Purpose and
Objectives1. 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
Basic Facts about
ClostridiumGram Positive Rods- short chains
Anaerobic
Produce endospores to survive
extreme conditions
Found in soil, mud, plants, animal
feces, and soured milk
5 groups
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
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
mudS1
S2
M
Dirt/mud samples
used –S1 S2 and M
Growth of BiofilmWhat is a biofilm?
Contained mix of
all soil and mud
samples in
deionized water
Allowed to grow
for 5 days
Biofilm after
growth
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
Rapid Identification of
Gram Negative Bacteria Eosin Methylene Blue
Agar
o Gram –
Differential for lactose
fermenters
Lactose fermentation
= dark colonies
EMB 1
EMB 2
API-20E Inoculation
Identification of…
o Family Enterobacteriaceae
o Other Gram negative bacteria
20 tests
API-20E inoculated tray
API-20E Results for Both Test
Strips
Serratia fonticola 40%
Enterobacter aerogenes 30%
Enterobacter cloacae 23.4%
Both API-20E trays after allowing to sit for ~48hrs
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
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
Motility Tests
Motility tests were negativeo S2M and S1M
Weak colonieso No growth from S1
Motility tests
after ~24 hrs
growth
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
Carbohydrate
Fermentation Glucose, Lactose, and Sucrose
Positive tests…
o color changes
o gas bubbles
Gas bubble present in Carb. Fermentation Test tube
S1 Carbohydrate Tests
Results
S1 Carb. Fermentation
test tubes (~48hr) – (a)
sucrose (b) glucose (c)
lactose
a b c
S1M Carbohydrate
Tests Results
S1M Carb. Fermentation
test tubes (~48hr) – (a)
sucrose (b) glucose (c)
lactose
a b c
S2M Carbohydrate Test
Results
S2M Carb. Fermentation test
tubes (~48hr) – (a) sucrose (b)
glucose (c) lactose
a b c
Results TableMotility Gelatinase Caseinase Sucrose Glucose Lactose
S1INC INC or
-- + GB GB
S1M - + - + GB +
S2M - + - GB GB GB
GB – formation of gas bubbles
INC - inconclusive
Identification of
SpeciesResults 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
Clostridium ramosumGroup III
Gelatinase not hydrolyzed
Non-motile
Positive glucose and lactose
White-glossy colonies
Optimal growth - 37 degrees
human and animal feces
Clostridium perenneGroup III
Gelatinase not hydrolyzed
Non-motile
Positive for Glucose and lactose
Irregular/round colonies
Glossy surface
Optimal growth – 30 degrees
Wound infections and feces
ClostridiumPutrefaciens
Group IV
Non-motile
Irregular shaped
Gelatinase hydrolysis
Caseinase negative
Glucose fermenter
Terminal endospores
STANKY!
ConclusionMany 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…
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.