Isolation of Bacteria

Introduction: Bacteria are the most abundant life form on Earth. They are found everywhere in the billions. Bacteria belong to the Kingdom Eubacteria and are prokaryotic. They all have a cell wall composed of peptides and complex sugars (non-cellulose). They are unicellular; however some grow in colonies or as filaments. Bacteria have very rapid reproductive rates; many copy themselves every 20 minutes! Identify 3 factors that can limit bacterial growth. _______________________________________ _____________________________________________________________________________ _____________________________________________________________________________ Bacteria have 3 general shapes: rod (bacilli), spiral (spirillum), and spherical (cocci). They can either be autotrophs (photosynthetic or chemosynthetic) or heterotrophs. Many heterotrophs are decomposers. Only about 1% of bacteria are pathogens (cause disease). Some bacteria can produce antibiotics, some are used in the food industry (vinegar, swiss cheese, yogurt), and some are used to clean-up pollutants in the environment. Many bacteria have mutualistic relationships with other organisms; i.e. E. coli in large intestine produce vitamins.

In this experiment you will collect samples from a lab surface, a chicken egg, uncooked chicken, uncooked hamburger, and from cooked rice, and transfer them to nutrient agar for growth. You will also plate out your E. coli and Staph epidermidis from the antibiotic resistant lab on selective media to determine which bacteria is gram positive and which is gram negative. The selective media used in the lab are MacConkey which supports the growth of gram negative bacteria, and Manitol Salt Agar (MSA) which supports the growth of gram positive bacteria.

Gram Positive (Bacillus cereus)

Gram Negative (Serratia marcescens)

Bacteria are divided into two broad categories: gram positive and gram negative. These subdivisions are based on the bacteria’s ability to be stained with the Gram stain. Gram positive bacteria stain blue and negative bacteria stain red. The difference in their ability to retain the stain is based on the structure of their cell wall. Bacteria have a complex cell wall constructed of a peptidoglycan layer. As the name implies, this wall consists of chains of repeating sugars cross linked with peptide bridges. Gram positive bacteria have a cell wall twenty times thicker than gram negative bacteria. Gram negative bacteria not only have a much thinner cell wall, but also have an outer membrane. This outer membrane blocks antibiotics, dyes and detergents from entering the cell. As a result, they are much more resistant to certain antibiotics like penicillin. (Ampicillin, choramphenicol and streptomycin are however effective.) The outer membrane is composed of a lipopolysaccharide called LPS. LPS is medically important because when released from bacterial cells free LPS is toxic to mammals. This endotoxin creates a wide spectrum of physiological reactions including the induction of a fever, changes in white blood cell

counts, leakage from blood vessels, and lowered blood pressure that can lead to vascular collapse and eventually shock. The differences between the cell walls of gram positive and gram negative bacteria influence the success of the microbes in their environments. The thick cell wall of gram positive cells allows them to do better in dry conditions because the thick wall reduces water loss. The outer membrane and its LPS help gram negative cells survive in the intestines and other host environments where moisture is available.

Examples of common gram-positive bacteria that infect humans and their shapes: Streptococcus (cocci) Staphylococcus (cocci) Bacillus (bacilli, protective spore) - causes anthrax and gastroenteritis Clostridium (bacilli, protective spore) - causes botulism, tetanus, and gangrene Corynebacterium (bacilli, no protective spore) - causes diphtheria Listeria (bacilli, no protective spore) - causes meningitis

Examples of gram-negative bacteria: Spirochetes (spiral-shaped) - causes syphilis, lyme disease Neisseria (cocci) - causes meningococcus, gonorrhea

Some gram positive bacteria can produce endospores (most commonly Bacillus and Clostridium). Endospores are metabolically inactive forms of bacteria that are surrounded by an extremely thick outer wall, and can survive for a very long time in nature, and return to a growing state when exposed to appropriate growing conditions. Endospores that were dormant for thousands of years in the great tombs of the Egyptian pharohs were able to germinate and grow when placed in appropriate medium. Endopores are resistant to heat (>100°C), radiation, many chemicals (i.e. acids, bases, alcohol, chloroform), and desiccation. The formation of an endospore is clearly a great advantage for these bacteria and enables them to endure extreme stress. At a later time, when conditions are favorable, they can reemerge and flourish. Endospores enable a species to spread easily from one suitable environment to another. Endopores are a particular problem in the food industry where great care must be taken to insure either the destruction of endospores or suitable preservation methods so that endospore-forming bacteria (and other microbes) cannot grow.

Materials:

Incubator 37oC sterile q-tips sterile bacterial loops

nutrient agar plates

MSA agar plate MacConkey agar plate

Procedures: Sampling a Surface and Food (Egg, Uncooked Chicken and Hamburger)

1. Use a sterile cotton swab to collect organisms from two surfaces of your choice. Do not touch the end you will use for collecting. Moisten the swab with a drop of water if the surface is dry.

2. Streak the swab or roll it gently across the surface of a nutrient agar plate. Close the dish promptly. Label the bottom of the dish with your initials and note the environment sampled.

3. Use a sterile swab to collect a sample from cooked rice, an egg or uncooked chicken or hamburger. Streak a new nutrient agar plate as above.

4. Incubate plates in incubator at 37oC for 24 hours. Observe and draw diagrams.

Sampling from Antibiotic Resistant Plates

1. Obtain a Manitol Salt Agar (MSA) plate and a MacConkey Agar plate.

2. Draw a line on the back of the plate, to divide it in half. Mark one half “E. coli” and mark

the other half “Soil”. Make sure to include your name.

3. Carefully open the E. coli plate and use a sterile swab to collect bacteria from the agar.

Close the plate.

4. Streak the MSA agar plate on the E. coli half.

5. Streak the MacConkey plate on the E. coli half.

6. Repeat procedure with the soil bacteria on both MSA and MacConkey agar.

7. Incubate plates in incubator at 37oC for 24 hours.

8. Record your observations

9. Using a smart phone or tablet, take a picture of each plate. Print out the pictures for

inclusion in your lab binder.

Food for thought:

1. What is the difference between gram positive and gram negative bacteria?

2. Which media selects for gram positive bacteria? Which selects for gram negative?

3. What organism and disease is associated with uncooked eggs and meat? How can these

diseases be prevented?

4. If a cutting board in the kitchen were contaminated with uncooked meat, what would be the most effective cleaning process? Why?

5. Why is it important to wash your hands? What is the difference between using regular soap, antibacterial soap and hand sanitizers? Considering what you know about antibiotic resistance, what would be the best cleanser to use?

Results/observations: