TITLE: ENUMERATION TECHNIQUE FOR VIABLE CELLS OBJECTIVE Counting the viable microorganisms by colony forming unit (CFU)

INTRODUCTION Counting bacteria is frequently important. For example, you may want to know the amount of bacteria in a sample such as the number of bacteria per millimetre of water in a swimming pool. Special techniques have been invented to enumerate bacteria, each with advantages and also disadvantages. There are three most common techniques namely plate count, direct count and turbidometric method. These counting of the number of colonies form are using colony forming unit (CFU) formula. Because it is not possible to be absolutely certain that each colony arose from an individual cell, the results are often referred or expressed in terms of colony forming unit (CFU) (Prescott L.M. et.al., 1999). Plate count is based on the premise that each viable bacterium produced a colony when growing on the agar plate. The material also known as the sample to be counted is suspended in a liquid and placed in an empty petri plate. The melted and cooled agar was poured to the plate and mixed with the inoculums. Each organisms produces a colony in the agar then can be counted after incubation period. The plate count is used very frequently but it has advantages and disadvantages. As for direct count, a suspension of bacteria is placed on the slide that has been ruled into squares and designed to hold a specific volume of liquid. By counting of bacteria that appear on the grid areas, the number of organisms can be calculated. Furthermore, this method is a little faster than plate count but it does have several drawbacks. Apart from these method, there is another method used that is turbidometric method. In this method of counting, a spectrophotometer is used to measure the turbidity or the optical density of bacteria in broth. The cloudier the broth means there are a lot of bacteria in it. Both direct and indirect counts are requiring that the organisms must uniformly disperse throughout the medium. If it is not uniformly dispersed, the result of the numbers of the count will not accurately represent the true microbial population in the entire sample. In some cases for example, the microorganisms were suspended in a liquid environment will commonly settle down to the bottom of the plate. In this case, such cultures must be resuspended again by swirling prior to the removal of a small volume for quantification. This isolation of pure culture method used can be spread plate and pour plate. Spread is an easy method to get a single colony by spread out the sample onto the surface of the agar medium. A small volume of a dilute microbial mixture is transferred to the centre of an agar plate and spread evenly with the L-shape rod (Prescott L.M et.al., 1999). For the 1

pour plate, the original sample is diluted several times to reduce the microbial population sufficiently to obtain separate colonies when plating. Then a small volume of several diluted samples are mixed with liquid agar that has been cooled and poured immediately into sterile dish. APPARATUS AND MATERIAL 1. Bunsen burner. 2. Universal bottle. 3. Agar plates. 4. L-shaped rod. 5. Micropipette METHOD SAMPLE PREPARATION 1. The amount of 10g (or ml) sample weighed on a bag stomacher and added 90ml saline solution to make a dilution factor of 10-1. 2. Sterilized pipette was used to transfer 1ml sample solution from 10-1 aseptically to other universal bottle containing 9ml saline solution. The serial dilution continued until it reaches the dilution factor of 10-6 and 10-7. A new pipette was used every new dilution was made. SPREAD PLATE TECHNIQUE 1. 0.1ml of the sample from the highest dilution factor was pipette onto the surface of the agar plate. The sample on the surface was then spread throughout the surface using the L-rod shaped. The L-rod shaped was first being sterilized by immersing it into ethanol a flame it into the fire. 2. The same step was repeated for the lower dilution. 3. The spread plate was then labelled and incubated for 24-36 hours. 4. After the period of incubation, the spread plate was observed and the colony formed was calculated. POUR PLATE TECHNIQUE 1. 1.0ml of sample from the highest dilution factor was transferred onto a sterilized empty Petri dish. The same step was done for the lower dilution factor. 2. Agar medium that was autoclaved and cooled was then poured aseptically onto the sample in the Petri dish until it covered all the bottom surface of the Petri dish. The 6. Ethanol. 7. Water bath. 8. Laminar air-flow. 9. Agar medium. 10. Food sample.

plate was then homogenized by making the 8 shape. it was then cooled down and solidified and incubated for 24-36 hours inverted. 3. After the period of incubation, do the calculation as usual. Result Figure 1: Spread Plate

From left to right: 10-2, 10-3

From left to right: 10-4, 10-5

Figure 2: Pour plate Pour plate

From left to right: 10-2, 10-3

From left to right: 10-4, 10-5

Table 1 the number of colony against the dilution factor for spread plate and pour plate Dilution factor 10-2 Spread plate Pour plate Average value colonies(pour plate and spread plate) UNC UNC 10-3 UNC UNC 10-4 202 97 10-5 149 52

Formula CFU value =

Since we only prepare one replicate for each plate dilution factor, therefore we did not average the colonies, 3

CFU value for spread plate at dilution factor of 10-4 = 2.02 x 107

CFU value for spread plate at dilution factor of 10-5 = 1.49 x 108

CFU value for pour plate at dilution factor of 10-4 = 9.7 x 105

CFU value for pour plate at dilution factor of 10-5 = 5.2 x 106

We also calculate the average colonies between pour plate and spread plate, CFU value for average colonies between spread plate and pour plate at dilution factor of 10-4 = 1.49 x 106

CFU value for average colonies between spread plate and pour plate at dilution factor of 10-5 = Discussion Enumeration of microorganism requires dilution of sample to achieve a population that is countable by the chosen method. Because it is difficult to count more than 3000 colonies on an agar plate, therefore it is necessary to dilute original bacteria culture before plate a known volume of the culture onto solid plate (Black, 2002). Generally, decimal or ten-fold dilutions are used for ease of calculation of final result. The serial dilution-agar plate (plate count) technique is based on the principle that when the material containing bacteria is cultured, every viable bacterium develops into a visible colony on a nutrient agar medium (Aneja, 2003). Based on result, the number of colonies in spread plate in dilution 10-2 and 10-3 is uncountable (UNC). However in spread plate of dilution 10-4, the number of colonies is 202, 4 1.0 x107

and in 10-5 is 149. For pour plate, the sample with dilution 10-2

and 10-3 both are

uncountable. Number of colonies in dilution factor dilution 10-4, the number of colonies is 97, and in 10-5 is 52. As for the calculation of CFU value, Since we only prepare one replicate for each plate dilution factor, therefore we did not average the colonies, For CFU value for spread plate at dilution factor of 10-4 is 2.02 x 107. While the CFU value for spread plate at dilution factor of 10-5 is 1.49 x 108. On the other hand CFU value for pour plate at dilution factor of 10-4 is 106. We also calculate the average colonies between pour plate and spread plate, CFU value for average colonies between spread plate and pour plate at dilution factor of 10-4 is 1.49 x 106 and CFU value for average colonies between spread plate and pour plate at dilution factor of 10-5 is 1.0 x107. For dilution factor of 10-2 1nd 10-3, the number of colonies are uncountable because the cell occur as clumps and stick as groups and it not easy to count is as a colonies. For the CFU value, it was calculated based on the number colonies that we observed and count under colony counter. However this is the estimation because mistake could happen during the experiment and the result might be not accurate as we think it is. From the result, we observed that the cell bacteria growing in aggregates. Some grow in clump or group form and some as single-cell. Therefore it difficult to counts the number of colonies. Therefore the there could be inaccuracy in determination of the colonies. The colony count methods provide an estimate number of viable microorganism in food according to the medium employed and the time and temperature of incubation. Microbial cell often occurs as clump or groups in food. Whereas shaking sample and dilution may uniformly distribute the clump of bacteria, this may not completely disrupt the clumps themselves. Mixing the initial dilution in a mechanical blender may provide better breakdown of clump. However, this does not ensure that the microorganism will be distributed as single cells. Consequently, each colony that appeared on the agar plates can arise from a clump of cells or from single cell and referred as colony forming unit. The failure of the microorganism to form visible colonies limits the accuracy of colony count method. This may result from deficiencies of nutrition, incubation temperature of unfavourable oxygen tension. In this experiment also, contamination may occur and cause the undesired microorganism to occur in agar. This can be cause by the dust circulation in 9.7 x 105 and CFU value for pour plate at dilution factor of 10-5 is 5.2 x

the lab or bacteria came from hand. Therefore it crucial to perform the experiment at laminar air flow to prevent contamination. There are advantages in using plate count techniques. The sensitivity of this technique is high and it allowed an inspection and positive identification of the organism counted. Plate count techniques is easy to perform and can be adapted to the measurement of populations of any magnitude. Since it is a sensitive technique, a small numbers or organisms can be counted. For instance, if a specimen contains a few as one bacterium per ml, one colony should develop up on the plating of 1 ml. However, plate count technique only living cells that develop colonies are counted. Clumps or chains of cells develop into a single colony. . A major disadvantage, however, is the time necessary for dilutions, platings and incubations, as well as the time needed for media preparation. Other limitation is colonies develop only from those organism for which the cultural condition are suitable for growth. Pour plate method plates are useful for quantifying microorganisms that grow in solid medium. It design to enumerate aerobic and facultative anaerobic bacteria in food, shellfish, water and dairy product (Messer et al., 1999). This method embeds colonies in agar. It is particularly useful for growing microaerophies that cannot tolerate exposure to oxygen in the air at the surface of the medium (Black, 2002). The disadvantage of this method is, the bacteria culture that pour with agar could heat-damaged, especially if the agar was pour when it still hot. This then result a smaller colonies that form inside the agar. In contrast with pour plate, instead of embedding microorganisms into agar, as is done with the pour plate method, in spread plate the liquid cultures are spread on the agar surface. An advantage of spreading a plate over the pour plate method is that cultures are never exposed to 45 oC (i.e. melted agar temperatures). Heat sensitive cells(ie. psychotroph) are not killed by the molten agar which may occur to an extent in the pour plate method. The spread plate eliminates the problem in pour plate method, because in spread plate case, the bacteria colonies will growth in surface of agar. This is result from the sample that spread on cooled agar surface evenly. The disadvantage of spread plate is there will be growth of more microbes and presence of more colony forming unit. In future experiment to get more accurate and reliable result, many of the error can be minimized. For instance, do a proper sterilization of diluents, media and equipment. Other than that, the sample measurement must accurate and the experiment must performed in

area that free from contamination. The sample should not be erratically mixing or shaking. Not only that, improper evaluation in counting and in computing count should not happen.