Isolation of

Clostridium

Ben Anderson & RichaBanthia

BIOL 3116Fall 2013

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

Analysis of Biofilm

Gram Stain Wet Mount

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

EMB – E. coli

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

Serratia fonticola

S. Fonticola

colonies on agarS. Fonticola

colonies on cheese

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

Streak Plates for Isolation

S2SI M SIM S2M

Colony Morphology

Round and

Irregular

Shiny

Opaque

Convex

Off-white

Streak plate for S2M sample

S1 Stain Analysis

Gram Stain

Endospore Stain 1 Endospore Stain 2

S1M Stain Analysis

Endospore Stain 1

Gram Stain

Endospore Stain 2

S2M Stain Analysis

Endospore Stain 1

Gram Stain

Endospore Stain 2

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.