..Counting Bacteria.. Made By: Duaa Mohammed EL- Boh. ID: 22042048. Supervised By: DR. Abdelraouf Elmanama.

Contents and Overview : we will see methods of counting: *Direct methods: - Counting chamber. - Coulter counter. - Colony counting (Viable Count): (How ,Advantages, Disadvantages) - Serial Dilution *Indirect methods.

If we need to count penciles we will say…. One. Two. Three If we need to count penciles we will say….. One.. Two .. Three .. .. .. .. But when we need to count bacteria this will be impossible ... so…. What???!!!

Importance : *Knowing how to count organisms and understanding their growth cycles is often important in treating infections. *By counting individual organisms and experimenting, we can determine how many it takes to cause disease. * Counting bacteria is also important in environmental microbiology; to control environmental conditions or enhance growth to obtain desired results.

please ... give me your attention..

**A single tiny drop of nutrient broth incubated overnight may contain 5 000 000 cells – this is a lot to count. **1cm3 may contain 108 cells.. so **In order to estimate numbers it is necessary to dilute the sample.

1- Counting Chamber (Hemocytometer) : *Direct methods : With direct methods we count individual cells or colonies that are assumed to have apart or arisen through the division of a single cell. 1- Counting Chamber (Hemocytometer) : The haemocytometer is a specialized microscope slide used to count cells. The centre portion of the slide has etched grids with precisely spaced lines. To get an accurate count there should be between 40 and 70 cells in a 1 mm square. If not you dilute or concentrate the cell suspension as necessary .

To get the final count in cells/ml, first divide the total count by 0 To get the final count in cells/ml, first divide the total count by 0.1 (chamber depth) then divide the result by the total surface area counted , then divide the result by 5 mm-squared, which is the total area counted (each large square is 1 mm-squared).

There are 1000 mm-cubed per ml, so you calculate cells/ml There are 1000 mm-cubed per ml, so you calculate cells/ml. Sometimes you will need to dilute a cell suspension to get the cell density low enough for counting. In that case you will need to multiply your final count by the dilution factor. Hemocytometer Counting using a counting chamber.

2- Coulter Counter : electronic counting (this machine detects the difference in current as individual microorganisms pass through a small orifice). It is Very fast , easy to use but; Very EXPENSIVE.

3- Viable count assays (Colony Counting) : Colony counting after plating dilutions of the sample onto growth medium. Standard plate counts using spread and pour plate techniques (cfu for “colony forming unit”) . This is the method we will be using to quantify our samples.

We will use two viable count assays: Count only those cells capable of growing Viable counts can be accomplished by such techniques as pour plating. Assumption each viable cell gives rise to a colony. We will use two viable count assays: 1- Spread plates A diluted sample is spread onto the surface of an agar plate 2- Pour plates Microorganisms are mixed with molten agar and poured into a petri dish.

*Advantages: *Disadvantages: - The method can be made to be very sensitive. - One count can be subsets of the population. *Disadvantages: - Colony-forming units may underestimate cell numbers because of clumping or chains of cells. - Counts require at least a few hours, usually overnight, for incubation.

4- Serial Dilution : *Why use Serial Dilutions? - Bacteria undergo exponential growth , thus many may be present in each sample. - In order to count the number of bacteria, each colony must be single and distinct. - The number of countable colonies per plate is 30-300 - Serial dilutions allow one to dilute a sample of bacteria to the point that the total number of bacteria can be counted on an agar plate.

How to Perform Serial Dilutions :

SERIAL DILUTION 1ml into 9ml = 1:10 dilution conc. 10-1 10-2 10-3 10-4 10-5 10-6

*Indirect Methods : Indirect methods often rely on the results of metabolic tests or other growth characteristics. And it’s to: - Measurement of metabolic activity. -Gas or Acid Production. - Turbidity using a spectrophotometer. -spectrophotometry, using a spectrophotometer .

These Indirect counts : - depend on the effects of the organisms to estimate their numbers. - As organisms grow they make the nutrient broth turbid. - This turbidity can be measured with a colorimeter - The more organisms the greater the optical density of the solution.

- The Coulter counter is a probe which measures the change in conductivity of a solution as a bacteria passes through a narrow gap. - Problem with INDIRECT COUNTS and DIRECT COUNTS is that they can not tell living cells apart from dead cells. - Advantage of INDIRECT COUNT is that the process can be automated.

*References : *http://sciencelinks.jp/j-east/article/200517/000020051705A0706126.php. *http://whyfiles.org/shorties/count_bact.html. *http://msucares.com/livestock/dairy/bacteria.html. *http://www.freesciencefairproject.com/biology/bacteria_counting.html. *http://www.disknet.com/indiana_biolab/b038.htm.