Methods
A. Isolation of Serratia marcescens
1. Obtain slices of bread and keep the bread moist using tap water.
2. Water the bread daily until bacterial growth is
observed on the bread. Serratia marcescens have a red-pigmented color
that distinguishes them from other bacteria.
3. Flame an inoculating loop and obtain a sample of
the reddish purple bacterial growth from the bread.
4. Using streak plate techniques, streak the sample
on MacConkeys agar plates containing 1%
lactose. MacConkeys media is selective for
Gram-negative organisms. Since Serratia marcescens are gram-negative and
lactose non-fermenting organisms, this organism will grow on MacConkeys agar plates.
5. Incubate MacConkey
plates at room temperature (25oC to 30oC) for 48 hours.
6. Observe plates for red-pigmented colonies.
7. If red-pigmented colonies are present, streak DTC
media plates, an enrichment media, with individual colonies obtained from MacConkeys agar plates.
8. Incubate streaked DTC plates at room temperature
(25oC to 30oC) for 48 hours.
9. Observe plates for growth. Colonies of
Serratia marcescens appear blue and purple on the DTC plates.
10. Conduct the following tests to confirm the
identity of Serratia marcescens:
a. Gram stain
b. Oxidase test
c. Catalase test
d. Motility agar test
e. Carbohydrate Utilization tests
- mannitol sugar tubes
- lactose sugar tubes
- glucose sugar tubes
f. Oxidation/Fermentation test
B. Enrichment Media Recipe
2. DTC media
- DTC media is an enrichment media that provides
nutrients to enrich the growth of Serratia marcescens.
Composition per liter:
Agar--------------------------------------20.0g
Pancreatic digest of Casein-----------15.0g
NaCl-------------------------------------5.0g
Papaic digest of Soybean meal-------5.0g
Deoxygibonucleic acid----------------2.0g
Toluidine Blue--------------------------0.1g
Cephalothin Solution------------------10.0ml
Cephalothin Solution:
Composition per liter:
Cephalothin-----------------------------1.0g
Preparation of Cephalothin
solution:
1. Add cephalothin to
distilled/deionized water and bring volume to
10.0ml. Mix thoroughly.
2. Filter sterile solution.
Preparation of DTC media:
1. Add components, except Cephalothin
to distilled/deionized water and bring volume to
990.0ml.
2. Mix thoroughly.
3. Gently heat and bring to a boil.
4. Autoclave for 15 minutes at 15psi pressure and
121oC. Cool to 45oC to 50oC.
5. Aseptically add sterile Cephalothin
solution.
6. Mix thoroughly.
7. Pour into sterile petri dishes.