Bacterial Growth Measurements: Viable Count

The purpose of this experiment is to apply viable count technique and determine amount of existence of bacterial contamination in milk sample by using same technique.

The number of microorganisms is a critical measurement for microbiological studies. For various purposes, cells are counted. These counting processes can show different techniques. According to media and conditions, these counting can use some methods; volume, mass or colonies etc. (1)
Viable count is a different method to count. Viable count can indicate living cells by separating death cells. Cells forming colonies into agar is basic for this method. Because of that, this technique is called plate count or colony count. For this, cells in a single colony have no connection with the other colonies. By this way, counting can have a better accuracy. (1, 2)
For preventing to clusters or low number of cells, a special statement is used for counting, named as colony forming units or shortly cfu. Cfu refers to a unit of the possibility of cells forming colony. Also basically two viable methods can be used spread plate and pour plate method. (1, 2)
Spread plate method, ~0.1 ml of bacterial solution is transmitted onto agar media and expended on all surface by Drigalski spatel or spreader. Only aerobic bacteria can grow after drying. (2, 3)
Pour plate method, 0.1 or 1 ml of bacterial solution is poured from pipette onto empty dishes and agar is poured directly onto bacteria. Previously melted agar form a solid media in plate. All mixture in plate is spread onto surface. (2, 3)
For getting better results, serial dilution is so necessary. If high concentrated bacterial solution is used, colonies can be so close to each other or colonies can be found on the top of each other. Dilution can put colonies farther from each other or form distances. On the other hand, too much diluted solution can form the other problem. Low number of colonies or colonies that are so away from each other can cause worse results and remove it from actual values.(3)

Material and Methods:
Before the experiment, 7 tubes including 9ml LB broth and E.coli culture were prepared. At the first step, 1 ml of bacteria after vortex was transmitted into initial 9ml-LB broth tube near flame and with sterilized micropipette. This became 10-1M solution and then 1 ml solution was taken from the 10-1M. 1 ml-solution was poured into second 9 ml LB broth tube. This was 10-2M same process was repeated until 10-7M solution. After preparing solution, 0.1 ml of sample was taken from each different solution and was transferred fresh agar by spreading through aseptic techniques. Same transferred was applied by pour method into fresh agars. For pour method, 1 ml samples from different concentrations were poured onto empty dishes. Agar media poured was spread onto samples at each dish. Each dish was labelled with dilution factor, date, and name of the method.
Same methods were used for milk sample, dilution processes until 10-6 as final. This step was applied for determining whether pastoralized milk or not. At diluting, vortex was used and also saline was added because for preventing denaturation of cells by difference osmatic pressure. All procedure was done according to aseptic techniques. The samples were labelled like previous methods.
Next day, the sample was observed and a spread method samples were counted.

Group I Deniz Keman Group II Ayhan Şen Group III Özgür Öztürk Group IV Öykü Özdemir Group V Deniz Uğur
Spread plate-E.Coli 10-4:TMTC 10-5 : 270
10-6 : 61 10-4 : TMTC
10-5 : TMTC
10-6 : 128 10-5:TMTC 10-6 :119
10-7 : <30 10-5:TMTC 10-6:85 10-7:4 10-3:TMTC 10-4:TMTC
Spread plate milk sample 10-1:TMTC 10-2:TMTC 10-3:TMTC 10-4 : TMTC
10-5 : error
10-6 : 10 10-4:0 10-5:0 10-6:0 10-1:0
10-2:0 10-3:0 10-2:TMTC 10-3:TMTC 10-4 :TMTC
Table 1- The result of cell counting after 24 hour- the experiment in different diluted solution of each group. TMTC: too much to count
Group 1: For 10-5, 270*1/10-5 =2,7*107 cfu/ml
For 10-6, 61*1/10-6 =6,1*107 cfu/ml
Group 2: For 10-6, 128*1/10-6 =1,28*108 cfu/ml
Group 3: For 10-6, 119*1/10-6 =1,19*108 cfu/ml
Group 4: For 10-6, 85*1/10-6 =8,5*107 cfu/ml

The aimed observation was to determine the number of bacteria by using colony forming unit, and that milk includes bacterial contamination or not.
At spread method, bacterial colonies could be seen easily. The solutions including high number of colonies have high concentration. It is an expected result because high concentration or high dilution factor causes that colonies are so close to each other. Generally it refers to more than 300 colonies. 10-5 and lower concentration have countable number of colonies until 10-7. Lowest concertation, 10-7, is a limit to count. Too low concentration can form a difficulty. Too low dilution can generate long distances between the colonies and it makes challenge to count. It usually is less than 30 colonies. Calculations can be used for rejection. But at spread plate method examinations, groups have enough data from comparing. The result of Group I is only used for this and it can be rejected because of different cfu/ml values. Only Group II and III have closer values between them.
Pour plate method didn’t give colonial bacteria expectedly. Pour method just showed that bacteria will grow in this media and this media can use for this experiment. Solutions includes at every dilution was showed.
The examination for milk has different results for each group. The results indicate that Group I, II and V include many bacteria in milk. On the other hand, the rest of the milk sample (Group III and IV) has been pasteurised before.

[1] Han, J.W.; Breckon, T.P.; Randell, D.A.; Landini, G. (2012). “The Application of Support Vector Machine Classification to Detect Cell Nuclei for Automated Microscopy” (PDF). Machine Vision and Applications. Springer. 23 (1): 15–24
[2] Goldman, Emanuel; Green, Lorrence H (24 August 2008). Practical Handbook of Microbiology, Second Edition  (Second ed.). USA: CRC Press, Taylor and Francis Group. p. 864
[3]  Daniel Y. C. Fung (2009). “Viable Cell Counts”. Bioscience International. Retrieved September 25, 2016.

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