FACULTY OF CIVIL AND ENVIRONMENTAL ENGINEERING DEPARTMENT OF WATER RESOURCES AND ENVIRONMENTAL ENGINEERING ENVIRONMENTAL ENGINEERING LABORATORY

LABORATORY INSTRUCTION SHEETS

SUBJECT CODE EXPERIMENT CODE EXPERIMENT TITTLE COURSE CODE

BFC3121 MA2 BACTERIA COUNT

KOD ETIKA PELAJAR (KEP)

JAB. KEJ. SUMBER AIR & ALAM SEKITAR FAKULTI KEJURUTERAAN AWAM & ALAM SEKITAR KUiTTHO
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___________________________ Tandatangan Pelajar Nama : _______________________________ No. Matrik :____________________________ Tarikh :________________________________

FACULTY OF CIVIL & ENVIRONMENTAL ENGINEERING DEPARTMENT OF WATER RESOURCES AND ENVIRONMENTAL ENGINEERING ENVIRONMENTAL ENGINEERING LABORATORY

SHORT REPORT

SUBJECT CODE CODE & EXPERIMENT TITLE COURSE CODE EXPERIMENT DATE NAME OF STUDENT NO.OF GROUP GROUP MEMBER 1. 2. 3. 4. 5. NAME OF LECTURER/INSTRUCTOR/ TUTOR DATE OF REPORT ACCEPTANCE MARKS DICIPLINE/ PARTICIPITATION RESULTS DATA ANALYSIS DISCUSSION TOTAL EXAMINERS COMMENT /25% /30% /30% /100% /15%

APPROVAL RECEIVE

FACULTY : CIVIL & ENVIRONMENTAL ENGINEERING DEPT : WATER RESOURCE & ENVIRONMENTAL ENGINEERING LAB : ENVIRONMENTAL ENGINEERING

PAGE NO.: EDITION: REVISION NO.: EFFECTIVE DATE:

1/5 MA2 03 01/12/2007 20/11/2007

EXPERIMENT : BACTERIA COUNT

AMMEND. DATE:

1.0 OBJECTIVE Students will be able to measure the bacteriological quality of water sample by performing total plate count.

2.0 LEARNING OUTCOMES At the end of the laboratory courses, students will be able 1. Become proficient at dilutions. 2. Become proficient at performing a standard plate count and determining bacterial counts in a sample. 3.0 THEORY Bacteria are remarkably adaptable to diverse environmental conditions: they are found in the bodies of all living organisms and on all parts of the earthin land terrains and ocean depths, in arctic ice and glaciers, in hot springs, and even in the stratosphere. Our understanding of bacteria and their metabolic processes has been expanded by the discovery of species that can live only deep below the earth's surface and by species that thrive without sunlight or in the high temperature and pressure near hydrothermal vents on the ocean floor. There are more bacteria, as separate individuals, than any other type of organism; there can be as many as 2.5 billion bacteria in one gram of fertile soil. Many studies require the quantitative determination of bacterial populations. The two most widely used methods for determining bacterial numbers are the standard, or viable, plate count method and spectrophotometric (turbidimetric) analysis. Although the two methods are somewhat similar in the results they yield, there are distinct differences. For example, the standard plate count method is an indirect measurement of cell density and reveals information related only to live bacteria. The spectrophotometric analysis is based on turbidity and indirectly measures all bacteria (cell biomass), dead and alive. The standard plate count method consists of diluting a sample with sterile saline or phosphate buffer diluent until the bacteria are dilute enough to count accurately. That is, the final plates in the series should have between 30 and 300 colonies. Fewer than 30 colonies are not acceptable for statistical reasons (too few may not be representative of the sample), and more than 300 colonies on a plate are likely to produce colonies too close to each other to be distinguished as distinct colony-forming units (CFUs). The assumption is that each viable bacterial cell is separate from all others and will develop into a single discrete colony (CFU). Thus, the number of colonies should give the number of bacteria that can grow under the incubation conditions employed. A wide series of -4 -10 dilutions (e.g., 10 to 10 ) is normally plated because the exact number of bacteria is usually unknown. Greater accuracy is achieved by plating duplicates or triplicates of each dilution. Increased turbidity in a culture is another index of bacterial growth and cell numbers (biomass). By using a spectrophotometer, the amount of transmitted light decreases as the cell population increases. The transmitted light is converted to electrical energy, and this is indicated on a galvanometer. The reading, called absorbance or optical density, indirectly reflects the number of bacteria. This method is faster than the standard plate count but is limited because 7 sensitivity is restricted to bacterial suspensions of 10 cells or greater.

PREPARED BY: BALKIS A. TALIP

SIGNATURE: DATE: 20 NOVEMBER 2007

FACULTY : CIVIL & ENVIRONMENTAL ENGINEERING DEPT : WATER RESOURCE & ENVIRONMENTAL ENGINEERING LAB : ENVIRONMENTAL ENGINEERING EXPERIMENT : BACTERIA COUNT

PAGE NO.: EDITION: REVISION NO.: EFFECTIVE DATE: AMMEND. DATE:

2/5 MA2 03 01/12/2007 20/11/2007

4.0 EQUIPMENTS & MATERIALS 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Petri plate Pipette Test tube Glass rod Bunsen burner Incubator Ethanol 95% @ methanol Sterilizer Microscope Bacteria medium: Peptone = 5g, Beef Extract=3g, Agar=15g, Distilled water=600 mL

5.0 PROCEDURES 5.1 Media preparation: Please prepare the nutrient media using the microbiology standard method.

5.2 Sample preparation: Prepare the serial dilution of the water sample using the appropriate
dilution factor.

5.3 Plating procedures: Using the pour plate and spread plate method.

FACULTY : CIVIL & ENVIRONMENTAL ENGINEERING DEPT : WATER RESOURCE & ENVIRONMENTAL ENGINEERING LAB : ENVIRONMENTAL ENGINEERING EXPERIMENT : BACTERIA COUNT

PAGE NO.: EDITION: REVISION NO.: EFFECTIVE DATE: AMMEND. DATE:

4/5 MA2 03 01/12/2005 20/12/2005

6.0 RESULTS & CALCULATIONS / ANALYSIS

Plating Method Pour Plate Spread Plate

Average Colony/plate 119 125

Dilution 1/10 190 200 1/100 175 180 1/1000 155 160 1/10 85 90
4

1/10 75 80

1/10 35 40

Total bacteria / mL

7.0 DATA ANALYSIS

1.

Show the calculation for each of the plating method and fill in the above table.

2.

Analyze the results by using the appropriate method. Explain your findings.

3.

State the systematic bias error that could occur during this experiment.

4.

Usually, the result shows different reading for both methods. However, in some cases, both methods produce the same result. Explain why the results are indistinguishable.

8.0 QUESTIONS Explain the meaning of phrases two times ten to the eight cells per mL in your own convenient terminology.

1.

2.

What the meaning of TNTC and the significant amount due to the TNTC? Give the formula for determining bacteria count

3.

Design the experiment for comparing the bacteria counts of water sample (tapwater, lake water, swimming pool water and rainbarrel water). Explain the different of bacteria count for each type of water sample?

4.

In many experiment there are 2 types of control used which are positive and negative control. Due to this experiment what is the suitable control?, How the control will effect to your finding?

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