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**ENUMERATION OF MICROORGANISMS**

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There are numerous occasions when it is necessary to either estimate or determine the number of bacterial cells. Determination of cell numbers can be accomplished by a number of direct or indirect methods. The methods include: 1- The standard plate count. 2- Turbidity. 3- Direct microscopic counts.

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**I-Standard Plate Count (Viable Counts) **

The number of bacteria in a given sample is usually too great to be counted directly. However, if the sample is serially diluted single isolated bacteria will form visible isolated colonies. We are determining the number of Colony-Forming Units (CFUs) in that known dilution. The number of colonies can be used as a measure of the number of viable (living) cells in that known dilution. I-Standard Plate Count (Viable Counts)

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**A viable cell: Is defined as a cell which is able to divide and form a population (or colony).**

Indirect viable cell counts, also called plate counts: 1- A viable cell count is usually done by diluting the original sample. 2- Plating aliquots of the dilutions onto an appropriate culture medium. 3- Then incubating the plates under proper conditions so that colonies are formed. 4- After incubation, the colonies are counted and from a knowledge of the dilution used, the original number of viable cells can be calculated.

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Materials 1- 6 tubes each containing 9.0 ml sterile saline plates of suitable media sterile 1.0 ml pipettes. 4- Pipette filler. 5- Turntable and bent glass rod. 6- Dish of alcohol. 1.0 Milliliter (ml) Pipette

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**Procedure A- Dilution of bacterial sample: Flame the sample flask.**

Insert the pipette to the bottom of the flask, and withdraw 1.0 ml of the sample. Flame the first dilution tube. Dispense the 1.0 ml of sample into the tube. Draw the liquid up and down in the pipette several times to rinse the pipette and to properly mix. Re-flame and cap the tube.

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Mix the tube thoroughly by holding the tube in one hand and vigorously tapping the bottom with the other hand.

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**Using the same procedure, aseptically withdraw 1**

Using the same procedure, aseptically withdraw 1.0 ml of the sample from the first dilution tube and dispense into the second dilution tube. Continue doing this from tube to tube.

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**B- Plate out on agar surface:**

Aseptically transfer 0.1ml from each of last 3 dilution tubes onto the surface of the corresponding plates. Note that since only 0.1 ml of the bacterial dilution is placed on the plate, the bacterial dilution on the plate is 1/10 the dilution of the tube from which it came. Place the bent portion of the glass rod on the agar surface and start to turn the plate at complete 360o. Repeat for each of the 3 plates. Incubate the 3 agar plates.

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**1- Using a Pipette to Remove**

Bacteria from a Tube. 2- Using a Vortex Mixer to Mix Bacteria Throughout a Tube.

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**3- Using a Pipette to Transfer Bacteria to an Agar Plate.**

4- Using a Bent Glass Rod and a Turntable to Spread a Bacterial Sample.

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**C- Results after incubation (Counting):**

1- Choose a plate that appears to have between 30 and 300 colonies. Sample 1/100,000 dilution plate (Figure a). Sample 1/1,000,000 dilution plate (Figure b). Sample 1/10,000,000 dilution plate (Figure c). 2- Count the exact number of colonies on that plate using the colony counter . 3- Calculate the number of CFUs per ml of original sample.

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Results (Figure a) (Figure b) (Figure c)

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**For accurate determination of the total number of viable cells: **

The total number of viable cells is usually reported as Colony-Forming Units (CFUs) rather than cell numbers. A plate having colonies is chosen because this range is considered statistically significant. If there are less than 30 colonies on the plate: small errors in dilution technique or the presence of a few contaminants will have a drastic effect on the final count. Likewise, if there are more than 300 colonies on the plate: there will be poor isolation and colonies will have grown together.

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**To determine the number of CFUs per milliliter (ml) of sample:**

Advantage: This method of enumeration is relatively easy to perform and is much more sensitive than turbidimetric measurement. Disadvantages: A major disadvantage, however, is the time necessary for dilutions, platings and incubations, as well as the time needed for media preparation. Only living cells develop colonies that are counted. Clumps or chains of cells develop into a single colony. Colonies develop only from those organisms for which the cultural conditions are suitable for growth. The number of CFUs per ml of sample = The number of colonies ( plate) X The dilution factor of the plate counted

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**A sample of E. coli diluted according to the above diagram**

A sample of E.coli diluted according to the above diagram. The number of colonies that grew is indicated on the petri plates. How many CFUs are there per ml in the original sample?

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II- Turbidity When you mix the bacteria growing in a liquid medium, the culture appears turbid. This is because a bacterial culture acts as a colloidal suspension that blocks and reflects light passing through the culture. The instrument used to measure turbidity is spectrophotometer.

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The ability of the culture to block the light can be expressed as either percent of light transmitted through the tube or the amount of light absorbed in the tube . The percent of light transmitted is inversely proportional to the bacterial concentration (The greater the percent transmittance, the lower the number of bacteria). The absorbance (or optical density) is directly proportional to the cell concentration (The greater the absorbance, the greater the number of bacteria.)

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**III- Direct Microscopic Count **

Petroff-Hausser counting chambers can be used as a direct method to determine the number of bacterial cells in a culture or liquid medium. It has squares 1/20 of a mm by 1/20 of a mm and is 1/50 of a mm deep. The volume of one square therefore is 1/20,000 of a cubic mm or 1/20,000,000 of a cubic centimeter (cc). In this procedure, the number of cells in a given volume of culture liquid is counted directly in microscope fields. The average number of cells per field is calculated and the number of bacterial cells ml-1 of original sample can then be computed.

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Procedure: Count the number of bacteria in five large double-lined squares. Divide by five to get the average number of bacteria per large square. This number is then multiplied by 20,000,000, since the square holds a volume of 1/20,000,000 cc, to find the total number of organisms per cc in the original sample. If the bacteria are diluted (such as by mixing with dye) before being placed in the counting chamber, then this dilution must also be considered in the final calculations.

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**Counting: number of bacteria per cc =**

the average number of bacteria per large double-lined square X the dilution factor of the large square (20,000,000) the dilution factor of any dilutions made prior to placing the sample in the counting chamber

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**Advantage of direct counts:**

Is the speed at which results are obtained. Disadvantage: Since it is often not possible to distinguish living from dead cells, the direct microscopic count method is not very useful for determining the number of viable cells in a culture.

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Counting Bacteria.

Counting Bacteria.

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