The document describes experiments conducted to isolate and identify Clostridium bacteria from environmental samples. Soil and mud samples were used to grow a biofilm. Gram-positive, spore-forming rods were isolated and identified through staining, biochemical tests, and carbohydrate fermentation profiles. The isolates were likely Clostridium species from Group III or IV based on morphology and tests, possibly Clostridium ramosum, Clostridium perenne, or Clostridium putrefaciens. Improved sampling and growth methods were suggested to obtain clearer results.

1. Isolation of
Clostridium
Ben Anderson & Richa
Banthia
BIOL 3116
Fall 2013

2. Purpose and
Objectives
1. Grow, collect and analyze
microscopic organisms from sample
biofilm
2. Enrich, isolate and identify Gram
negative organisms using rapid
identification – API-20E.
3. Enrich, isolate and characterize
organisms from the genus Clostridium

3. Basic Facts about
Clostridium
Gram Positive Rods- short chains
Anaerobic
Produce endospores to survive
extreme conditions
Found in soil, mud, plants, animal
feces, and soured milk
5 groups

4. Infamous Species of
Clostridium
 Clostridium Botulinum
o Produces neurotoxins that can cause muscular
paralysis
o Botulism
 Clostridium difficile
o Can cause severe cases of diarrhea by taking over
the normal flora of the gut
 Clostridium tetani
o Produces tetanospasmin which causes severe
muscle spasms
o Tetanus

5. Environmental
Samples
 Obtained 2 soil and
1 mud samples
 S1 – Soil sample 1
 S2 – Soil sample 2
M – mud
 S1M – Mix of S1 and
mud
 S2M – Mix of S2 and
mud
S1
S2
M
Dirt/mud samples
used –S1 S2 and M

6. Growth of Biofilm
What is a biofilm?
Contained mix of
all soil and mud
samples in
deionized water
Allowed to grow
for 5 days
Biofilm after
growth

7. Analysis of Biofilm
Gram Stain Wet Mount

8. Rapid Identification of Gram
Negative Bacteria
Inoculated LSL
with mud
Incubated at 37°
for 24 hour period
Streaked onto EMB
plates
LSL broth 1
– visible
growth
LSL broth 2
– visible
growth

9. Rapid Identification of
Gram Negative Bacteria
 Eosin Methylene Blue
Agar
o Gram –
 Differential for lactose
fermenters
 Lactose fermentation
= dark colonies
EMB 1
EMB 2

10. EMB – E. coli

11. API-20E Inoculation
API-20E inoculated tray
 Identification of…
o Family Enterobacteriaceae
o Other Gram negative bacteria
 20 tests

12. API-20E Results for Both Test
Strips
Both API-20E trays after allowing to sit for ~48hrs
 Serratia fonticola 40%
 Enterobacter aerogenes 30%
 Enterobacter cloacae 23.4%

13. Serratia fonticola
S. Fonticola
colonies on agar
S. Fonticola
colonies on cheese

14. Environmental Isolation of
Clostridium
 Prepared Yeast Extract in
minimal salts Broth and
agar plates.
 5 different tubes inoculated
with soil and mud
o S1, S2, M, S1M, S2M
YEMS broth with
mixing rod
YEMS broth separated
and enriched with
samples

15. Generation and Maintenance
of Anaerobic Conditions
Pasteurization
Mineral Oil
Anaerobic Jar
GasPak EZ
Indicator Strips
Anaerobic jar with
GasPak EZ,
indicator strip and
plates
Broths with
mineral oil layer

16. Streak Plates for Isolation
SI S2 M SIM S2M

17. Colony Morphology
Round and
Irregular
Shiny
Opaque
Convex
Off-white
Streak plate for S2M sample

18. S1 Stain Analysis
Gram Stain
Endospore Stain 1 Endospore Stain 2

19. S1M Stain Analysis
Endospore Stain 1
Gram Stain
Endospore Stain 2

20. S2M Stain Analysis
Endospore Stain 1
Gram Stain
Endospore Stain 2

21. Motility Tests
 Motility tests were negative
o S2M and S1M
Weak colonies
o No growth from S1
Motility tests
after ~24 hrs
growth

22. Gelatinase & Caseinase
Tests
GELATINASE
 Positive for S2M
and S1M
 Negative for S1
CASEINASE
 Negative for all
three samples
Gelatinase test after ~48 of growth (a)S2M
(b)S1 (c)S1M
a
b
c

23. Carbohydrate
Fermentation
 Glucose, Lactose, and Sucrose
 Positive tests…
o color changes
o gas bubbles
Gas bubble present in Carb. Fermentation Test tube

24. S1 Carbohydrate Tests
Results
S1 Carb. Fermentation
test tubes (~48hr) – (a)
sucrose (b) glucose (c)
lactose
a b c

25. S1M Carbohydrate
Tests Results
S1M Carb. Fermentation
test tubes (~48hr) – (a)
sucrose (b) glucose (c)
lactose
a b c

26. S2M Carbohydrate Test
Results
S2M Carb. Fermentation test
tubes (~48hr) – (a) sucrose (b)
glucose (c) lactose
a b c

27. Results Table
Motility Gelatinase Caseinase Sucrose Glucose Lactose
S1
INC INC or
- - + GB GB
S1M - + - + GB +
S2M - + - GB GB GB
GB – formation of gas bubbles
INC - inconclusive

28. Identification of
Species
Results not always conclusive
o poor growth and isolation
Group III or IV
o Terminal endospores
Group III: Negative Gelatinase
o S1
Group IV: Positive Gelatinase (most
likely)
o S1M/S2M

29. Clostridium ramosum
Group III
Gelatinase not hydrolyzed
Non-motile
Positive glucose and lactose
White-glossy colonies
Optimal growth - 37 degrees
human and animal feces

30. Clostridium perenne
Group III
Gelatinase not hydrolyzed
Non-motile
Positive for Glucose and lactose
Irregular/round colonies
Glossy surface
Optimal growth – 30 degrees
Wound infections and feces

31. Clostridium
Putrefaciens
Group IV
Non-motile
Irregular shaped
Gelatinase hydrolysis
Caseinase negative
Glucose fermenter
Terminal endospores
STANKY!

32. Conclusion
Many results inconclusive
o Poor growth and isolation
To improve our results, obtained samples
from a greater variety of sources
including soured milk, deeper soil
samples
 Unhappy species…

33. References
Bergey, David H., and John Holt. Bergey's Manual of
Determinative Bacteriology. Philadelphia:
Lippincott, 2000. 552-72. Print.
Murdoch, D. A. "Gram Positive Anaerobic Cocci." Clinical
Microbiology Reviews 11th ser. (1998): 81-120. NCBI.
National Institute of Health. Web. 21 Sept. 2013
Sturges, W. S., and E. T. Drake. "A Complete Description of
Clostridium Putrefaciens."Journal of Bacteriology 14
(1927): 175-79. NCBI. National Institute of Health. Web.
21 Sept. 2013.
Sullivan, Karen, Dr. General Microbiology Laboratory Manual
Louisiana State University. 8th ed. Plymouth: Hayden
McNeil, 2013. Print.