The aim of this laboratory hour was to provide both theoretical knowledge and practical skills about the process of counting different organisms present in a specific sample.
Process of counting can be realized by using either direct or indirect method. There are some factors that affect the counting, such as: pipetting, optimized incubation time, maintenance of homogeneity and a proper amount of the mixture .
Direct method includes two different techniques: total cell count and viable count, where the only difference between them is that in the viable count, only the cells that have the potential to give rise to new colonies are counted, while the total cell count technique involves counting both living cells and dead cells. Viable count is known also as plate count or colony count method Viable count technique is unable to give every time accurately the total number of the viable cells, because of the possibility it exists that the groups of cells get disrupted before being plated or the cells to get clumped and form a colony together. In order to take in account the possibility that the colony forming unit can contain one or more cells, the number of the viable cells is calculated as colony forming unit (cfu). Viable count can be realized either by spread plate method or pour plate method .
Serial dilution of bacterial culture is a crucial process in the process of enumeration a sample with unknown number of bacteria and it is realized by diluting the sample either with broth, saline solution or phosphate buffer in the ratio 1:9. The range of the number of colonies is 30-300 and it is an optimized value, as a number less than 30 colonies does not fulfill the statistical requirements, while a number bigger than 300 would cause colonies to compete for nutrition and could also lead to the suppress of the colony growth .
MATERIALS & METHOD
Dilution of Bacteria Cultures for Spread Plate and Pour Plate Techniques
- Bunsen burner
- Pipettes & tips
- E. coli culture
- Test tubes consisting of LB broth
1 ml from the Eppendorf that holds overnight cultured E. coli is taken by using the micropipette. Tube where 9 ml of broth is positioned is opened and sterilized through the flame of the Bunsen burner. Then 1 ml of E. coli is poured inside the broth and the tube`s mouth is sterilized again through the flame. Tube is vortexed and labeled with the specific data about the experiment. The procedure of dilution is repeated 7 times by using each time 1 ml from the tube that was diluted and vortexed in the preceding step.
Pour Plate Technique
- Water bath
- Diluted E. coli culture
- Pipettes and tips
- 15 ml LB agar in tubes
- Petri dishes
Initially, 1 ml of the diluted sample of E. coli (10-4, 10-5, 10-6, 10-7) is positioned in the Petri dish. Afterwards, 15 ml melted agar that was molten in the water bath at a temperature of 50OC, is added in the Petri dish. The plate is covered and moved over the bench in order that the samples inside get swirled. After that, the Petri dish is positioned there till the solidification of agar occurs and then placed in the incubator for 24 hours at a temperature of 37OC.
Spread Plate Technique
- Drigalski spatula
- Bunsen burner
- Pipettes and tips
- LB agar plates
- Diluted E. coli cultures
From the diluted samples (10-4, 10-5, 10-6, 10-7) 0.1 ml is taken by using the micropipette and it is thrown into the agar plate. The Drigalski spatula is first sterilized inside the alcohol and later through the fire. After it cools down, this spatula is used to spread the inoculums throughout the area of Petri dish. The plate is covered and placed over the bench for a short period of time so that the inoculums get absorbed by the agar. Afterwards, the Petri plate is positioned upside down and incubated for 24 hours under the temperature of 370C.
Standard Plate Count of Milk
- LB agar plates
- Pipette and pipette tips
- Drigalski spatula
- Tubes consisting of 9 ml saline solution
1 ml from the milk sample is taken by micropipette and poured inside the tube that contains 9 ml of saline solution whose mouth was sterilized through the Bunsen burner. The sample is vortexed for a short period of time. This process is repeated by transferring 1 ml of sample of the preceding step`s culture to the next buffer till the dilution of 10-4. After the dilution procedure is over, the dilutions 10-1, 10-2, 10-3, 10-4 plates are spread by using Drigalski spatula. The plates are covered and positioned over the bench for a short period of time till the inoculums are absorbed by agar. Afterwards, the plates are placed in incubator for 24 hours under the temperature of 37oC.
|Plate count about each dilution factor||Cigdem` s group
|Ayhan` s group
|Nergiz` s group
|Oyku` s group
|Deniz` s group
Table 1: Serial dilutions of the samples of raw milk and pasteurized milk and series of plates inoculated with diluted cultures
|Plate count||314; 0; 43||32; 0; 0; 1||11; 2; 0; 2; 0; 0||0; 1; 0; 4; 0; 0||0; 3; 0||0; 2; 0|
|Colony-forming units||(314+43)/2 x1/10-2=
1.79 x 104
|32 x 1/10-3=
3.2 x 104
|0 (as no one of the values are in the range [30;300]||0 (as no one of the values are in the range [30;300]||0 (as no one of the values are in the range [30;300]||0 (as no one of the values are in the range [30;300]|
Table 2: CFU calculations of the standard plate count of the milk by using the formula [CFU= Plate Count x Dilution Factor]. The values are represented either as duplicate or triplicate, because of the experiment performed by the different lab groups.
|Colony-forming units||>300 x 1/10–4=
More than 3 x 106 colony forming units
|>300 x 1/10-5=
More than 3 x 107 colony forming units
|135 x 1/10-6=1.35 x 108||As it is not in the range [30;300] it is not calculated|
Table 3: CFU`s calculations of the serial dilutions of samples inoculated by the spread plate technique
Figure1: The representative image of bacteria inoculated by the spread plate technique with diluted factors 10-4, 10-5, 10-6, 10-7. It is seen that as the dilution factor decreases, number of bacteria decreases too.
Figure 2: The representative images of the growth of bacteria in the standard plate count of the raw milk of Ayhan` s group ordered according to the dilution factor, specifically: 10-2; 10-3; 10-4; 10-5. It is seen that there is a decrease in the growth of bacteria proceeding from the left to right samples.
Figure 3: The representative image of the growth of bacteria inoculated by the pour plate technique. The used diluted samples are 10-4, 10-5, 10-6, 10-7. The growth of bacteria increases proceeding from the left to the right samples.
By comparing the data from the standard plate count of the raw milk and pasteurized milk, it is seen that there is a relationship between the number of bacteria and whether the milk is raw or pasteurized. In the pasteurized milk, the plate count leaded to a number of 0 bacteria. Additionally, in the raw milk it is seen that as dilution factor`s value decreases, the number of bacteria decreases. In the calculations of the colony forming units, most of the calculations are not performed because the values of bacteria are outside the range [30; 300].
In the pour plate method, it is observed that most of the bacteria have accumulated in the upper part of the medium and this occurs, because they have a high requirement of oxygen. The other part has accumulated in the down part of the medium, because they are micro-aerophiles and do not need lots of oxygen.
In the spread plate method, it is seen that as the dilution factor decreases, number of bacteria decreases too. The reason for that is that as the dilution process is made the ratio of the sample to the medium continues to proceed in this form: 1/100, 1/1000, 1/10000.
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