Lab 5: Using the negative stain to determine true morphology
10/18/10
I. Purpose
To determine the true morphology of bacteria by staining the outside of an organism that has not been
heat fixed.
II. Materials and methods
As stated in the Microbiology 1 lab manual, pages 34-36 with the following exceptions: Nigrosin was
used as a staining agent instead of eosin, Proteus vulgaris was used instead of Proteus mirabilis.
III. Results
Escherichia coli
Proteus vulgaris
Rhodospirillum rubrum
Magn: 1000x
1000x
1000x
Stain: Negative
Negative
Negative
Shape: bacillus
bacillus
vibrio
Size:
3-4m
2-2.5m
singles, palisades
singles
1-1.5m
Arrang: singles, palisades
The bacteria all appeared clear against a dark background. Size reflects the average size of the
organism, or the size of a representative organism that appeared visually to be of average size. P.
vulgaris was larger, but thinner, than E. coli. R. rubrum was not uniformly vibrio in shape. The majority
of cells appeared to be vibrio, though some were only slightly curved. All of the slides were prepared
from 48h broth cultures.
IV Discussion
In the preparation of the simple stain, and most other bacterial stains, the slide is heat fixed. This
process causes protein to coagulate and can visually reduce the size of the bacteria so that true size is
less apparent. When comparing the size of the E. coli observed in this lab, for example, with E. coli
prepared by simple stain in the previous lab, the E. coli in this lab appeared almost twice as large. The
same is true of P. vulgaris and R. rubrum. One concern is that some of these organisms may also
produce capsules. If so, then the presence of a capsule in the negative stain would make the organism
appear larger than its actual size. We will have to perform a capsule stain with the organisms to
determine if the size we obtained in the negative stain is in fact the true size of the organism.
V Conclusions
By preparing negative slides that are not heat fixed and that stain the area surrounding the organism
rather than the organism itself, we may be able to get a better idea of its true shape and size. Heat can
significantly distort the shape and size of an organism and may lead to false identification.