with the acidic stain nigrosin is shown in Figure 3-84.
✦ 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 ex- ample 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 size of M. luteusmeasured with the negative stain to its size as determined using a simple stain.
✦ Materials
Per Student Group
✦nigrosin stain or eosin stain
✦clean glass microscope slides
✦disposable gloves
✦compound microscope with oil objective lens and ocular micrometer
✦immersion oil
✦lens paper
✦bibulous paper or paper towel
✦recommended organisms:
⽧Micrococcus luteus
⽧Bacillus cereus
⽧Rhodospirillum rubrum
Procedure
1 Follow the Procedural Diagram in Figure 3-85 to prepare a negative stain of each organism.
2 Dispose of the spreader slide in a disinfectant jar or sharps container immediately after use.
3 Observe using the oil immersion lens. Record your observations in the chart on the Data Sheet.
4 Dispose of the specimen slide in a disinfectant jar or sharps container after use.
3-6 Negative Stains
Background is stained Negatively charged cell
Apply acidic stain (Negative Chromogen )
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3-83 CHEMISTRY OFACIDICSTAINS✦Acidic stains have a negatively charged chromogen (●ⳮ) that is repelled by negatively charged cells. Thus, the background is colored and the cell remains transparent.
3-84 A NIGROSINNEGATIVESTAIN(X1000)✦Notice that the Bacillus megateriumcells are unstained against a dark background. The circular objects are bubbles.
References
Claus, G. William. 1989. Chapter 5 in Understanding Microbes—A Laboratory Textbook for Microbiology. W. H. Freeman and Co., New York.
Murray, R. G. E., Raymond N. Doetsch, and C. F. Robinow. 1994. Page 27 in Methods for General and Molecular Bac teriology,edited by Philipp Gerhardt, R. G. E. Murray, Willis A. Wood, and Noel R. Krieg. American Society for Micro biology, Washington, DC.
1. Begin with a drop of acidic stain at one end of a clean slide. Be sure to wear gloves.
3. Take a second clean slide, place it on the surface of the first slide, and draw it back into the drop.
2. Aseptically add organisms and emulsify with a loop. Do not over-inoculate and avoid spattering the mixture.
Sterilize the loop after emulsifying.
4. When the drop flows across the width of the spreader slide...
5. ...push the spreader slide to the other end.
Dispose of the spreader slide in a jar of disinfectant or sharps container.
6. Air dry and observe under the microscope.
Do NOT heat fix.
3-85 PROCEDURALDIAGRAM: NEGATIVESTAIN✦Be sure to sterilize your loop after transfer, and to appropriately dispose of the spreader and specimen slides.
✦ Theory
The Gram stain is a differential stain in which a decolori - zationstep occurs between the appli cation of two basic stains. The Gram stain has many variations, but they all work in basically the same way (Figure 3-86). The pri- mary stainis crystal violet. Iodine 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 (of variable composition—
generally alcohol or acetone) whereas Gram-positive cells are not. Gram-negative cells can thus be colorized by the counterstainsafranin. Upon successful completion of a Gram stain, Gram-positive cells appear purple and Gram-negative cells appear reddish-pink (Figure 3-87).
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 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 (Figure 3-88). The alcohol/acetone in the decolorizer extracts the lipid, making the Gram-negative wall more porous and in capable of retaining the crystal violet–
iodine complex, thereby decolorizing it. The thicker peptidoglycan and greater degree of cross-linking (because of teichoic acids) trap the crystal violet–iodine complex more 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 tech- nique. The decolorization step is the most crucial and most likely source of Gram stain inconsistency. It is possible to over-decolorizeby leaving the alcohol on too long and get reddish Gram-positivecells. It also is
Differential and Structural Stains
Differential stains allow a microbiologist to detect differences between organisms or differences between par ts of the same organism. In practice, these are used much more frequently than simple stains because they not only allow determination of cell size, morphology, and arrangement (as with a simple stain) but 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 impor tant 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.✦
E X E R C I S E
3-7 Gram Stain
Cells are transparent prior to staining.
Crystal violet stains 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.
Gram- negative
cells
Gram- positive
cells
3-86 GRAMSTAIN✦After application of the primar y stain (cr ystal violet), decolorization, and counterstaining with safranin, Gram-positive cells stain violet and Gram-negative cells stain pink/red. Notice that cr ystal violet and safranin are both basic stains, and that the decolorization step is what makes the Gram stain dif ferential.
possible to under-decolorizeand produce purple Gram- negativecells. 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 dries to a faint haze on the slide.
Until correct results are obtained consistently, it is recommended that control smears of Gram-positive and Gram-negative organisms be stained along with the organism in question (Figure 3-89). As an alternative control, a direct smear made from the gumline may be Gram-stained (Figure 3-90) with the expectation that both Gram-positive and Gram-negative organisms will be seen. Over-decolorized and under-decolorized gum- line direct smears are shown for comparison (Figures 3-91 and 3-92). Positive controls also should be run when using new reagent batches.
Known
Gram + Unknown Known Gram -
?