LaboronExerceo nd Siig 1 Beco Mapolny [6 Nepnve Sing Coon, 212 EXERCISE Negative Staining Materials per Student 24- to 48-hour tryptic soy broth cultures of Bacillus subtilis (ATCC 6051), Micrococcus Iueus (ATCC 9341), and Spirilum volutans (ATCC 19554) Dorner’s nigrosin solution, India ink, or eosin blue clean microscope slides inoculating loop immersion oil, microscope lens paper and lens cleaner wax pencil Bunsen burner Learning Objectives Bach student should be able to 1, Understand the reason for the negative staining procedure 2, Stain three different bacteria using the negative staining procedure Suggested Reading in Textbook 1, Dyes and Simple Staining, section 2.2 Pronunciation Guide Bacillus subtilis (bah-SIL-lus sub-til-us) Micrococcus luteus (mny-kr0-KOK-us LOO-(ee-s) Spirillum volutans (spy-RIL-lum VOL-u-tans) 32 = Because Why Ate the Following Bacteria Used in This Exercise? es of Bacillus subilis (L. subtilis, slender) and Spirillum volutans were used in exercise 6, students are now familiar with the rod-shaped morphology of these bac- teria, Tous, these same bacteria can be used ta ilustrate the negative staining technique. One new bacterium hae been added to broaden the student's awareness of bacterial mor- phology by way of negative staining. Micrococcus luteus luteus, golden yellow) isa spherical cell, 09 fo 18 wn in diameter, occurting in pais, tetrads, or iregular clusters but not chains. It is nonsporing and seldom motile. The bacterium is easy to culture since it grows on simple ‘medium and forms yellow, yellowish green. or orange colonies. M. luteus occurs primarily on mammalian skin and in soil but ean be easily isoated from food products and the air, ST Medical Application ‘Treponema pallidum is the sprochete that cases the sexually teansmited disease syphilis. Tis bacterium isa very delicate cell hat is easly distorted by heat-fixing: thus, negative stain- ing is the procedure of choice in the elinical laboratory. Principles Sometimes it is convenient to determine overall bacte- rial morphology without the use of harsh staining or hheat-fixing techniques that change the shape of cells, ‘This might be the case when the bacterium does not stain well (e.., some of the spirochetes) or when itis desirable to confirm observations made on the shapeind Sng Microbiology. Fh Eon 1 acti Mepology | 6 Negri Sning Tretia tian Copano. 212 Figore 61 India Ink Stain of Bails meqatrium (2,000). Notice the dik background around the cea acter el i and size of bacteria observed in cither a wet-mount or hanging drop slide. Negative staining is also good for viewing capsules (see exercise 11), Negative, indirect, or background staining is achieved by mixing bacteria with an acidic stain such as nigrosin, India ink, or eosin, and then spreading out the mixture on a slide to form a film. The above stains ‘will not penetrate and stain the bacterial cells due to repulsion between the negative charge of the stains and the negatively charged bacterial wall. Instead, these stains ether produce a deposit around the bacte- ria or produce a dark background so that the bacteria appear as unstained cells with a clear area around them (figure 6.1). Procedure 1. With a wax peneil, label the left-hand comer of three glass slides with the names of the respective bacteria. 2. Use an inoculating loop to apply a small amount ‘of bacteria o one end of a clean microscope slide (igure 6.24). 3. Add 1 to 2 loops of nigeosin, India ink, or eosin solution to the bacteria (figure 6.26) and mix thoroughly. 4, Spread the mixture over the slide using a second slide. The second slide should be held at a 45° angle so that the bacteria-nigrosin solution Figure 62 Negative Staining Procedure and Thin Smear Preperation. ineculstng oop © 1-2arops of bactrleutre ~ ‘hangs ste 1-2.ops efron » (ert Sa) am Second se (65 ange wh @ Song edge ot tae tetet AO [oer spreads across its edge (figure 6.2c). The slide is ‘then pushed across the surface ofthe first slide in ‘order to form a smear that is thick at one end and thin atthe other (figure 6.24), This is known as a thin smear. Negative Staining 33Tater 1 acti Mepology | 6 Negri Sning Tete vest Laboror Exercsosin an Suing Corr, 202 Microbioopy.Fth Eon 5. Allow the smear to air dry (figure 6.2¢). Do not heattix! 6, With the low-power objective, find an area of the ‘smear that is of the optimal thickness for ‘observation, 77, Use the oil immersion lens to observe and draw the three bacterial species in the report for exercise 6. 4 Bacterial Morphology and Staining