1. Faculty of Medicine and Health Sciences Microbiology Lab Second semester 2014 prepared by: Mohammad Al-Qadi E-mail: m.qadi@najah.edu

2. Methods and techniques used for the enumeration of bacterial cells Lab # 7

3. Introduction Dilution Why we need to dilute? Ten fold dilution? Dilution factor?

4. Growth measurements techniques 1. Cell count: - Using microscope - Electronic Particle counter - Colony count 2. Cell mass: - Using bacterial dry weight, - Measuring of cell nitrogen - Turbidity 3. Cell activity: -Biochemical activity to the size of population

5. Direct Microscopic Method PROCEDURE 1. Prepare 10 folds serial dilution in sterile water or saline as mentioned above 2. Using a Pasteur pipette, fill the chamber of a Petroff-Hausser counting chamber with a drop from one of the tubes with a high dilution factor. 3. Place a coverslip over the chamber 4. Place a drop of immersion oil on the coverslip 5. Bring the squares into focus using oil immersion lens 6. Count the number of bacterial cells in each of the designated areas for counting 7. Calculate the average number of bacterial cells per area 8. Calculate the number of bacterial cells per 1 ml or the original sample 1mm X 1mm X 0.1mm =0.1 cubic mm = 0.1 micro litter Calculating equation: The number of counted bacterial cells X 10 X 1000 X dilution factor of the used tube

6. counting chamber Stain?

7. THE PLATE COUNT (VIABLE COUNT) The number of bacteria in a given sample is usually too great to be counted directly. However, if the sample is serially diluted (10 folds serial dilution) and then plated out on an agar surface in such a manner that every single isolated bacterial cell forms a visible isolated colony. Achieved by: Spread plate technique? Streak plate technique? Pour plate technique

8. Plate Count PROCEDURE 1. Prepare 10 folds serial dilution as mentioned earlier (10 dilution tubes) 2. Using automatic pipette, inoculate 0.1 ml from each of the dilution tubes into Trypticase Soy agar plate. 4. Sterilize the glass spreader by dipping the bent portion in a dish of alcohol and igniting the alcohol with the flame from your burner. Let the flame burn out) 3. Using the sterilized glass spreader, spread the solution over the surface of the plate 4. Sterilize the glass spreader 5. Repeat for each plate. 6. Incubate the inoculated plates upside down in a 37°C incubator corresponding to your lab section for over night. 7. Count the number of the grown colonies on a plate that has 30-300 colonies 8. Calculate the number of CFU/ml in the original sample.

9. Serial ten-fold dilution (a series of 1:10 dilutions)

10. Plate Count Therefore, when doing the plate count technique, generally, we are determining the number of Colony-Forming Units (CFUs) in that known dilution. Recall that, the bacterial sample is diluted by factors of 10 ( 10 fold serial dilution). About 0.1 ml from each of the diluted samples is plated on agar. After incubation over night, the number of colonies on a plate showing between 30 and 300 colonies is determined. Equation to calculate the number of CFU/ml of the original sample : Number of counted colonies X 10 X dilution factor (of the sample you cultured) Note: For a more accurate count it is advisable to plate each dilution in duplicate or triplicate and then find an average count.

11. Reporting The technique is based on the principle that each viable organism will grow into one colony Cells present in culsters as ( Staphlococci & Streptococci ). May be clumped together. For that reason, reporting as Colony-Forming Units (CFUs) /ml ( colony forming unit ) rather than # of bacteria/ml is more accurate.

12. Note A direct count of a bacterial population only detects the total number of organisms and includes both dead and living organisms While a viable count with the use of a pour plate culture procedure estimates the number of living bacteria in a population.

13. 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. Within limits, the light absorbed by the bacterial suspension will be directly proportional to the concentration of cells in the culture. By measuring the amount of light absorbed by a bacterial suspension, one can estimate and compare the number of bacteria present. Drawing curve !! The instrument used to measure turbidity is a spectrophotometer.