Monerans

Supplies

Equipment . Autoclave bag for disposal of cultures Compound microscopes Dissecting microscopes Oil immersion microscope Immersion oil Gram’s stain kit with crystal violet and sofranin Alcohol lamps Sterile water Diamond pencils Sterile 0.85% saline, packaged 99 ml per bottle Bacterial loops 25% acetone in isopropyl alcohol

Materials ~ TDC culture medium for Azotobocter Thioglycollote medium India ink 70% ethanol Microscope slides and coverslips Test substances, such as antibiotics, metal salts, or the kingdom Monera Livingcultures 2. To illustrate some of the techniques in determinative Anabaena microbiology Bacillus megaterium Pseudomones fluorescens related organisms. The largest of these categories is the

Prepared slides for demonstration Composite of bacterial types (cocci, bacilli, and spirilla Escherichio coli The diversity of organisms is staggering. In order to or- Gloeocapso ganize information about the living world, biologists group Oscillatoria Merismopedio (for demonstration) Azotobocter sp. Nutrient agar with 0% and 6% NaCI in petri dishes phylum several classes, and each class several orders.

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Diversity and Phylogeny

Within the orders are families composed of several genera that are further subdivided into species.

Each type of organism is ultimately identified in this hierarchical classification system by two names, a binomial that includes the genus and a species name, both written in Latin and italicized. All binomials are unique; the same name is never given to two different species.

About 30 years ago, it was common to think of all organisms as belonging either to the plant or to the animal kingdoms. Bacteria, blue-green algae, and fungi were placed in the plant kingdom, and protozoa were considered animals. The primary difference between the kingdoms was how nutrients were procured. Plants were autotrophs, making their own food in photosynthesis, except for degenerate forms, such as bacteria and fungi. Animals were heterotrophs, acquiring their food by ingestion of plants or other animals. However, this system was too simplistic and taxonomically grouped organisms that were not necessarily closely related.

In recent years, the five kingdom classification scheme has been widely accepted (fig. 1 4. 1). In it, living organisms are grouped into one of the following kingdoms:

Monera-- prokaryotic organisms, such as the bacteria and blue-green algae.

Protista --eukaryotic organisms that exist as single cells or as colonies of single cells. Food is obtained by ingestion, absorption, or photosynthesis.

Fungi --nonmotile, multinucleate eukaryotic organisms with absorptive nutrition and mycelial organization.

Plantae- —nonmotile, eukaryotic multicellular organisms Chloroplasts, with photosynthetic nutrition, walled cells, and chloroplasts.

Animalia-- motile, eukaryotic multicellular organisms having cells without’ walls and ingestive nutrition.

This exercise covers the kingdom Monera. The monerans are split into three groups:

( 1 ) Archaeobacteria(ancient bacteria),

(2) Eubacteria (true bacteria), and

(3) Cyanobacteria (blue-green algae).

All are prokaryotes; they lack a nucleus and membranous cytoplasmic organelles, such as vacuoles, mitochondria, and chloroplasts. All groups have cell walls composed of peptidoglycans, and are often surrounded by a gelatinous sheath. Their DNA is arranged in circular molecules and is not complexed with histones to form chromosomes as in the four other kingdoms.

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Most bacteria are heterotrophs and depend on other organisms for food. Some are parasitic and cause diseases, such as pneumonia and syphilis, but others are saprophytes and break down organic matter, thus recycling elements, such as carbon, nitrogen, and phosphorus in the environment. A few bacteria and all blue-green algae are autotrophs and make their own food by photosynthesis. Many bacteria and blue-green algae can fix nitrogen converting atmospheric nitrogen gas into nitrate or organic nitrogen compounds. No eukaryotes can fix nitrogen, so all other living organisms depend on the monerans to provide nitrogen in a form that can be used in making organic

Lab Instructions You will study representatives of the Monera in today’s lab period and explore some of the techniques used in microbiology.

Bacteria are among the smallest living organisms and are very difficult to observe through the finest light microscopes, let alone those that are normally found in teaching laboratories. Therefore, the identification of bacteria is based on a combination of characteristics. One characteristic used is cell shape. Some bacterial cells are cocci, small spheres, while others are rod-shaped bacilli or cork-screw-shaped spirilla.

Look at the composite slide of bacterial cell types that your instructor has set up as a demonstration on a microscope with an oil immersion objective. Objectives such as this allow magnifications of up to 1,000X. Compare these to the scanning electron micrograph of bacteria in figure 2. 1 . Sketch the basic shapes of bacteria below.

Gram Staining

Bacteria are also identified by their stainability with certain dyes. One such test is Gram’s stain. It contains crystal violet, a dye that binds with components of the cell walls of some, but not all, bacteria. Bacteria that retain Gram’s stain when washed with alcohol are said to be Gram positive, whereas those that lose the stain are said to be Gram negative. This is a primary tool in bacterial identification.

(a) Flame a loop before and after use.

(b) Remove a bacterial colony.

(C) Spread the colony in drop of water. Allow to air dry

(d) Kill cells by three passes through a flame.

(e) Place the slide over a beaker and cover with several drops of crystal violet.

(f) Wash off excess stain.

(g) Repeat staining with iodine solution and wash.

(h) Wash slide with acetone-isopropyl alcohol.

(I) Counterstain with safranin and wash.

(j) Blot the slide and allow to air dry.

and the other

You will perform the Gram’s stain on two 18- to 24- hour cultures of bacteria available in the laboratory.

One culture contains Bacillus megaterium, and one Pseudomonas fluorescens. Both species are large, but one is Gram positive and the other Gram negative. You should determine which bacterium is which by using the following procedures (fig. 14.2):

1 . Wash a microscope slide with soap and water to remove oils. Dip the slide in a beaker of alcohol and let it air dry.

2. 2. If you start with colonies from petri plates, put two tiny drops of distilled water on the slide. If you start from a liquid culture, water is not needed. Flame a bacterial loop and let it cool. Dip the loop into the culture or scoop part of a colony off the agar plate surface. Use the loop to spread one species of the bacteria evenly on one third of the slide and the other species on another third with a blank area between. Let the slide air dry at room temperature. Gently heat the slide by passing it through a low flame three times. This makes the cells adhere to the surface of the slide.

3. After the slide is cool, use a diamond pencil to put a B on the side of the slide that has the smear of Bacillus and a P on the Pseudomonas side. Put the slide on top of a beaker and flood the surface with Gram’s stain. After one minute, carefully pick up the slide and wash off the stain with a gentle flow of tap water.

Be Careful! This stain is very difficult to remove from your skin and clothing.

4. Now the slide should be flooded with an iodine reagent that enhances color development. After one minute, gently wash the slide again with tap water.

5. 5. Take a squeeze bottle containing 25% acetone in isopropyl alcohol and gently squirt it on the surface of the slide until the solvent running off the slide is colorless. Wash the slide with tapwater.

6. Flood the surface of the slide with safranin, a counterstain that helps you see Gram-negative cells. After 30-60 dseconds, wash off the counterstain with water.

7. Blot the excess at the edge of the slide onto paper toweling and allow the slide to air dry. Examine with a high dry objective, or an oil immersion objective if available.

Which species of bacterium is Gram positive?_____________ Which is negative?_______________

Sketch a few of the cells below and describe in word how they look.