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 bacteriaDeinococcus 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

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 dilutions (e.g., 10-4 to 10-10) 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 sensitivity is restricted to bacterial suspensions of 107 cells or greater.

EQUIPMENTS & MATERIALS

Test tube

Petri plate

Pipette

Glass rod

Stirring hot plate

Incubator

Microscope

Agar Bacteria = 15g

Meat Bacteria = 15g

Peptone Bacteria = 15g

PROCEDURES

Procedures of preparing nutrient media

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

Dilution procedures

1. A clean, sterile, dry pipette used to remove 0.1mL from the bacteria sample and blew into the 9.9mL 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. The pipette discharged into the used jar for later cleaning. Notice tube#1 now contains 1/100 the concentration of bacteria in the original sample because 0.1mL is 1/100 of 10mL. Since nearly 0.1mL of liquid may cling to the outside of the pipette, you must wipe the pipette with Kleenex or toilet paper before inserting the pipette into tube#1.

2. Another clean, sterile, dry pipette used to remove 0.1mL from tube#1, wipe pipette, blow contents of pipette into tube#2, and continue blowing bubbles for a second or two for good mixing. 3. Using another clean, sterile, dry pipette remove 0.1mL from tube#2, wipe, blow contents of pipette into tube#3, continue blowing bubbles for a second or two for mixing. 4. The same procedures repeated until tube#6. The diagram below referred for better understanding. 5. Our tubes labeled with the dilution factor as to notice the bacteria content in the tubes.

Note: There are many types of pipettes, and you are advised to use blow out pipette, that is indicated by a frosted ring on the pipette at the top end.

0.1 mL

0.1 mL

0.1 mL

0.1 mL 0.1 mL

0.1 mL test tubes contain 9.9 mL dilution fluid test tubes contain 9.0 mL dilution fluid water sample 1.0 mL

water sample

1/10

1/100

1/10

1/10

1/10

1/10

Spread Plate test method

1. A clean, dry, sterile pipette used to remove 0.1mL of diluted sample from each test tube into six different petri plates contain sterile agar. 2. The petri plate closed. 3. All the petri plates placed inside the incubator for 24 hours with a temperature of 37oC.

Pour Plate test method

1. A clean, dry, sterile pipette used to remove 0.1mL of diluted sample from each test tube into six different petri plates. 2. The agar poured into the plates, and waited until the agar to solidify. 3. The petri plate closed. 4. All the petri plates placed inside the incubator for 24 hours with a temperature of 37oC.

Methods of counting bacteria

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