1. Counting Bacteria

2. 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

3. 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

4. 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

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

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

7. Counting Chamber

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

10. 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

14. 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

15. Viable Cell Count –
Living Cells Only

16. 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

17. 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

18. 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

20. 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

22. 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

23. 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.

24. Serial Dilution

26. 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

29. 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.

30. 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.

33. 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