Counting Bacteria

Serial Dilutions Because of their very small size, counting the number of bacteria in a sample can be difficult Although direct counts are possible with a microscope, they require a lot of time and expertise

Why Count the Number of Bacteria?? Drug Companies: How effective drugs are in killing microorganisms? Hospitals: How many MO in CSF, blood, urine Public Health: - Contaminants in food, milk, water

Total Cell Count The most common method of enumerating the total microbial cells (dead and alive) The direct counting of cell suspension in a counting chamber of known volume using a microscope

Total Cell Count – Both living and dead MO Disadvantage: Unknown potential for continuing infection Advantage: Toxins can come from both living and dead MO

Total Cell Count – Both living and dead MO Neubauer counting chamber Special slide & cover slip, microscopic marks on slide, known area

Counting Chamber

Counting Chamber Use special pipette for dilution Count specific areas of chamber, multiply by dilution factor

Instrumentation * Spectrophotometer (Light Meter) Each single MO stops some light from passing through a special test tube. One side light source, then test tube containing MO, other side light meter. Needle reading from scale, then conversion table telling # of MO in test tube

Coulter Counter Another method for counting cells An electronic instrument “electronic eye” Has a tiny tube (one cell diameter) that lets through only one cell at a time

Viable Cell Count – Living Cells Only

Viable Count – Living Cells only An easier method is to spread bacteria over a wide area (nutrient agar plate) and count the number of colonies that grow If the bacteria are spread out enough, each bacterial cell in the original sample should produce a single colony

Serial Dilutions Bacterial samples must be diluted considerably to obtain reasonable counts To determine the number of cells in a bacterial culture carry out serial dilutions

Serial Dilutions Bacterial cell numbers are usually very high in your original sample Plating out this sample, undiluted, would lead to the creation of a bacterial lawn Bacterial Lawn - many individual bacteria colonies that are all growing next to or on top of one another

Serial Dilutions Bacterial cell numbers need to be reduced This is done by repeatedly diluting the amount of bacteria you have in your sample A small amount of bacteria sample is mixed with a diluent solution (sterile broth or water), and then successive dilutions are made

Serial Dilutions A small amount of each of the diluted bacteria samples is then spread onto an agar plate The numbers of bacterial colonies that grow on each plate are counted

Serial Dilutions Work backwards using multiplication with the "dilution factor” Dilution Factor - the number of times that you have diluted the bacteria sample with the diluent solution Make a determination of the numbers of bacteria in your original sample.

Serial Dilution

Serial Dilutions *Make serial dilutions *Spread a small amount from each tube onto agar plate *Culture bacteria for a couple of days *Count colonies Each living MO = 1 Colony

Membrane Filtration Membrane filters can also be used to determine the bacterial numbers. In this method cells are filtered onto membrane filter which is then placed over a nutrient agar plate.

Membrane Filtration The methodology is similar to conventional total plate counts. Membranes have a printed millimeter grid and colonies can be counted under a binocular microscope.

Factors essential for bacterial growth --proper nutrients --proper environment (space, temperature) --No inhibiting factors Factors that could inhibit bacterial growth --lack of nutrients --lack of proper environment --presence of an inhibiting factor