Lab Report. Serial Dilution

Introduction It is difficult to know and imagine the size of microorganisms, since they are too small to be seen under the naked eyes. The size of microorganisms is about 1/10th the size of a typical human cell, and measured in the scale of one millionth of a meter known as micrometer [1]. Moreover, it is often important to know not only what kinds of microorganism are present in a given or obtained sample but also to know the number of microorganism in sample which is difficult to count under the naked eyes because of their small size. In addition, worldwide the numbers of microorganisms in a given sample are required to know in certain aspects such as dairy industries, water treatment, food manufactures, and importantly in investigation and study of diseases [2]. Because of above reasons, variety of methods had been developed for quantitative enumeration of microorganisms such as direct microscopic counts, electronic cell counters, chemical methods, spectrophotometric analysis and serial-dilution agar plate analysis [3]. Direct microscopic counts are performed by placing a bacterial suspension in a counting chamber, and require the use of specialized slide called the Petroff-Hausser counting chamber [3]. Generally, there are two types of direct microscopic counts such as Petroff-Hausser method and breed method. Petroff-Hausser method is performed to calculate the total cell of microorganism in small sample by using the glass slides in square form with fixed volume as shown in figure 1 and 2.

Figure 1.Petroff-Hausser counting chamber

Figure 2.Petroff-Hausser counting chamber grid

There advantages of this method are quick and easy count of cells and cheap cost. However, there is no different cells calculation for live and dead cells. Moreover, the cells that own too small size is difficult to count. Also, the sample needs to be free from debris and food extraction. Breed method is mainly used to assess the quality of milk by giving the number of bacteria and leucocytes in milk. This method also does not discriminate between viable and dead cells as in Petroff-Hausser method [4]. A Coulter counter is an example of electronic cell counters, and has one or more micro channels in order to separate two chambers containing electrolyte solutions. After passing of

fluid containing cells via each microchannel, some particles causes change to the electrical resistance of liquid [5]. Thus, the resistance is recorded enumerating the number of cell flowing through the orifice [3]. The structure and principle of Coulter counter is shown below:

Figure 3.Coulter counter and its principle

The chemical methods and spectrophotometric analysis are also used for counting the number of cells. However, they are limited by high cost of chemicals and low sensitivity to microbial suspension respectively [3]. Almost all of the above listed methods are limited in distinguishing between viable and dead cells [3]. However, serial dilution-agar plate technique is used in order to count only the viable cells. Serial dilution is a series of dilutions used to determine the concentration or titer of a substance [6]. Once organism is diluted out by certain dilution factor, it is allowed to inoculate on suitable nutrient media in Petri dished. After the incubation of plates, the number of cell can be counted. Although, plates suitable for counting must contain colonies not fewer than 30 which is called as too few to count (TFTC) nor more than 300 which is called as too numerous to count. Then, visible for naked eyes colonies are calculated and represented as colony forming units (CFU). Finally, the population of original sample is known by multiplying the CFU to corresponding dilution factor [3]. The main purpose of this experiment was to use of serial dilution-agar plate technique in order to determine the number of cells in bacterial culture. It was expected that the number of colonies in each A and B agar plate would be close to each other. However, it is impossible to suggest the exact number of cells in each plate.

Materials and method Cultures: 24-to-48-hour nutrient broth culture of Escherichia coli Media: six 20-ml nutrient agar deep tubes, seven sterile 9-ml water blanks

Equipment: Hot plate, water bath, thermometer, test tube rack, Bunsen burner, sterile 1-ml serological pipettes, mechanical pipetting device, sterile Petri dishes, glassware marking pencil, turntable, bent glass rod, beaker with 95% alcohol, manual hand counter Procedure: E. coli culture tube was labelled as 1, and seven tubes with 9-ml water blanks as 2 through 8. Then, six Petri dishes were labelled as 1A, 1B, 2A, 2B, 3A, and 3B. First of all, 1 ml of bacterial suspension was aseptically transferred to tube 2 by sterile fresh pipette. This step was repeated for tubes 3, 4, 5, 6, 7, 8. Secondly, 0.1 ml of “tube 5” suspension was transferred to plate 1A with fresh pipette. 1 ml of “tube 6” suspension was transferred to plate 1B, and 0.1 ml to plate 2A with sterile pipette. 1 ml of “tube 7” suspension was transferred to plate 2B, and 0.1 ml to plate 3A. Then, 1 ml of “tube 8” suspension was transferred to plate 3B with fresh pipette. Finally, diluted bacterial cultures in plates were spread by bent glass rod on the whole surface of agar plate. Results Plate

Dilution factor

ml of dilution plated

Final dilution on plate

Number of colonies

1A 1B 2A 2B 3A 3B

105 105 106 106 107 107

0.1 ml 1.0 ml 0.1 ml 1.0 ml 0.1 ml 1.0 ml

10-4 10-5 10-5 10-6 10-6 10-7

TNTC TNTC TNTC TNTC 118 116

Bacterial count per ml of sample (CFU/ml) TNTC TNTC TNTC TNTC 1.18 x 109 1.16 x 109

Average count per ml of sample (CFU/ml) TNTC TNTC TNTC TNTC 1.17 x 109 1.17 x 109

Answer to review questions 1. Serial dilution-agar plate technique counts only viable cells whereas other methods count both viable and dead cells. 2. Dilution is the process which involved making the solution weak or less concentrated. Dilution factor is the amount of solvent required to reach a given concentration of solution for a given volume. 3. Advantages: only viable cells are counted; it allows isolation of discrete colonies which can be then subcultured into pure cultures. Disadvantages: overnight incubation is needed before colonies develop on agar surface; there may be errors in dilution or plating. 4. 0.1 ml x 10-6 = 1 x 10-7- dilution factor. Then, 56 x 107 = 5.6 x 108 cells/ml 5. 305 colonies – TNTC (too numerous to count) 15 colonies – TFTC (too few to count)

6. Chemical methods may be used to measure increases in protein concentration and DNA production. Measurement of certain metabolic parameters can be used to enumerate the bacterial population. For example, aerobic cells: increase in the amount of oxygen uptake shows the increasing number of aerobic cells; anaerobic cells: rate of carbon dioxide production is related to increased growth of anaerobes. 7. After the dilution of water sample, there were colonies fewer than 30 (TFTC) or more than 300 (TNTC).

Reference list: 1. "Microbe Size - Boundless Open Textbook." Boundless. Accessed November 11, 2016. https://www.boundless.com/microbiology/textbooks/boundless-microbiologytextbook/microscopy-3/looking-at-microbes-28/microbe-size-238-4283/. 2. "Quantitative Analysis of Microbes – Bacterial Counts." Accessed November 11, 2016. http://biolabs.tmcc.edu/Micro Web/BacterialCounts.pdf. 3. Cappuccino, James G., and Natalie Sherman. Microbiology: A Laboratory Manual. San Francisco: Pearson/Benjamin Cummings, 2011. 4. "MIKROBIOLOGI DASAR Methods for the Microbiological Examination." Accessed November 11, 2016. http://syarifahsari.lecture.ub.ac.id/files/2013/05/analisamikrobiologi.pdf. 5. "Coulter Counter." Wikipedia. Accessed November 11, 2016. https://en.wikipedia.org/wiki/Coulter_counter.

6. "Serial Dilution-Agar Plate Procedure to Quantitate Viable Cells." Microgroup3s Blog. 2014. Accessed November 11, 2016. https://microgroup3.wordpress.com/2014/03/30/serialdilution-agar-plate-procedure-to-quantitate-viable-cells/. Figure 1.KG, BRAND GMBH CO. "Liquid Handling Station." Laboratory Instruments, Liquid Handling, Life Science: BRAND GMBH CO KG. Accessed November 11, 2016. http://www.brand.de/uploads/tx_dkdcolumns/counting_chamber2_01.jpg. Figure 2. Corporation, Steven J. Kusnerus K-COM. "Counting Chambers Petroff-Hausser Counter." Counting Chambers Petroff-Hausser Counter. Accessed November 11, 2016. http://www.hausserscientific.com/products/petroff_hausser_counter.html. Figure 3. "Coulter Counter & Coulter Principle." Coulter Counter & Coulter Principle. Accessed November 11, 2016. http://samhs.org.au/Virtual Museum/Medicine/Lab_and_other_tests_except_xray/Coulter-counter/Coulter-counter.htm.