NT20203 FOOD MICROBIOLOGY
SEMESTER II SESSION 2015/2016
FACULTY OF FOOD SCIENCE & NUTRITION
UNIVERSITY MALAYSIA SABAH
PRACTICAL 5:
ENUMERATION TECHNIQUES FOR VIABLE CELLS
NAME: SAZLINAWATIE BINTI ALADIN
MATRIC NUMBER: BN14110167
LECTURER: DR. CHYE FOOK YEE
GROUP: MONDAY, 3
PRACTICAL 5: ENUMERATION TECHNIQUES FOR VIABLE CELLS
Introduction
Plate count can be done by using either the pour plate method or the spread plate method. In the pour-plate method, 0.1 to1.0ml of the final dilution is transferred into a sterile petri dish and then overlaid with melted nutrient agar that has been cooled to 50ºC. Agar is still in liquid formed at this temperature. The petri dish gently swirled to mix the bacteria with the liquid agar. At times the agar harden, the individual cells are fixed in place and after it incubate the colonies form are distinguishable.
In the spread-plate method, 0.1 to 0.2ml of final dilution transferred directly onto a plate contain a solidified nutrient agar medium. As the name itself, the solution spread over the surface of the agar with a sterilized bent glass rod /sterile glass L-shaped rod.
In both of the methods, the plates incubated for a specific time period to allow the colonies to form and then counted. By knowing how much the sample was diluted prior to being plated, along with the amount of the dilution used in plating, the concentration of the viable cells in the original sample can then be calculated. Cells attached to one another form a single colony and counted as a single cell or colony-forming unit (Nester, Anderson, Roberts, Martha, 2007).
Microbiological test can either be quantitative or qualitative (Bell, Neaves and Williams, 2005). To measure the number of the viable bacteria is under the quantitative test. Quantitative test, as the name itself quantity known as enumeration (counting) test. This test used for estimation of the number of a particular type of microorganism present in a certain quantity of sample (Bell, Neaves and Williams, 2005). Enumerations of viable bacteria number consist of two types of technique which are colony count and most probable number (MPN) tests.
Colony count is defined as the test that estimates number of cells present in the sample by counting the visible developed colonies. Colony count test are based on the growing of microorganisms on a solid agar surface and allow them to develop into colonies are visible to naked eye. The colonies formed will then counted to estimate the number of cells that are present in the sample. Colony count expressed as the number of colony forming units (CFU) per ml or gram of the sample.
OBJECTIVE
To learn the enumeration technique for viable cells using pour plate method and spread plate method.
To determine how to prepare or perform serial dilution techniques.
To determine how to perform total viable count using colony counter.
MATERIALS AND EQUIPMENTS
Analytical balance
Measuring cylinder
Blue-cap bottle
Deionized water
Dried agar media
Autoclave
Water bath
10g food sample (fermented bambangan)
Stomacher plastic bag
Peptone water
Stomacher
Lamina air flow
Micropipette
Disposable plastic micropipette tips
Universal bottle
Sterile Petri dish
Petri dish containing agar plate
Sterile glass L-shaped rod
Incubator
Colony counter
Marker pen
White stickers
Bunsen burner
Lighter
METHODS
A) Culture media preparation (agar medium)
To prepare broth media, 2.625 g of dried agar media was weighed using an analytical balance in a blue-cap bottle.
Using a measuring cylinder, 150 ml of deionized water was transferred into a blue-cap bottle.
The blue-cap bottle was sealed and shook to dissolve the powder of dried media.
The blue-cap bottle was labelled with group number and autoclaved at 121°C, 15 psi for 15 minutes.
After 15 minutes, the bottle were placed in water bath and later used for colony selection in pour plate method.
B) Sample dilution (done with laminar air flow)
10 g of food sample (fermented bambangan) was weighed in a stomacher plastic bag and added with 90 ml of Peptone water and homogenised for 30 seconds by using stomacher. This operation dilutes the sample to a tenth and the resultant suspension is, therefore, referred to as the 10-1 dilution.
After that, the solution of food sample (10-1 dilution) immediately taken to the laminar air flow for the next dilution.
With micropipette and sterilized micropipette tips, 1 ml sample of 10-1 dilution was transferred aseptically into a universal bottle containing 9 ml of Peptone water. This operation dilutes the 10-1 dilution to 10-2 dilution.
The solution was shook to homogenise the dilution.
Step 3 and 4 was repeated 2 times in every new solution of from the lowest (10-1) to higher (10-3) dilutions to achieve up to 10-4 dilution.
The micropipette tip were ejected and replaced with new ones for each time transferring the solutions to another universal bottle.
Each of the universal bottles was labelled as of the number of dilution.
C) Pour plate method (done with laminar air flow)
Using a new and same tip throughout this activity, starting with the highest dilution (10-4), the universal bottle containing sample on one hand was held, the cap was unscrewed and the lip was flamed.
Holding a micropipette on the other hand, 1.0 ml of sample was pipette out.
The bottle lip was flamed and the cap was replaced.
The pipette sample was transferred into a labelled empty sterile Petri dish.
Step 1-4 were repeated into another labelled empty sterile Petri dish.
Step 5 was repeated twice using 10-3 dilution and then 10-2 dilution into every new Petri dish.
The autoclaved agar medium was cooled at room temperature to 40-50°C.
The blue-cap bottle was unscrewed and the lip was flamed.
The plate lid was opened slightly and the medium was poured directly on top of the sample in the Petri dish until the bottom surface of the dish was covered up.
The blue-cap bottle lip was flamed and the cap was replaced.
The plate was rotated like number ‘8’ to homogenise the sample and agar medium. Care was taken not to get agar on the lid of the Petri dish.
The agar was allowed to set.
Step 7-12 was repeated on the other 5 Petri dishes.
All the 6 Petri dishes were incubated in inverted position at 37°C for 24-26 hours.(why inverted position
Colonies on each Petri dish was counted and the results were calculated.
D) Spread plate method (done with laminar air flow)
Using a new and same tip throughout this activity, starting with the highest dilution (10-3), the universal bottle containing sample on one hand was held, the cap was unscrewed and the lip was flamed.
Holding a micropipette on the other hand, 0.1 ml of sample was pipette out.
The bottle lip was flamed and the cap was replaced.
The pipette sample was transferred onto the middle of the surface of agar medium a labelled agar plate.
Step 1-4 were repeated into another labelled agar plate.
The pipetted sample was spread over the surface of the agar medium using sterile glass L-shaped rod by flaming on Bunsen burner and cooled down.
Step 5-6 were repeated for 10-2 dilution and then 10-1 dilution into every new labelled agar plate.
All the 6 Petri dishes were incubated in inverted position at 37°C for 24-26 hours.
Colonies on each Petri dish was counted and the results were calculated.
RESULTS
Pour plate
Dilution factor 10-2 10-3 10-4
A 224 91 15
B 224 74 4
Average 224 82.5 9.5
Total viable count (Average of colonies within a dilution factor)/(dilution factor) X 1/(plating volume)
224/〖10〗^(-2) X 1/1.0
= 2.24 X 104 CFU/ml 82.5/〖10〗^(-3) X 1/1.0
= 8.25 X 104 CFU/ml 9.5/〖10〗^(-4) X 1/1.0
= 9.5 X 104 CFU/ml
Table 1: Colony counts obtained by pour plate with different dilutions factor.
From the table 1, two of the pour plates with dilution factor 10-2 and 10-3 yields colonies between the ranges of 30-300 colonies. Based on the calculation, the colony count is the highest at the lowest dilution (10-2) and the lowest for the highest dilution (10-4).
Spread plate
Dilution factor 10-1 10-2 10-3
A 600 246 16
B 244 163 1216
Average 422 204.5 *contaminated
Total viable count (Average of colonies within a dilution factor)/(dilution factor) X 1/(plating volume)
422/〖10〗^(-1) X 1/1.0
= 0.442 X 104 CFU/ml 204.5/〖10〗^(-2) X 1/1.0
= 2.045 X 104 CFU/ml N/A
Table 2: Colony counts obtained by spread plate with different dilutions factor.
From the table 2, one of the spread plates with dilution factor 10-2 yields colonies between the ranges of 20-250 colonies. Based on the calculation, the colony count is the highest at the lowest dilution (10-1) and the lowest for the highest dilution (10-2). For dilution factor 10-3, the colony count for plate A cannot be calculated due to the contamination of the spread plate. While, the colony count for plate B is more than the range of 20-250 colonies therefore the enumeration of bacteria using colony count is not possible.
DISCUSSIONS
Based on the result from the table 1, two of the pour plates with dilution factor 10-2 and 10-3 yields colonies between the ranges of 30-300 colonies. Based on the calculation, the colony count is the highest at the lowest dilution (10-2) and the lowest for the highest dilution (10-4). The total viable counts for pour plate with dilution factor 10-2 is 2.24 x 104 CFU/ml, dilution factor 10-3 is 8.25 x 104 CFU/ml and dilution factor 10-4 is 9.5 x 104 CFU/ml.
From the table 2, one of the spread plates with dilution factor 10-2 yields colonies between the ranges of 20-250 colonies. Based on the calculation, the colony count is the highest at the lowest dilution (10-1) and the lowest for the highest dilution (10-2). For dilution factor 10-3, the colony count for plate A cannot be calculated due to the contamination of the spread plate. While, the colony count for plate B is more than the range of 20-250 colonies therefore the enumeration of bacteria using colony count is not possible. The total viable counts for spread plate with dilution factor 10-1 are 0.442 x 104 CFU/ml, dilution factor 10-2 is 2.0450 x 104 CFU/ml and for dilution factor 10-3 CFU cannot be counted since it is contaminated.
In this experiment, the CFU/ml unit used instead of CFU/g because the sample used in the test is in the form of liquid and not solid. For the spread plate method in lowest dilution are chosen to calculate the CFU of this method because it is more accurate estimation where it can be made in colony count.
There are several precaution steps needed to follow in this practical, one of it is the micropipette need not to be sterile each time before and after transfer the material as its tip is already autoclaved. The tip of micropipette has to be change each time before transfer solution to avoid contamination. Besides, the micropipette should be only held vertically to prevent any contamination of it. All of the procedures in this practical should be done in lamina air flow to prevent any contamination at any stages. Also, make sure the micropipette is not over-pressed in order to get an accurate amount of dilution.
In addition, the diluted food samples must homogenize perfectly and antibacterial presence in the original sample before transfer into agar plate. Besides, serial dilution is used purposely to dilute the solution so that over-crowded of the organisms will not happen in the agar and result in spreader or too numerous to count (Tortora, Funke & Case, 2004).
As for the preparation of culture media/ nutrient medium, in which the agar is kept liquid by holding it in the water bath at about 50°C is poured over the sample, which is then mixed into the medium by gentle agitation of the plate. When the agar solidifies, the plate is incubated. With the pour plate technique, colonies will grow within the nutrient agar (from the cells suspended in the nutrient medium as the agar solidifies) as well as on the surface of the agar plate. This technique has some drawbacks because some relatively heat-sensitive microorganisms may be damaged by the melted agar and will therefore be unable to form colonies (Tortora, Funke & Case, 2004). Thus, it is necessary to wait until the agar to cool down to warm temperature before pouring on the top of the inoculums. If the temperature of the molten agar is still high, it can be kill the inoculums and the test cannot proceed. However, the agar should not wait until the temperature the agar will solidify. It is important to beware of the temperature of the agar. Thus, is causing a problem during handling with the agar.
Another precautions needed to be taken is to avoid to spoil the agar during spreading. While for the spread plate, the inoculums has to be make sure that it is spread evenly on the agar surface. Much attention has to give as the agar is very easy to be break by the rod.
Conclusion
In conclusion, an enumeration technique for viable cells which involves serial dilutions, plating and counting was used in this practical. As a result by using colony counter, the total viable counts for pour plate with dilution factor 10-2 is 2.24 x 104 CFU/ml, dilution factor 10-3 is 8.25 x 104 CFU/ml and dilution factor 10-4 is 9.5 x 104 CFU/ml. While, the total viable counts for spread plate with dilution factor 10-1 are 0.442 x 104 CFU/ml, dilution factor 10-2 is 2.0450 x 104 CFU/ml and for dilution factor 10-3 CFU is contaminated therefore it cannot be counted. The accuracy and precision of the bacterial enumeration results depend upon the competency and accuracy of the analyst when performing the technique.
References
Bell. C., Neaves. P., Williams. A. P. (2005), Food Microbiology and Laboratory Practise, Blackwell Publishing.
Black. J. B. (2002), Microbiology Principle and Explorations 5th edition, USA: John Wiley & Sons, Inc.
Eugene W. Nester, Denise G. Anderson, C. Evans Roberts, Jr., Martha T. Nester. 2007. Microbiology Human Perspective. The McGraw-Hill Companies, Inc: United States of America