The Determination of Microbial Numbers Objectives

Objectives:

* Practically every phase of microbiology requires method for measuring microbial numbers.

* Study the theoretical relationship of one bacterial cell, or clump of cells.

* Study the effect of dilution to the bacteria growth.

* Determine the cell masses of a culture in order estimates the total cellular protoplasm per milliliter of culture.

* To learn both quantitative plating methods which are spread plate and pour plate to measure the number of bacteria.

* To understand the measurement for the number bacteria by performing plate and dilution count.

Result and Observations:

Part I: Spread Plate

Unlabelled sample – Dilution factor 10-1

Sample A – Dilution factor 10-2

Sample B – Dilution factor 10-3

Sample C – Dilution factor 10-4

Observation:

According to the observation, the result is showed that the colonies of E.coli cultures are too numerous to count via normal visible with density diminish from sample A to sample C. As a result, we get the accurate number of colonies for each plate in the experiment doesn’t count for calculation. For the unlabelled plate sample that was showed off, we discover that Whitish strands of colonies were observed apart from the usual circular whitish colonies which are produced by E.coli bacteria.

Part II: Pour Plate

Sample A – Dilution factor 10-4

Sample B – Dilution factor 10-6

Sample C – Dilution factor 10-6

Sample D – Dilution factor 10-6

Sample E – Dilution factor 10-9

Observation:

According to the observation during the experiment, we can only do the measurement of colonies in the plate of sample A. The result that we can get from the plate A is 40 colonies. The dilution factor of plate A is at 10-4. Those colonies observed are present in a form of circular whitish as appearance with a small degree of overcrowding at several regions around of the plate. However, the rest of the dilution factor plate of sample gave too little colonies which are unacceptable range according to the viable count theory. Therefore, colonies that form surrounding the perimeter of the plate are impossible to measure.

Calculation:

For plate A of 10-4 dilution,

= 40 � 10 000

= 400 000 bacteria mL-1

Discussions:

In the experiment, a few methods were introduced to make us convenient in direct measure the microbial growth. The approach we are using called viable count which means a direct counting method in which only viable cells are counted. Viable counts can be accomplished by such techniques as:

* pour plating

* spread plating

* most probable number method

However, we are needed to apply the method of pour plating and spread plating in the experiment. A viable cell is one that is able to divide and form offspring. We use viable count to measure the number of cells in the sample capable of froming colonies on the visible appropriate culture medium. So, the viable count is often called the plate count or colony count. The main hypothesis made in this type of counting procedure is that each sample which is viable cell can grow visible and divide to yield.

For the pour plate method, a known volume (usually 0.1-1.0 ml) of culture is pipetted into a sterile Petri plate. Melted agar medium is then added and mixed well by gently shake the plate on the bench top. Because the sample is mixed with the molten agar medium, a larger volume can be used than with the spread plate. However, with this method the organism to be counted must be to briefly withstand the temperature of molten agar (~45-50 �C). In this method, colonies will form throughout the plate, and just on the agar surface as in the spread plate method. The plate must therefore be examined closely to make sure all colonies are counted.

Whereas, in the spread plate method, a volume (usually 0.1 ml or less) of an appropriately diluted culture is spread over the surface of an agar plate using a sterile glass spreader. Streaking in this technique is done using a bent glass rod. Bacterial suspension is placed in the center of the plate using a sterile pipet. The glass rod is sterilized by first dipping it into a 70% alcohol solution and then passing it quickly through the Bunsen burner flame. The burning alcohol sterilizes the rod at a cooler temperature than holding the rod in the burner flame thus reducing the chance of you burning your fingers.

When all the alcohol has burned off and the rod has air-cooled, streak the rod back and forth across the plate working up and down several times. Unlike streaking for isolation, you want to backtrack many times in order to distribute the bacteria as evenly as possible. Turn the plate 90 degrees and repeat the side to side, up and down streaking. Turn the plate 45 degrees and streak a third time. Do not sterilize the glass rod between plate turnings. Cover the plate and wait several minutes before turning it upside down for incubation. This will allow the broth to soak into the plate so the bacteria won’t drip onto the plate lid. The plate is then incubated until the colonies appear, and the number of colonies is counted. It is important that the surface of the plate be fairly dry so that the spread liquid soaks in. volumes greater than 0.1ml are rarely used in this method because the excess liquid does not soak in and may cause the colonies to coalesce as they form, making them difficult to count.

For the diluting sample suspensions before plating, during the spread plate and pour plate methods steps, it is important that the number of colonies developing on the plates not to be too large. On crowded plates some cells may not form colonies, and some colonies may fuse, leading to erroneous measurements. It is also essential that the number of colonies not be too small, or the statistical significance of the calculated count will be low. The usual practice, which is the most valid statistically, is to count colonies only on plates that have between 30 and 300 colonies. Moreover, it is usual to determine the incubation condition (medium, temperature, time) that will give the maximum number of colonies of a given organism and then use these conditions throughout.

In the experiment, we start Part I spread plate method with prepare three dilution tubes and four nutrient agar plates and make sure that the step of labeling are done on the each tube. Label the tubes with the dilution factor 10-1 10-2and 10-3 and the plates 10-1 to 10-4. Using a sterile pipette remove 1.1ml of the water sample. Pipette 0.1 ml on the agar surface of plate 10-1 and the remaining 1.0 ml to the 10-1 dilution tube. This tube now contains 1 ml of the original sample diluted ten times: thus 1.0 ml of this dilution is equivalents to 0.1 ml of the original sample. Later on, mix thoroughly by shaking vigorously.

Repeat the same procedure to obtain the other two dilution factor tube which is 10 and 10. After obtained it, remember to shake it vigorously to ensure all the bacteria have equally spread to all over the tube. Finally, transfer the dilution tube to the agar surface plate which is labeled accordingly and remember do the most important step to spread the sample on the plate. During this step, a sterile glass is required to use. In this technique, the glass spreader is sterilized by dipping it in alcohol, and the alcohol is burned off. Allow the spreader too cool. Lastly, spread the sample over the surface of the agar plate by revolving the plate on desk. The main reason to dipping it to alcohol is to wash the sterile glass spreader without any foreign microbial and burned off with the fire is to ensure all the microbial bacteria have killed. Well, spread all the agar plate surface of every dilution factor using the same technique and incubate the Petri plates at room temperature in an inverted position for 2 days.

In the part II of the experiment, as usual, we do the step of label four 9.9 ml water blanks: 10-2, 10-4, 10-6 and 10-7. Then perform labeling step for five sterile Petri plates (on the bottom) with: (A) 0.1 ml of 10-4, (B) 1 ml of 10-6, (C) 0.5 ml of 10-6, and (D) 1.0 ml of 10-36 and (E) 0.1 ml of 10-7. After that, remove 0.1 ml of water sample and transfer it to the dilution blank labeled “102” and mix the 10-2 dilution by tightly capping the blank and shaking the mixture 25 times. We need to ensure that shake it 25 times in order to confirm that all the microbial bacteria have spread equally over the whole tube to make the result more accurate.

Then with a fresh 1 ml pipette, aseptically transfers 0.1 ml of the 10-2 dilution to the dilution blank labeled 10-4 and shake it 25 times. So these steps will be repeated for every dilution factor tube which has labeled accordingly just now. After we perform the latter step, now is the most critical step in the experiment which is pouring the sample. First, we need to remember to wipe the liquid off the outside of the melted agar tubes before pouring the agar into the plates. Aseptically add the agar in each tube into each of the five plates. We should make sure that the latter step undergo as soon as possible before the agar become solidifies in the room temperature. After you have added the molten agar, please ensure that cover the plate and rotate the plates gently to get good mixing. Make sure the every sample didn’t splash onto underside of the Petri plate lid. Finally let the plates cool until hardened. Tape your plates together and incubate in Petri plates at room temperature in an inverted position for 2 days.

Based on the observation for part I which is spread plate, the result shows that the colonies for each plates of sample with different dilution factor are far too many and crowded. With the viable count theory, the range for viable count is only 30 to 300 colonies are acceptable. However, based on our experimental result the numbers of colonies has denied the viable count theory because the number of colonies is over the limited range of the theory. This is because it is unreliable for us to count it in accurate number. According to the theory of viable count, we can just ignore the measurement for those plates of sample are over 300 colonies and vice versa. However, there is also a limited range for the colonies too less or below 30 colonies. We will include into the ignorance because it also disobey the viable count theory. With the colonies are far too less, it will become imprecise and significant statistical error might occur in this case.

Based on the observation on part II, the result is showing that the colonies of almost plates of sample with different dilution factor are far too less to count because they are not included between the limited range of the viable count theory. However, only the plate A with the dilution factor 10-4 obeys the viable count theory which stated count only the plate is 30 to 300 colonies. From the calculation to determine the number of bacteria or colony forming units per mL of the original sample, we obtained 400 000 of cfu mL-1. This figure represents the number of bacteria present in a milliliter of the original sample. For other dilution factor plate, all result and the number of colonies are far too less and disobey the viable count theory. Therefore, we have to ignore it. In those dilution plates which are far too less and unacceptable is maybe due to the mistake in the process of pouring the sample to Petri plates. As we seen in the result, the distribution of the colonies are sometimes colonize certain area of the Petri plate. I think the main reason is cause by uneven mixing of the molten agar. To enhance the accuracy, I think we should duplicate the result and compare it with other group to get best average reading of the colonies for each plate with different dilution factor.

The number of colonies obtained in a viable count experiment depends not only on the inoculums size and its viability, but also on the suitability of the culture medium and the incubation condition. The colony number can also change with the length of incubation. For example, if a mixed culture is used, the cells deposited on the plate will not all develop into colonies at the same rate; if short incubation time is used, fewer than maximum number of colonies will be obtained. Furthermore, the size of colonies often varies. If some tiny colonies develop, they may be missed during the counting. With pure cultures, colony development is more synchronous process.

Viable counts can be subject to rather large errors for several reasons. These include pipetting inconsistencies, in homogeneity of the sample, insufficient mixing and several other factors. Hence, if accurate counts are too obtained, great care and consistency must be taken in sample preparation and Pipetting, and replicate plates of key dilutions must be prepared. Note also that two or more cells in a clump will form only a single colony. So if many clumps are present in sample, a viable count of that sample may be erroneously low. To more clearly state the results of a viable count, the date are often expressed as the number of colony forming unit (cfu) obtained rather than as the number of viable cells (since a colony-forming unit may contain one or more cells).

Despite the difficulties associated with viable counting, the procedure gives the best information on the number of viable cells in a sample and so is widely used in many areas of microbiology. For example, in food, dairy, medical, and aquatic microbiology, viable counts are employed routinely. The method has the virtue of high sensitivity, as a sample containing only a single viable cell can in theory be counted. This feature allows for sensitive detection of microbial contamination of food products or other materials. Moreover, the use of selective culture media and growth conditions in viable counting procedures allows for the counting of only particular species in a mixed population of microorganisms present in the sample.

Conclusion:

* The number of colonies on plate A in part II is 40 and the calculation yields a concentration of 400 000 bacteria mL-1 of the original sample.

* The plate count method is suitable to determine the number of microbial number but is not so accurate because we just assume the colonies as viable cells.

* The plating count theory is assumed that each colony arises from one cell.

* The viable count are counting the living cell and able to divide and form offspring.

Reference:

* http://www.textbookofbacteriology.net/growth.html

* http://www2.hendrix.edu/biology/CellWeb/Techniques/microspread.html

* http://www.biotopics.co.uk/microbes/tech3.html

* http://biology.clc.uc.edu/fankhauser/Labs/Microbiology/Meat_Milk/Pour_Plate.htm

* http://www.mansfield.ohio-state.edu/~sabedon/biol4038.htm

* Biology of Microorganism, ELEVENTH Edition, Micheal T.Madigan, John M.Martinko, Prentice Hall, page144-146.