Growth of Chromobacterium violaceum on R2A and Tryptic Soy Agar

Wenfa Ng
Department of Chemical and Biomolecular Engineering, National University of Singapore
Email: ngwenfa771@hotmail.com
Abstract
Chromobacterium violaceum (ATCC 12472), a Gram-negative, rod-shaped bacterium capable
of synthesizing hydrogen cyanide, was cultivated on R2A and Tryptic Soy Agar (TSA) at 30
o
C for understanding the relative growth performance of the bacterium on different agars.
Experiment results revealed that C. violaceum could grow well on both agars with the
generation of small, round, purple colonies. Such morphology was similar to those obtained
during growth of C. violaceum on LB Lennox agar. However, amount of purple violacein
pigment secreted was significantly higher during growth on TSA agar compared to R2A agar.
Swarming motility was not observed on both agars during growth of C. violaceum. Overall,
relatively high cell density of C. violaceum could be supported by R2A and TSA agar, but TSA
would be a more preferable growth medium for the bacterium if violacein production is a
subject of study.
Keywords: violacein, round colonies, purple pigment, Chromobacterium violaceum, R2A agar,
Tryptic Soy Agar,
Subject areas: biochemistry, cell biology, microbiology, biotechnology,

Background and summary

Microbes exhibit different growth behaviour when cultivated in different growth
medium. Thus, it is important to cultivate a microbial species in different growth medium for
understanding its relative growth performance (and thus, metabolic programme), which holds
important implications to the understanding of experiment observations in future more in-depth
research.

Chromobacterium violaceum (ATCC 12472) is a Gram-negative, rod-shaped soil

bacterium that secretes a violet pigment known as violacein in addition to hydrogen cyanide.1
Its growth performance on R2A and Tryptic Soy Agar (TSA) was assessed via streak plate
inoculation of the bacterium on both agars. Specifically, growth of C. violaceum on R2A agar
generated small, purple, round colonies without signs of swarming motility after 2 days of
incubation at 30 oC (Figure 1).

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 a) b)

Figure 1: Growth of Chromobacterium violaceum on R2A agar after 2 days of incubation at

30 oC. a) Main agar plate, b) Close-up of the same plate showing small, round, purple colonies.

Similarly, growth of C. violaceum on TSA agar generated small, round, purple

pigmented colonies after 2 days of incubation at 30 oC. However, the purple pigment was
darker, which suggested increased secretion of the pigment during growth on TSA agar (Figure
2). No swarming motility was observed.

a) b)

Figure 2: Cultivation of C. violaceum on Tryptic Soy Agar at 30 oC for two days. a) Main agar
plate, b) Close-up of the same plate showing small, round, purple colonies similar to those of
C. violaceum on R2A agar.

Overall, C. violaceum could grow well on both R2A and TSA agar with the generation
of round, small colonies with the secretion of a purple violacein pigment. No swarming motility
was detected. Between the two agar, C. violaceum secreted less violacein pigment during
growth on R2A agar possibly due to the low nutritional content of the agar compared to the
much richer TSA agar. Both agar were able to support relatively high cell density of C.
violaceum.

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Materials and Methods
Materials
R2A agar and Tryptic Soy Agar were purchased from Merck and used as is. LB Lennox
medium was purchased from Difco and used as is. Composition of R2A agar was [g/L]: yeast
extract, 0.5; Proteose Peptone, 0.5; Casein hydrolysate, 0.5; Glucose, 0.5; Starch soluble, 0.5;
Sodium pyruvate, 0.3; K2HPO4, 0.3; MgSO4, 0.024; Agar, 15.0. Composition of Tryptic Soy
Agar was [g/L]: Pancreatic digest of casein, 15.0; Papaic digest of soya bean, 5.0; NaCl, 5.0;
Agar, 15.0. Composition of LB Lennox medium was [g/L]: Tryptone, 10.0; Yeast extract, 5.0;
NaCl, 5.0.

Cultivation of Chromobacterium violaceum on agar

Stock cultures of Chromobacterium violaceum was prepared in 40% glycerol and stored at -70
o
C prior to use. To prepare a seed culture, one glycerol stock culture was used as inoculum for
100 mL of LB Lennox medium in 250 mL glass conical flask and incubated at 30 oC and 230
rpm rotational shaking. After 24 hours of incubation, sterile inoculating loop was used in
retrieving a small amount of inoculum for streak plate inoculation of R2A agar and Tryptic
Soy Agar plates. Inoculated agar plates were incubated at 30 oC.

Reference
1. The complete genome sequence of Chromobacterium violaceum reveals remarkable and

exploitable bacterial adaptability. Available at:

http://www.pnas.org/content/100/20/11660.full. (Accessed: 20th October 2017)

Conflicts of interest
The author declares no conflicts of interest.

Author’s contribution
The author designed and performed the experiments, analysed the data and wrote the
manuscript.

Funding
The author thank the National University of Singapore for financial support.