EXERCISE 4

SPOREFORMERS IN FOOD

Sporeformers are capable of forming spores around themselves, which allow the organism to
survive in hostile environmental conditions

Predominant sporeformers: Bacillus and Claustridium

Observation of ropiness
Medium: Dextrose Tryptone Agar (DTA) used for isolation of mesophilic or
thermophilic spoilage microorganisms from food
Components:

Dextrose CHO source

Tryptone N source

Organism: Bacillus subtilis mesophile

-

Gram +, catalase positive bacteria commonly found in soil

(+) for ropiness

Theoretical result: Grey-white, vesicle-like, dry and wrinkled

Test for ropiness: Dip a wire loop in the growth

Pull it up

(+) result: thread-like strands dripping

Detection of flat-sour spores:

Organism: flat-sour spores thermophilic bacteria that can cause flat-sour
spoilage in low acid canned food (flattening/deformation of cells)

Medium: Dextrose Tryptone Bromcresol Purple Agar (DTBPA)

 used for cultivating thermophilic flat-sour microoorganisms associated with

food spoilage
Components:

Dextrose CHO source

Tryptone Nitrogen source
Bromcresol purple pH indicator
acidic

Purple neutral

Yellow

Boiling (5 min.) to kill vegetative cells

Theoretical result: round, opaque central spot with yellow halo

Theoretical
(Flour)

Experimental (Sugar)

Experimental

Sample Calculations:
Sugar =

20 g
g
=0.2 ( 10 mL ) =2 g sugar
100 ml
ml

Assume flat sour counts: 2 mL/plate

Data:
Plate 1 = 13, Plate 2 = 51, Plate 3 = 76

FS counts=140 FS

/2g (5) =

700FS/10g
Flour =

11 g
0.1 g
FS counts flour ( 10 ) = FS counts/ 10 g
( 1mL )=
=

11 g+ 99 mL mL
0.1 g

0.1 g
FS counts flour ( 10 ) = FS counts/ 10 g
( 0.1mL )=

mL
0.01 g

FS counts (0.1 mL+1 mL)/10 mL

2

Enumeration of Bacillus cereus

Medium: Mannitol Yolk Polymyxin Agar (MYPA) + Egg yolk emulsion +
Polymyxin B
for the enumeration of Bacillus cereus in food samples
Agar Components:

Peptone and Beef extract N, C and vitamin source (will be utilized, increases
pH)
D-mannitol CHO source (will not be utilized)
Sodium chloride maintains osmotic environment
Phenol red pH indicator

Egg Yolk emulsion (1:1 saline solution + egg yolk) produces Lecithin
Polymyxin B inhibits non-B. cereus organisms
Food sample: Sugar
Organism: Bacillus cereus: Gram (+), rod-shaped, facultative anaerobic
Mannitol (); pink colonies

Produces Lecithinase which hydrolyzes Lecithin; forms a zone of white

precipitation around the colonies (halo)
Theoretical result: rough and dry colonies with a violetred background and a halo
of white dense precipitate

Theoretical

Experimental

Detection of Thermophilic Anaerobes not producing hydrogen sulfide

Medium: Cooked Meat Medium (CMM) used to detect thermophilic anaerobic
microorganisms (50-55C)
Components:

Beef heart and Proteose peptone N, C and vitamin source

Dextrose sufficient energy source, but low enough to not accumulate toxic
metabolites
Sodium chloride maintain osmotic balance
2% water agar provides anaerobic condition; prevents reabsorption of O 2
Boiling serves the same purpose of water agar
Theoretical results: Turbidity and/or presence of gas bubbles, cheesy odor, agar is
pushed upwards

Food samples:
A. Sugar

B. Flour

Detection of sulfide spoilage organisms

Medium: Sulfite Agar + Fe source (rusty nail)
for detecting thermophilic, H2S-producing anaerobes, particularly in foods
Agar Components:

Tryptone C, N, mineral and vitamin source

Sodium sulfite upon reduction, produces H 2S

Fe source combines with any dissolved oxygen in the medium and provides an
anaerobic
environment,
no gas
Food sample: sugar
Theoretical result: blackened spherical areas

Theoretical

References:

Experimental

http://www.scienceprofonline.com/science-image-libr/sci-image-libr-bacterialcolonies.html
http://www.neogen.com/Acumedia/pdf/ProdInfo/7741_PI.pdf
http://www.neogen.com/Acumedia/pdf/ProdInfo/7110_PI.pdf
https://prezi.com/60kqfodi5eav/sporeformers-in-food/
http://www.neogen.com/Acumedia/pdf/ProdInfo/7340_PI.pdf
https://www.sigmaaldrich.com/content/dam/sigmaaldrich/docs/Fluka/Datasheet/86128dat.pdf