Students enrolled in the Microbiology course (BIOL223) at The College of the Holy Cross are required to complete a final project in which they create their own media and enrich for bacteria from two different genera, one of moderate difficulty and another of advanced difficulty. For my project, I enriched for members of the genus Alteromonas (moderate) and of the genus Methylobacterium (advanced).

1. By Matthew Laird ‘15

2. I. Introduction
II. The Organisms
I. Methylobacterium
I. Physiology
II. History
III. Habitat
IV. Ecological Importance
V. Applications
VI. Medium
VII. The Inoculum
VIII. Incubation Conditions
IX. Results
X. Interpretation of Results

3. II. Agar Decomposing Bacteria
(Alteromonas)
I. Physiology
II. Medium
III. Inoculum
IV. Incubation Conditions
V. Results
VI. Interpretation of Results
III. Summary

4. • For my final enrichment project, I
decided to isolate two microbial
organisms, one being
Methylobacterium, because I was
interested in its physiology. The
second was sea agar decomposing
bacteria (Alteromonas), which I
chose because of the close proximity
of my home to ocean water.

6. • The genus Methylobacterium is
composed of a variety of pink-
pigmented facultatively
methylotrophic (PPFM) bacteria.
• They are capable of growing on one
carbon compounds such as formate,
formaldehyde, and especially
methanol as a sole carbon source.

7. • Members of Methylobacterium are
strict aerobes and gram-negative,
though sometime gram-variable.
• Are mainly rods, occasionally
branched and exhibit polar growth.
• All strains are motile by a single sub
polar/lateral flagellum.
• Contain citrate synthase.

8. • Their characteristic pink pigment is
nondiffusible, nonfluorescent, and
most likely a carotenoid.
• Are catalase and oxidase positive.
• Have a complete tri-carboxylic acid
cycle when grown on complex
organic substrates.

9. • Urease is produced by all strains.
• All strains are Indole negative and TSI
negative (do not produce H2S).
• Usually Nitrate-Reduction negative,
though some strains are positive.
• The fatty-acid composition of PPF
cells is largely composed of mono-
unsaturated acid chains.

10. • Sensitive to antibacterial agents:
kanamycin, gentamycin, albamycin,
streptomycin, framycetin, and
especially tetracyclines.
• Resistant to antibacterial agents:
cephalothin, nalidixic acid, penicillin,
bacitracin, carbenicillin, colistin
sulfate, polymixin B, and
nitrofurantoin.

11. • The first strain of Methylobacterium
was discovered in 1913 by Polish
microbiologist Kazimierz Bassalik.
• This strain was isolated from a piece
of an earthworm.
• However, the genus was not studied
excessively until the 60’s/70’s when
interest in its one-carbon
assimilation pathways peaked.
Bassalik
circa 1928

12. • There presently exists twenty-two validated
species of the genus Methylobacterium.
• They are: Methylobacterium adhaesivum; M.
aminovorans; M. aquaticum; M.
chloromethanicum; M. dichloromethanicum;
M. extorquens; M. fujisawaense; M.
hispanicum; M. isbiliense; M. lusitanum; M.
mesophilicum; M. nodulans; M.
organophilum; M. podarium; M. populi; M.
radiotolerans; M. rhodesianum; M. rhodinum;
M. suomiense; M. thiocyanatum; M. variabile;
M. zatmanii.

13. • Members of the genus
Methylobacterium are ubiquitous
and are thus found in a variety of
habitats.
• Habitats include freshwater, lake
sediments, leaf surfaces, rice grains,
“air”, hospital environments, and in
pharmaceutical preparations such as
face creams.

14. • Since members of Methylobacterium
are strict aerobes, they can be isolated
from almost any freshwater source
where dissolved oxygen is present.
• One species, M. organophilum, a
methane-oxidizer, can only be found
during the summer months in the
upper stratified layers of lake, where
methane is present.

15. • Methylobacterium may play an
important ecological in the carbon
cycle in nature.
• Methylobacterium strains have been
localized as endosymbionts within
cells in the buds of Scotch pine
(Pinus sylvestris).

16. • Methylobacterium forms a strong
cohesive mat in fuel/water
interfaces, such as those that t occur
in storage tanks for middle distillate
fuel-oils for heating or diesel
engines. These chemofilms promote
biofilm formation, which present
potential problems for filters that
may lead to engine failure.

17. • Methylobacterium have the potential
to produce single-celled proteins
from methanol, however, their
bioconversion ratios do not stack up
to those of other methylotrophs.
• Because of this, no immediate uses
of this genus in this way is seen in
the immediate future.

18. • The pink carotenoid pigment in
Methylobacterium strains has been used
commercially as a dye as well as a colorant
in food.
• It appears that Methylobacterium has some
sort of connection with vehicular emissions,
showing that these organisms are able to
grow on the polycyclic aromatic
hydrocarbons found in exhaust emissions.
• This suggests that these organisms might be
used in the future as biological monitors of
vehicle pollution.

19. • Because of the ability of
Methylobacterium to grow on
methanol as sole carbon and energy
source, can use methanol mineral
salt medium (MMS) to isolate
bacteria.
• Both MMS plates and broth can be
and were made.

20. • Methanol Mineral
Salts Medium
• Chemicals:
– The following
are added per
liter (L):
(1-2% of sterile
Methanol is
added to medium
after autoclaving.)
Ingredient: Amount:
K2HPO2 1.20 g
KH2PO2 0.62 g
CaCl2●6H20 0.05 g
MgSO4●7H2 0.20 g
NaCl 0.10 g
FeCl3●6H20 1.0 mg
(NH2)2SO4 0.5 ɥg
CuS04●5H20 5.0 ɥg
MnSO4●5H20 10.0 ɥg
Na2MOO4●2H20 10.0 ɥg
H3BO4 10.0 ɥg
ZnSO4●7H20 70.0 ɥg
CoCl2●6H20 5.0 ɥg

21. • Because chemicals in blue are added
in such small amounts (ɥg/L), a stock
solution of 100 mL was made.
• This stock solution was then added
to the 1L medium solution to obtain
the correct concentration of each
chemical.

22. • Looking at the chemicals in the MMS
media, the only carbon source is the
methanol added after autoclaving.
• Thus, only methylotrophs, those
organisms which can utilize
methanol as a sole carbon source
should be able to grow on the
medium.

23. • Inoculum:
• I previously stated that
Methylobacterium can usually be
isolated from almost any freshwater
source with dissolved oxygen.
• Thus, my inoculum was collected
from Middle River, a fresh body of
water here in Worcester.

26. • MMS plates were streaked for
isolation with the inoculum and then
placed in an incubator at 30°C,
optimal growth temperature.
• MMS tubes were placed in the
shaker with caps loosely tightened to
aerate the medium.

27. • Plates:

28. • Broth:

29. • Gram Stain:

30. • Fermentation Tests:
• Results: negative for
Glucose, Lactose, and
Sucrose.
• This is expected, since
Methylobacterium is an
obligate aerobe.
G L S

31. • TSI Test:
• Previously, I stated that
Methylobacterium strains
were TSI negative (not producing
H2S).
• Result: not negative, but also
no H2S either, since no black
precipitate.

32. • Indole Tests:
• Previously, I stated that
Methylobacterium strains
were all Indole negative.
• Result: negative test,
consistent with literature.

33. • Nitrate Reduction Test:
• Previously, I stated that
Methylobacterium strains are
usually Nitrate-Reduction
negative, though some strains
are positive.
• Result: positive for nitrate
reduction, so consistent with
literature.

34. • Citrate Test:
• Previously, I stated that all
Methylobacterium strains
contained the enzyme
citrate synthase.
• Result: positive citrate test,
consistent with literature.

35. • Urease Test:
• Previously, I stated that all
Methylobacterium strains were
urease positive.
• Result: positive urease test,
consistent with literature.

36. • Catalase Test:
• Previously, I stated that
Methylobacterium strains are
catalase positive.
• Result: negative test, not consistent
with literature.

37. • Oxidase Test:
• Previously, I stated that
Methylobacterium strains are
oxidase positive.
• Result: negative test, not consistent
with literature.

38. • Many of the physiology tests matched
that of the literature, while others did
not or were ambiguous.
• The fact that the colonies were not
their characteristic pink color leads me
to believe that I did not isolate a
member of the genus of
Methylobacterium, but rather another
methylotroph with a similar physiology.

40. • The genus Alteromonas:
• Aerobic, Gram-negative, non-
fermentative, heterotrophic, motile,
non-pigmented, Pseudomonas-like
bacteria able to decompose algal
polysaccharides such as agar.

41. • Because of the ability of these
organisms to depose agar, sea water
agar (SWAGAR) plates were made to
isolate bacteria.
• The following are added per
liter (L).
Ingredient: Amount:
Sea Water 500 mL
Agar 15 mL
Distilled H20 500 mL

42. • Inoculum:
• Inoculum was
sea water taken
from Craigville
Beach on Cape
Cod in a town
called
Centerville, near
where I live.

43. • Plates:

44. • Fermentation: glucose, lactose, sucrose
all negative.
• TSI: yellow/yellow. No H2S precipitate.
• Indole: negative test.
• Nitrate Reduction: negative for nitrate
reduction and nitrite reduction.
• Citrate: negative
• Urease: negative

45. • The characteristic depressions that
were found in the plates leads me to
believe that I have in fact isolated a
member of the genus Alteromonas.
• The physiology tests also go to
confirm this belief.

46. • The organism that I isolated in the
first part is likely not a member of
the genus Methylobacterium, but
rather another methylotroph will
similar physiology.
• The organism that I isolated in the
second part is very likely to be a
member of the genus Alteromonas.