Isolation and Identification of Clavibacter michiganensis Soil Bacteria in Flagstaff, Arizona
Stephanie A. Wamble
BIO 305W section 003
Bo Stevens
13 November 2017
INTRODUCTION
Bacteria impact plants in the environment in symbiotic, commensalism, and pathogenic
forms. Plant pathogens are the common cause of stunted crop growth (Jahr et al. 1999).
Environmental bacteria have provided humans and animals with the nutrients it needs, however,
when pathogens are introduced into a population it can be detrimental to plant and human health
(Mooney and Drake 2012). The infection of plants is difficult to detect until the late stages of
plant growth (Metzler et al. 1997). The detection and identification for plant microflora is greatly
advanced by phylogenetic inference methods (Joseph et al. 2003). This method allows for the
appraisal of soil and plant microflora based on the 16s rRNA sequencing and classifies the
organisms by their heritable traits.
With ribosomal DNA-analysis methods, the ability to classify the bacteria by their rRNA
without the need of a cultivable broth and increases the amount of bacterial diversity that can be
seen throughout the soil (Joseph et al. 2003). By studying the rRNA of bacteria in multiple
locations, biologists can then link the DNA sequences to the possible environments that they can
thrive in. HOW ARE ENVIRONMENTAL BACTERIA IMPORTANT: INCLUDE SPEC.
DETAILS. BACTERIA IMPROVE PLANT GROWTH RESTATE FINAL PP NOT WITH
SPEC BACTERIA AND INCLUDE CROPS IN GENERAL
The purpose of this study was to isolate, characterize, and identify unknown species of
bacteria collected from soil in Flagstaff, Arizona. Using the morphological and physiological
characteristics of the unknown species of soil bacteria were then compared with characteristics
of known bacterial species using Bergey’s Manual of Systematic Bacteriology (Holt et al. 1994).
Bacteria, such as the environmental isolate, grow more readily in common using common lab
methods, which have been used to characterize bacteria for decades (Sait et al. 2002). Therefore,
it was expected the environmental isolate would be identified.
MATERIALS AND METHODS
An unknown environmental isolate (EI) was collected by using a sterile cotton swab to
gather bacteria from woodchips on the topsoil layer in Flagstaff, Arizona. Using the cotton swab,
streaks were made onto trypticase soy agar (TSA) plates. Three isolation streaks were used to
isolate and obtain a pure sample of the EI. Purity was confirmed by preforming a wet mount and
a Gram stain. When streaking the TSA slants a zig-zag streak was used. All bacteria and
controls used for the staining and biochemical tests were grown on TSA plates. Slides made were
visualized under bright field at 1000x magnification with oil immersion.
The test for storage conditions exhibited the conditions at which the EI was best stored at.
Using two screw-top TSA slants, the EI was used to do a singular streak in each. The two TSA
slants were stored at room temperature for 24 hours with the cap unscrewed half a turn from a
fully tightened position to allow for oxygen. Once both tubes had growth the caps were
tightened, and one was keep out at room temperature, and the other was stored within the
refrigerator for two weeks. Using a subculture of each slant onto two push cap TSA slants, the
tubes were incubated at room temperature for 24 hours. Comparisons between the two slants
were made to conclude the results. If the slant from4 degrees Celsius regrew more abundantly
then that storage condition would be morefavorable to the unknown. If the slant from room
temperature grew more abundantly then that storage condition would be more favorable to the
unknown.
The simple stain was used to see the cell size, structure, and formations in more detail. A
dry mount of the EI was heat fixed and methylene blue was applied as the primary stain. A
positive result would be the appearance of cells colored blue, there is no negative for this stain.
In order to discern the EI based on the size of their peptidoglycan layer and membrane
types a Gram stain was applied. Using two control organisms, Escherichia coli, Gram-negative,
and Bacillus megaterium, Gram-positive, a dry mount was prepared with the EI on the same
slide. The dry mount was heat fixed and the primary stain, crystal violet, was applied. Gram’s
iodine was used as a mordant, the slide was decolorized with 70% ethanol. Gram positive
organisms remaining purple and decolorizing any Gram-negative cells. The counterstain safranin
was then used todye any Gram-negative bacteria.
To determine if the EI had capsular material surrounding the bacteria cells, a capsule
stain was conducted. The slide was prepared by emulsifying the bacteria with Congo red and air
dried. The slide was covered with Maneval’s stain and rinsed. When the cells are dyed against a
dark background, capsules would appear clear showing a halo around the cell producing a
positive result. A negative result will be indicated by a dark background with dyed cells
appearing with no clear halo surrounding them.
The acid-fast stain was used to identify if the EI produced mycolic acids. The acid-fast
stain was prepared by heat fixing a dry mounted slide of Mycobacterium smegmatis, the positive
control organism, and thenegative control organism Bacillus megaterium. The slide was steamed
with the primary stain carbol fuchsin. The slide was decolorized with 70% ethanol and counter
stained with methylene blue. A positive result would be indicated by a cell color of purple. A
negative result would be indicated by a blue cell color resulting from the counterstain methylene
blue.
The purpose of the endospore stain was to determine if the unknown had the ability to
produce spores. For this stain, a stress plate of the EI and the positive control was used. Using
Bacillus megaterium as a positive control and the EI, the endospore stain was prepared by heat
fixing a dry mounted slide. The slide was steamed with malachite green then decolorized with
70% ethanol and counter stained with safranin. A positive result will have the appearance of
green spore capsules and a bacterial cell color of pink. A negative result would be just the
appearance of pink cells with no presence of green.
The catalase test was used to test if the EI had the presence of the catalase and peroxidase
enzyme which converts hydrogen peroxide to water and oxygen gas. Staphylococcus epidermidis
was used as a positive control and Streptococcus lactis for the negative control. The EI was
combined with a drop of 3% hydrogen peroxide on a slide. Immediately after the application of
