Bacterial Growth

Figure 1. Bacterial Growth Curve Figure 2. Determination of Generation Time In this lab you will estimate numbers of bacteria by the two most widely used methods: viable plate count and spectrophotometric analysis. In liquid culture, the medium appears more and more cloudy as the bacteria increase in number by division. A tube of bacteria will tend to reflect light so that less light is transmitted through the tube. A spectrophotometer can measure the amount of light passing through the tube, or conversely the amount of light absorbed. These measurements of turbidity or optical density (OD) are not direct measurements of bacterial numbers, but an indirect measurement of cell biomass that includes both living and dead cells. As the bacterial cell population increases, the amount of transmitted light decreases, increasing the absorbance reading on the spectrophotometer. (Fig. 3,4). If one takes readings of the same culture over time, the absorbance readings will increase as the cell number increases. Figure

Flask on left contains sterile medium; flask on right medium inoculated with E. coli the day before. Note the turbidiy in second flask due to bacterial cells. This can then be graphed to show the growth curve for the particular conditions being tested. There are some limitations with this method, though. A growth curve that includes the lag, log, and stationary phase will take several hours to complete and the relationship between cell number and absorbance will begin to deviate from linearity at high cell densities. Generally an absorbance reading or O.D. of 0.8 is about as high as one should try to measure. Gram Stain Most bacteria are characterized by having not only a cell membrane but also a cell wall which lies outside of the cell membrane. This cell wall is composed mostly of peptidogycan and helps to maintain osmotic pressure and the cell’s characteristic shape. Some taxonomic groups of bacteria also have an outer membrane that is attached to the peptidoglycan by small lipoprotein molecules (Fig. 5). This difference in outermost cell structure is the basis for classification of bacteria by a differential staining technique known as the Gram stain. Gram-positive cells (those without an outer membrane) stain purple in the procedure, gram-negative cells (which have the outer membrane) stain red or pink. The usual first step in any bacterial identification is the determination of whether or not it is a G+ or G- bacterium. A sample of the bacteria in question is first stained with the primary cationic dye crystal violet. Since most bacteria carry a net negative charge at pH 7 they pick up the dye. At this point morphological features such as relative size, shape, and characteristic arrangement of cell groups can be seen. A mordant (in this case Gram's iodine) is then added to form a tighter complex between the stain and the cells . To remove any excess stain or stain that has not adhered to the cell, a decolorizing agent is then added(ethanol). At this point gram-positive cells are purple and gram-negative cells are colorless. Cells are then stained with a counterstain (safranin). Gram-negative cells will pick up the counterstain and appear red or pink. In this lab you will be given an unknown bacterium that you will be asked to identify as either gram positive or negative. Outer