The aim of this experiment was to provide general knowledge related to the direct microscopic count, other indirect methods of counting and application skills about performing them.
The cells` s count can be performed either in a direct or indirect way. Direct method or direct microscopic count can be performed either in samples positioned in slide or samples in liquid. In the case of the samples of liquid, a counting chamber (hemocytometer) whose main usage`s principle the counting of cells in the upper and left part avoiding the counting of the ones in the right and the bottom part. The chambers that are most commonly used are Petroff-Hausser or Neubauer one. The indirect methods enable the counting of the cells by measuring some cell`s characteristics that increase proportionally with the number of the cells, such as turbidity. Turbidity is the scientific term to describe the cloudiness that is caused inside a solution, because the microorganisms present scatter the light and its measurement is performed by spectrophotometer. Additionally, the absorbance is the magnitude of the light that is absorbed by the microorganisms in the solution and has an inverse relationship with turbidity .
Optical density is the term used to describe the absorbance in a specific wavelength and one of the most widely used values in microbiologic experiments is OD600 (absorbance in the wavelength of 600 nanometers) and those experiments include: determination of the optimal time about inducing a culture in the protein expression protocols or about their harvesting and preparation and the concentration of inoculums about MIC (minimum inhibitory experiments) .
Growth curve is the representation of the phases that a microorganism undergoes. There are 4 phases, specifically: lag, exponential, stationary and the death phase. The lag phase acts as a preparatory phase for the microorganisms to synthesize their genetic material, enzymes and molecules that the cell will need about division in the exponential phase. During the exponential phase, the microorganisms divide in case they find the appropriate conditions: such as optimal temperature, pH and stable nutrition. The stationary phase enables the remaining of the number of cells in the sample constant, because the number of the cells that die balances the number of the cells that divide. Death phase happens when the cell division stops and it occurs in an exponential mode .
MATERIALS & METHODS
Direct Microscopic Count
- Neubauer Chamber
- S. cerevisiae
- Pipette & Tips
- Bunsen Burner
Initially, the Bunsen burner is opened. Afterwards, the dilution process is performed. Firstly, 1 ml sample is put inside 9 ml H2O inside an Eppendorf tube and then 1 ml is taken with pipette from this tube and put inside another tube containing 9 ml H2O. The suspension of bacteria is placed in the Neubauer chamber following the principle of the capillary action and over it a cover-slip is placed. The measurement was performed under the microscope in the magnitude of 10X.
- Tubes containing LB
- E. coli
- Spectrophotometer tubes
- Pipette & Tips
Initially a specific task was given to be performed by each group and our group`s task was 2% inoculums with shaking and without aeration. About preparation of 20 ml sample of E. coli, 400 µl of E. coli was taken by pipette and placed inside the and 19.6 ml of LB medium. Afterwards, tube was placed in the vortex for approximately one minute. In one of the cuvettes, 2 ml of blank was positioned, while in the other one 2 ml sample was positioned. Before putting tubes in the spectrophotometer, 15 minutes of time were required for it to get the appropriate heat. In the spectrophotometer, firstly the blank values were measured under the wavelength of 600 nm.
|First count||Second count||Third count||Fourth count|
Table 1: Counting results of the direct microscopic count technique under the microscope
Direct Microscopic Count
Total cells counted = (38+39+40+44)/4 = 40
Dilution factor= 10–2
# of squares= 4
Total cells = total #cells x (dilution factor/# of squares) x 10,000 cells/ml
Total cells = 40 x (10-2/4) x 10,000 = 100 cells
(2% with shaking and aeration)
(2%inoculums with shaking & without aeration)
(20%inoculums with shaking & aeration)
(20%inoculums with shaking & without aeration)
(2%inoculums without shaking with aeration)
(20%inoculums without shaking with aeration)
Table 2: OD measurements of the bacterial growth after the 2nd hour, 18th hour and 24th hour reciprocally. The unit of measurement is abs.
The reason blank was used in this experiment was as it has the ability to dissolve the specimen, it can absorb the same wavelength with the solution needed to be analyzed. Blank solution consists of everything present in the sample solution except of the substance that needs to undergo an analysis .
There is a direct relationship between increase of OD during a specific amount of time and the growth curve. According to the visual representation of all the groups` s graphs it is seen that the graphs follow this order: lag phase, exponential phase, but none of them has undergone death phase. Probably if spectrophotometer values were taken after the 24th hour, the results would demonstrate a big decrease in OD, leading to the conclusion that as time passes, more of the cells present start to undergo death.
In Oyku teacher`s group, a sharp increase of the slope of the exponential growth `s part is observed. A high slope demonstrates that a higher number of cells have undergone a specific phase. As this sample was under the effect of shaking and aeration, the bacteria had appropriate conditions to grow. Aeration affects the growth of aerobic bacteria positively. E. coli is a facultative anaerobe, so this makes it to shift between respiration and fermentation. According to the graph, aeration and shaking have positively supported its graph. The importance of shaking is to support the aeration and nutrition availability and to eliminate the possibility of bacteria settling in the bottom of the flask . Probably some of other samples who had undergone other conditions related to aeration, concentration and shaking, have a sharper increase in the slope of the graphs due to the small concentration of the inoculum. If an inoculums` s concentration bigger than 2% was used, rather than 2%, the growth curve would be different, demonstrating a sharper increase during the exponential growth.
In Ayhan teacher`s group where the sample was under the conditions of 2% inoculums concentration, shaking and non-aeration, the growth curve exhibits a smaller slope than the first case (Oyku) and probably the reason to it can be, because the aeration in the first case had helped bacteria divide in a faster rate.
In Deniz teacher`s group, the highest slope is observed according to the others and the reason after that is because of its conditions that were nearest to the optimum conditions related to the others. The 20 % inoculums` s concentration in this sample had contributed in a big number of cells and aeration and shaking as explained above, have positively affected the growth.
In Cigdem teacher`s group, where the only condition differing from Deniz teacher`s group experiment was the absence of aeration, a decrease in the slope was observed related to Deniz group`s graph, because non-aeration can lead to the inhibition of the growth of cells, but compared to the samples of 2% inoculums, a greater rate of growth was seen.
In Nergiz teacher`s group, the slowest rate of lag phase was observed, that means that cells, need more time than other cells to start dividing. This could have probably occurred, because of the low level of inoculums` s concentration and the lack of shaking that in case it would be done, could provide the cells with more oxygen and also would help the cells in the bottom part not to compete with each other for nutrition or oxygen, but freely grow.
In Elvan teacher`s group where the only difference between the Deniz teacher`s group was the lack of the shaking, the second highest slope was observed. This was probably due to the lack of shaking.
Additionally, in the direct microscope count, the value calculated seemed reliable, because a high value was expected to develop, and the same occurred in the experiment performed.
- http://www.microbiol.org/resources/monographswhite-papers/measurement-of-cell-concentration-in-suspension-by-optical-density/ retrieved 07.04.2016 from