Team:Newcastle/Results/Chemotaxis/AgarGrowth

Model

Alternative Roots

Characterising Bacterial Growth on Solid Media

Rationale

In order to understand chemotaxis on agar, it was important to understand how the selected bacteria (Azospirillum brasilense, Azorhizobium caulinodans and Herbaspirillum seropedicae) grow on solid media in standard laboratory conditions. This measure would serve as a control to which experimental plates would be compared to. Familiarisation of bacteria also allows identification of abnormal behaviour and contamination.

Experimental Approach

Each species was grown on 1% agar. The type of agar utilised was selected from DSMZ suggestions which states which media is appropriate for each species [1-3]. Overnight culture for each species were then spread on 3 individual agar plates which were labelled and incubated at the DSMZ suggested temperature for 24-48hours (depending on the species). More details about out experimental design can be found on the protocol page.

Results

Species (Strain)	Colony Pigmentation	Colony Morphology	Points of Interest
Azorhizobium caulinodans (ORS571)	White	Regular form, Typically raised, Entire margin	Colonies rarely grow to a measurable size when grown at 30˚c on YEB media after 24 hours
Azospirillum brasilense (SP245)	Orange/Pink	Non-slimy, Regular and round form, Entire margins	Both immature and dead colonies lack the orange/pink pigment, Colonies wrinkle with age
Herbaspirillum seropedicae (Z67)	Cream/Light Green	Circular or Irregular form (occasionally rhizoid), Raised elevation, Shiny	Colonies took on a different morphology depending on how the media was innoculated; stab-innoculation lead to rhizoid form while spreading leads to circular/irregular form

Table 1: Qualitative analysis of Azorhizobium caulinodans, Azospirillum brasilense, Herbaspirillum seropedicae colonies grown on solid media.

Behaviour on agar was observed from two aspects: colony size after 24 hours and colony morphology. A. caulinodans colonies (Figure 1) often did not grow to a measurable diameter within 24 hours at 30˚C on YEB media. Due to this, the average diameter of these colonies was measured after 48 hours instead. Colonies of A. caulinodans possess a regular form and are generally raised in elevation; while flat colonies existed, they were in minority. All colonies had a light cream pigmentation which was consistent in colour from the entire-shaped margin to the centre. Larger colonies were typically <2mm whilst smaller colonies, which were much more numerous, count not accurately be measured by the chosen technique.

A. brasilense (figure 2) was very distinguishable when grown on agar due to its distinctive orange/pink pigmentation. Colonies of this species are white opaque but begin to turn orange as they mature, as they mature they also become harder to remove from the plate. Dead colonies lose their pigmentation in addition to becoming extremely hard to remove from the plate without damaging the agar. As such, there may be a relation between colony age and adhesion to the agar; however, this is work for future investigation. The colonies often began to wrinkle with age.

A. brasilense colonies were generally the largest colony after 24 hours at 37˚C, with an average diameter of 3mm. The colonies were non-slimy and round with entire borders. As the margins are less mature, there exists a white to orange/pink gradient.

Finally, H. seropedicae colonies took on different forms depending on how the plate was inoculated. If the plate was stab-inoculated, the colony took a rhizoid appearance (figure 3). On the other hand, if the culture was spread across the plate then it would typically take a circular or irregular form (figure 4). Colonies possessed a green-cream pigmentation and were raised from the surface. Most colonies were shiny and typically 1.5mm in diameter after 24 hours at 30˚C