The effect of atmosphere and inoculum size on Staphylococcus epidermidis biofilm density

Abstract number: 1134_03_407

Cargill J.S., Upton M.


Staphylococcus epidermidis is a potential cause of nosocomial infection, and its pathogenicity is related to its ability to form a biofilm. The aim of this study was to assess the variation in biofilm density of Staphylococcus epidermidis strains (including the reference strain RP62A) at different concentrations and in different atmospheres.


Staphylococcus epidermidis strains were cultured overnight in typtone soya broth with an additional 0.25% w/v glucose (TSB). Sterile flat-bottomed polystyrene 96-well cell culture plates were prepared with 100 ml of fresh TSB in 10 columns. 100 ml of overnight culture was added to an empty column, and serial dilutions performed in the TSB containing wells, with 100 ml discarded from the final dilution. One column was left blank as a reference. The plates were then incubated at 36°C for 20 hours before the wells were emptied by pipette, washed three times in 200 ml phosphate-buffered saline and air dried. They were then stained with 0.4% crystal violet solution for ten minutes before rinsing under gently running water. Biofilm density was measured as the optical density at 450 nm (OD > 0.12, weak positive; OD > 0.24, strong positive). 200 ml of 1:100 dilutions of overnight culture were added to the columns of 2 96-well plates. One set was incubated in normal atmosphere and the other in a 5% carbon dioxide enhanced atmosphere. Biofilm density was assessed as above. Each experiment was repeated three times.


For most strains the biofilm density of the serial dilutions remained approximately constant at dilutions above 1:4, although some showed a gradual reduction in density above this level. One strain showed a pronounced drop in biofilm density between 1:8 and 1:32 dilution. Eight of fifteen strains showed biofilm positive results above 1:4 dilution in all three repeats.

Nine of fifteen strains showed increased biofilm densities in a normal atmosphere while four strains showed increased density in when grown in the presence of elevated carbon dioxide. Two strains did not show statistically significant (Mann–Whitney test) overall differences.


This study shows that the biofilm density of some Staphylococcus epidermidis strains are affected by the dilution, and hence inoculum size, and the atmosphere. The results vary in a strain dependant manner.

Session Details

Date: 01/08/2007
Time: 00:00-00:00
Session name: XXIst ISTH Congress
Location: Oxford, UK
Presentation type:
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