PaCES/HIMB Summer Program in Environmental Science

Microbiology Lab

ProkaryotesBIOLOGY 171L1

Introduction

Prokaryotes (bacteria) make a vast heterogeneous group of very small unicellular organisms. They are distinguished from the eukaryotes (all other living things) because of the following cellular characteristics.

1.Their genetic material (DNA) is not enclosed within a membrane (nucleus);

2.They lack other membrane-bounded organelles;

3.Their DNA is not associated with histone proteins (special chromosomal proteins found in eukaryotes);

4.Their cell walls, if present, almost always contain the complex polysaccharide peptidoglycan (as opposed to cellulose in green plants and chitin in fungi); and

5.They usually divide by binary fission (most eukaryotic cells divide by mitosis).

Prokaryotes include eubacteria (“true” bacteria) and archaeobacteria. The majority of bacteria belong to the eubacteria. In contrast, while appearing similar to the eubacteria, the archaeobacteria are so different chemically from the eubacteria that they may represent a different kingdom of life. The thousands of species of bacteria are differentiated by many factors, including morphology (shape), chemical composition (often distinguishable by specific staining reactions), nutritional requirements, biochemical activities, and source of energy (sunlight or chemicals).

Typical Prokaryotic Cell Structures

Prokaryotic cells appear simpler than eukaryotic cells. However, one must remember that these “simple” cells contain everything they need to sustain life. Unfortunately, our microscopes do not provide enough resolution to see the details of a typical prokaryotic cell. However, if we could see these details, then they may appear as presented in Figure 1 below. The functions of each of these structures are also described in the text that follows.

Plasma Membrane

The plasma membrane maintains the internal integrity of the cell by regulating what goes in and out of the cell.

CELL WALL

The cell wall, found in nearly all bacteria, is a semi-rigid structure that maintains the general shape of the cell. It protects the fragile plasma membrane from rupturing due to osmotic stress. It also provides anchorage for flagella.

The cell wall of bacteria is composed of a network of a polysaccharide (= complex carbohydrate) called peptidoglycan. Differences in the amount and arrangement of peptidoglycan in the cell wall form the basis for classifying bacteria as gram-positive or gram-negative. Gram-positive bacteria retain the dark purple color of crystal violet dye, while gram-negative bacteria do not. Thus gram-negative bacteria must be stained with another stain (e.g., the counterstain safranin) to be visualized when using the gram staining procedure (you will learn how to use this procedure in this laboratory activity).

ProkaryotesBIOLOGY 171L1

Figure 1. Diagrammatic representation of a typical bacterium illustrating structures.

Microbiology LabBIOLOGY 124L1

Antibiotics are chemicals that prevent cell wall formation in bacteria. When bacteria are exposed to antibiotics they cannot multiply and they eventually burst due to osmotic disruption.

CAPSULE

The capsule (or glycocalyx), which is not found in all bacteria, is a gelatinous material surrounding the cell wall. The presence of a capsule may be important in the ability of a bacterium to cause disease. The capsule inhibits your body’s defense system from attacking the bacterium.

FLAGELLUM

The flagellum (pl. flagella) is a whip-like structure that propels the bacterium. Some bacteria have many flagella. Others lack them entirely.

CYTOPLASM

The cytoplasm is the fluid interior of the cell. It is about 80% water and contains proteins, carbohydrates, lipids and other materials. The cytoplasm is where much of the chemistry of life takes place.

Ribosomes

The ribosomes are granules of RNA (ribonucleic acid) and protein that participate in translating the genetic message into functional proteins (e.g., enzymes or structural proteins).

Nuclear Area (Nucleoid)

The nuclear area contains a single circular molecule of double-stranded DNA (deoxyribonucleic acid). The DNA contains the genetic instruction set that carries all the information required for the cell’s structures and functions.

Size, Shape and Arrangement of Bacterial Cells

Bacteria vary greatly in size, shape and the arrangements of their cells. Most bacteria range from 0.2 to 2.0 µm in diameter and from 2 to 8 µm in length. They have a few basic shapes: spherical coccus, rod-shaped bacillus and spiral. Some of the various shapes and arrangements for these cells are presented in Figure 2.

Microbiology LabBIOLOGY 124L1

Figure 2. Some common shapes and arrangements of eubacteria.

Microbiology LabBIOLOGY 124L1

Modes of Nutrition

All cells need energy and materials to survive and reproduce. Some cells are autotrophic because they can make their own organic molecules (e.g., sugars, amino acids and lipids) from inorganic precursors (e.g., carbon dioxide and water). Autotrophic metabolism requires a source of energy in the form of either light energy (photoautotrophy) or chemical energy (energy released from certain chemical reactions such as the conversion of sulfide to sulfate; such organisms are said to exhibit chemoautotrophy). Photoautotrophic organisms, such as prokaryotic cyanobacteria and eukaryotic green plants, carry out this process through photosynthesis. Chemoautotrophic organisms, such as hydrothermal vent bacteria from the deep ocean floor, carry out this process through chemosynthesis.

Most eubacteria are heterotrophic in that they cannot manufacture their own organic molecules from inorganic precursors. Thus they must obtain these organic molecules from their surroundings. They either live off dead organic matter (saprophytes) or derive their nutrients from living hosts (parasites).

In this laboratory exercise we will do the following activities:

become familiar with the diversity of prokaryotes from living specimens and prepared slides;

learn how to carry out the Gram Stain procedure and observe preparations using oil immersion optic; and

Procedure

A.Macroscopic Observations of Bacteria Colonies

Pick at least two colonies from the plates you prepared previously. Choose colonies that are clearly separate and distinct from other colonies. Note the following characteristics of the colonies chosen (see also Figure 3):

  • Whole Shape Of Colony
  • Edge/Margin Of Colony
  • Color
  • Opacity Of Colony

Transparent (clear), opaque, translucent (almost clear, but distorted vision–like looking through frosted glass), iridescent (changing colors in reflected light)

  • Elevation Of Colony
  • Surface Of Colony

Smooth, glistening, rough, dull (opposite of glistening), rugose (wrinkled)

  • Consistency

Butyrous (buttery), viscid (sticks to loop, hard to get off), brittle/friable (dry, breaks apart), mucoid

  • Emulsifiability Of Colony

Is it easy or difficult to emulsify? Does it form a uniform suspension, a granular suspension, or does not emulsify at all?

  • Odor

Absent or present? If it has an odor, what does it smell like?

Prepare a table (Table I) that summarizes these characteristics for each colony

Figure 3. Some bacteria colony characteristics.

B.Microscopic Observations of Bacteria Colonies

The Gram stain procedure is used to visualize the morphology (coccus, bacillus, or spirillum) of individual bacteria cells, determine their cell arrangements (individual, diplo-, tetrad, staphylo-, or strepto-, etc.), and distinguish the two major types of cell wall chemistry (Gram positive versus Gram negative). Gram staining will be demonstrated to you in class. You will also be provided with a handout that describes this procedure.

Using the two bacteria colonies selected, perform the Gram stain procedure and determine the morphology, arrangement, and Gram staining properties of the bacteria. Include these observations in Table I described above. Use of the microscopes will be explained and demonstrated to you in class.

Draw labeled diagrams of these bacteria.

Lab Summary

1.Write a short Introduction that describes the major objectives of this lab activity.

2.Prepare a table (Table I) summarizing your observations on the bacteria colonies observed. The table should include information about the color and texture of the whole colony, Gram staining properties of the bacteria from the colony, and information about the shape and arrangement of the bacteria. Include your diagrams of the bacteria with your summary.

3.In Figures 1 & 2, draw diagrams illustrating the microscopic structure of these bacteria.

4.Write a short conclusion about what you learned from this lab activity.