Microbiology Lab

Name: Loreena Alyacoub | 2010204

Experiment: Enumeration of Microorganisms, Sample preparation, and Total count

 Objective:

1. To learn the different enumeration techniques and the techniques used to count the number of
microorganisms in a sample.
2. To have more practice in serial dilutions and calculations.

 Introduction:

For unicellular microbes like bacteria, reproduction of cells reproduces the entire organism.
As a result, microbial growth is identical with microbial reproduction. And to determine rates of
microbial growth and death it’s necessary to enumerate microorganisms and determine their
numbers.

Dilution is the process of a decreasing the concentration of a solute in solution, usually

simply by mixing with more solvent. In a cereal dilution, a series of sequential dilutions are
performed to dilute a food sample in a diluent saline. In this case, cereal dilutions are usually
tenfold, but you can use any dilution factor that's convenient. By sequentially diluting the food
sample, you reduce the bacterial concentration in each dilution.

Viable counts: give the potential of microbial deterioration of food that individual living cells
develop, during incubation in the appropriate medium, into visible discrete colonies which can be
counted. And to calculate rates of microbiological growth and death, microorganisms must be
counted and the goal is to obtain a dilution that produces an easily quantifiable number of CFU
once plated. The number of CFU and the total volume that was plated can be used to determine
the bacterial concentration in the original food source. Only Numbers between 30 to 300 CFU.

 Materials:

1. Petri dishes
2. Agar plates
3. Colony counter
4. Plate count agar
5. Dilution bottle and tubes each containing buffer or ringer’s.

 Methods:

1. Total counts (Breed’s methods, Howard mold count method).

2. Viable counts (standard plate count or pour plate , spread plate method).
  Procedure:

1. Using a marker, mark the tubes and the petri dishes.

2. Using a pipette transfer 1mL of food homogenate to tube 1.Discard the pipette into cylinder
provided and mix it.
3. Using a fresh pipette, for each subsequent transfer proceed as in step 2 above, each 1mL from
tube 1 to tube 2 then from tube 2 to tube 3 etc..

 The dilution factor then will be 101, 102…… 107 and the volume of the original inoculum per ml
of each dilution will be 10-1, 10-2…… 10-7

4. In the petri dishes, transfer 1mL of the corresponding dilution.

5. Pour into each petri dish 10 mL of plate count agar previously molten and maintained in 45 c
water bath.
6. Move the petri dishes gently in the bench for six lines in a clockwise circle and repeat in anti-
clockwise, back and forward..
7. Record the results after after inserting the plates in the incubator for 48 hours.

 Results:

Figure 1 Figure 2 Figure 3 Figure 4

Figure 1: can’t be counted, more than 300

Figure 2: almost 205, less than 300
Figure 3: counted 42, more than 30
Figure 4: counted 13, less than 30

 Discussion:
 As it shown in the figures above, the first figure dilution is mixed and the process is repeated to
continually dilute the previous dilution. As a result, multiple dilutions are created and the
concentration of the food sample in the diluent becomes less and less with each new dilution.
The most concentrated dilution is termed the ten to the negative one dilution and the second most
is considered the ten to the negative two dilution and so on.

 Conclusion:

Bacterial enumeration and strain isolation by plating requires manageable concentrations of target
organisms. Successful plating is therefore contingent upon serial dilution.
For the plates with more than 300 of colonies won’t be used in the calculations because they are
hard to count. Counts under 30 are not considered representative of the sample and over 300 CFU
colonies tend to grow into each other and cannot be accurately counted.

 References:

 Lab manual.
 Videos posted in the e-learning.