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INTRODUCTION

Microorganisms, such as bacteria and yeast, are present within all human environments without being conspicuously recognizable to the naked human eye. Environmental swabs were taken of 11 variable locations, were isolated, and were then grown over a 48 hour incubation period in agar media for optimum colony presence. Colonies were gram stained in order to morphologically categorize bacteria by shape and gram negative or positive status.

Bacteria consist of only a single cell which is amazingly complex and fascinating group of creatures. Bacteria have been found that can live in temperatures above the boiling point and in cold that would freeze your blood. They "eat" everything from sugar and starch to sunlight, sulfur and iron. There's even a species of bacteria—Deinococcus radiodurans— that can withstand blasts of radiation 1,000 times greater than would kill a human being.

Bacteria fall into a category of life called the Prokaryotes (pro-carry-oats). Prokaryotes' genetic material, or DNA, is not enclosed in a cellular compartment called the nucleus. Bacteria and archaea are the only prokaryotes. All other life forms are Eukaryotes (you-carry-oats), creatures whose cells have nuclei. Bacteria can be found virtually everywhere. They are in the air, the soil, and water, and in and on plants and animals, including us.

OBJECTIVE

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

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

Many studies require the quantitative determination of bacterial populations.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 earth–in land terrains and ocean depths. and this is indicated on a galvanometer. 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 spectrophotometric analysis is based on turbidity and indirectly measures all bacteria (cell biomass). plate count method and spectrophotometric (turbidimetric) analysis.5 billion bacteria in one gram of fertile soil. the final plates in the series should have between 30 and 300 colonies. That is.g. There are more bacteria. Although the two methods are somewhat similar in the results they yield. called absorbance or optical density. the amount of transmitted light decreases as the cell population increases. By using a spectrophotometer. This method is faster than the standard . the number of colonies should give the number of bacteria that can grow under the incubation conditions employed. in arctic ice and glaciers. For example. or viable.. than any other type of organism. The transmitted light is converted to electrical energy. Thus. 10-4 to 10-10) is normally plated because the exact number of bacteria is usually unknown. as separate individuals. 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. Fewer than 30 colonies are not acceptable for statistical reasons (too few may not be representative of the sample). 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). there are distinct differences. in hot springs. dead and alive. The two most widely used methods for determining bacterial numbers are the standard. the standard plate count method is an indirect measurement of cell density and reveals information related only to live bacteria. indirectly reflects the number of bacteria. The reading. A wide series of dilutions (e. there can be as many as 2. 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. and even in the stratosphere. The assumption is that each viable bacterial cell is separate from all others and will develop into a single discrete colony (CFU).

EQUIPMENTS & MATERIALS Test tube Petri plate Pipette Glass rod Stirring hot plate Incubator .plate count but is limited because sensitivity is restricted to bacterial suspensions of 107 cells or greater.

beef extract. For the Spread Plate test. agar and distilled water mixed in 600mL beaker and boiled. the nutrient media poured into half of the six petri plates.Microscope Agar Bacteria = 15g Meat Bacteria = 15g Peptone Bacteria = 15g PROCEDURES Procedures of preparing nutrient media 1. Note: All the agar preparation procedures should be performed under laminar flow to keep the samples sterile. The agar cooled up to 40-50oC. Use gloves to prevent contamination of the samples . 2. 3. Peptone.

The pipette discharged into the used jar for later cleaning. you must wipe the pipette with Kleenex or toilet paper before inserting the pipette into tube#1. sterile.9mL of dilution fluid (normally deionizer/distilled water) in tube#1 and mixed thoroughly by blowing lots of bubbles with the pipette for a couple seconds.Dilution procedures 1.1mL from the bacteria sample and blew into the 9. Since nearly 0. dry pipette used to remove 0. Notice tube#1 now contains 1/100 the concentration of bacteria in the original sample because 0. A clean.1mL of liquid may cling to the outside of the pipette. .1mL is 1/100 of 10mL.

sterile. All the petri plates placed inside the incubator for 24 hours with a temperature of 37oC.0 mL water sample 1/10 1/100 1/10 3 1/10 4 1/10 5 1/10 6 Spread Plate test method 1. 2.1 mL 0. .1mL from tube#2. 0. and continue blowing bubbles for a second or two for good mixing. sterile pipette used to remove 0. 5. A clean. and you are advised to use blow out pipette. 4. continue blowing bubbles for a second or two for mixing.1mL from tube#1.1 mL 0. dry pipette used to remove 0.1 mL 0.1 mL 0. that is indicated by a frosted ring on the pipette at the top end. The petri plate closed. blow contents of pipette into tube#2. Using another clean. The same procedures repeated until tube#6. Another clean.1mL of diluted sample from each test tube into six different petri plates contain sterile agar. Our tubes labeled with the dilution factor as to notice the bacteria content in the tubes.2. dry. dry pipette remove 0.1 mL 0. wipe pipette. The diagram below referred for better understanding. sterile. 3. Note: There are many types of pipettes.1 mL test tubes contain 9. 3.0 mL dilution fluid water sample 1. blow contents of pipette into tube#3.9 mL dilution fluid test tubes contain 9. wipe.

Pour Plate test method 1. 4. A clean. and waited until the agar to solidify. All the petri plates placed inside the incubator for 24 hours with a temperature of 37oC.1mL of diluted sample from each test tube into six different petri plates. . dry. 3. The agar poured into the plates. The petri plate closed. 2. sterile pipette used to remove 0.

. The petri plate placed on the counting chamber.Methods of counting bacteria 1. 3. the petri plates took out. After being incubate for 1 day. The bacteria colonies on the culture were counted. 2.