Faculty of Medicine and Health Sciences

Microbiology Lab

Second semester 2014

prepared by: Mohammad Al-QadiE-mail: m.qadi@najah.edu

Methods and techniques used for the enumeration of bacterial cells

Lab # 7

Introduction

• Dilution

• Why we need to dilute?

• Ten fold dilution?

• Dilution factor?

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

Direct Microscopic Method

PROCEDURE1. Prepare 10 folds serial dilution in sterile water

or saline as mentioned above2. 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 coverslip5. Bring the squares into focus using oil

immersion lens6. Count the number of bacterial cells in each of

the designated areas for counting7. Calculate the average number of bacterial cells

per area8. 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

counting chamber

Stain?

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

Plate Count

PROCEDURE1. 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 spreader5. 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.

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

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.

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.

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.

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.

Thank you