Laboratory work № 4 Structure of bacteria and yeasts
Purpose of work:
To familiarize with the structure of bacteria, mushrooms and yeasts.
Materials and equipment:
Collection of cultures should to recultivate each 2-3 months on fresh nutrient media: bacteria and actinomycetes - on meat peptone agar, barmy and mold mushrooms on a wort-agar. 2 - 3 days before classes cultures recultivate in test tubes (bacteria and actinomycetes) or Petri dishes (mushrooms) from calculation after a bottom of a test tube of each culture and one-two Petri dishes on group of students of 10-15 persons. Microscopes and all accessories are necessary for classes; slide and cover glasses; bacteriological loops (needle); microscopic needles; a fresh 5 %-s' solution of fuchsine
Cells from animals, plants, and fungi are eukaryotes (Greek for "true nucleus"), whereas bacteria and the blue-green algae belong to the prokaryotes (Greek for "primitive nucleus"). In addition to lacking a nucleus and other organelles, prokaryotes use a smaller ribosome, the 70S ribosome, and in most bacteria, a mesh like peptidoglycan cell wall surrounds the membranes to protect it against the environment. Bacteria can survive and, in some cases, grow in hostile environments in which the osmotic pressure outside the cell is so low that most eukaryotic cells would lyse, at temperature extremes (both hot and cold), with dryness, and with very dilute and diverse energy sources. Bacteria have evolved the structures and functions to adapt to these conditions. Several of these distinctions provide the basis for antimicrobial action.
Bacteria are a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria have a wide range of shapes, ranging from spheres to rods and spirals. Bacteria are present in most habitats on Earth, growing in soil, acidic hot springs, radioactive waste, water, and deep in the Earth's crust, as well as in organic matter and the live bodies of plants and animals, providing outstanding examples of mutualism in the digestive tracts of humans, termites and cockroaches. There are typically 40 million bacterial cells in a gram of soil and a million bacterial cells in a millilitre of fresh water; in all, there are approximately five nonillion (5×1030) bacteria on Earth, forming a biomass that exceeds that of all plants and animals.
Bacteria can be distinguished from one another by their morphology (size, shape, and staining characteristics) and metabolic, antigenic, and genetic characteristics. Although bacteria are difficult to differentiate by size, they do have different shapes. A spherical bacterium, such as Staphylococcus, is a coccus; a rod-shaped bacterium, such as Escherichia coli, is a bacillus; and the snakelike treponeme is a spirillum. In addition, Nocardia and Actinomyces species have branched filamentous appearances similar to those of fungi. Some bacteria form aggregates such as the grapelike clusters of Staphylococcus aureus or the diplococcus (two cells together) observed in Streptococcus or Neisseria species.
The Gram stain is a powerful, easy test that allows clinicians to distinguish between the two major classes of bacteria and to initiate therapy. Bacteria that are heat-fixed or otherwise dried onto a slide are stained with crystal violet; this stain is precipitated with Gram iodine, and then the unbound and excess stain is removed by washing with the acetone-based decolorizer. A counterstain, safranin, is added to stain any decolorized cells red. This process takes less than 10 minutes.
For Gram positive bacteria, which turn purple, the stain gets trapped in a thick, cross-linked, meshlike structure, the peptidoglycan layer, which surrounds the cell. Gram negative bacteria have a thin peptidoglycan layer that does not retain crystal violet stain, so the cells must be counterstained with safranin and turned red. A mnemonic device that may help is "P-Purple-Positive." Gram stain is not a dependable test for bacteria that are starved (e.g., old or stationary phase cultures).
Bacteria that cannot be classified by Grain stain include mycobacteria, which have a waxy outer shell and are distinguished with the acid-fast stain, and mycoplasmas, which have no peptidoglycan.
Figure 2. Major features of prokaryotes and eukaryotes.
Bergey's Manual of Determinative Bacteriology
Commonly termed Bergey's Manual
Describes the majority of bacterial species identified by scientists so far.
Provides descriptions for the colony morphologies of each bacterial species.
Basic Elements in Identifying Colonies
Form - What is the basic shape of the colony? For example, circular, filamentous, etc.
Elevation - What is the cross sectional shape of the colony? Turn the Petri dish on end.
Margin - What is the magnified shape of the edge of the colony?
Surface - How does the surface of the colony appear? For example, smooth, glistening, rough, dull, rugose, etc.
Opacity - For example, transparent (clear), opaque, translucent (almost clear, but distorted vision, like looking through frosted glass), iridescent (changing colors in reflected light), etc.
Chromogenesis - For example, white, buff, red, purple, etc.
Consistency
Butyrous (butter-like)
Viscous or stringy (a portion of it may come off the agar surface with the transfer needle)
Rubbery (whole colony comes off the agar surface with the transfer needle)
Dry, brittle or powdery (colonies that break when touched by a needle)
Odor
Sweet
Putrefactive
Fruity
Figure 3. Comparison of the Gram positive and Gram negative bacterial cell walls. A, a Gram positive bacterium has a thick peptidoglycan layer that contains teichoic and lipoteichoic acids. B, a Gram negative bacterium has a thin peptidoglycan layer and an outer membrane that contains lipopolysaccharide, phospholipids, and proteins. The periplasmic space between the cytoplasmic and outer membranes contains transport, degradative, and cell wasll synthetic proteins. The outer membrane is joined to the cytoplasmic membrane at adhesion points and is attached to the peptidoglycan by lipoprotein links.
Figure 4. Gram stain morphology of bacteria. A, the crystal violet Gram stain is precipitated by Gram iodine and is trapped in the thick peptidoglycan layer in Gram positive bacteria. The decolorizer disperses the Gram negative outer membrane and washes the crystal violet from the thin layer of peptidoglycan. Gram negative bacteria are visualized by the red counterstain. B, bacterial morphologies.
Figure 5. Bacteria and Bacilli. Highly magnified.
What Can Grow on a Nutrient Agar Plate?
Bacteria
Each distinct circular colony should represent an individual bacterial cell or group that has divided repeatedly.
Being kept in one place, the resulting cells have accumulated to form a visible patch.
Most bacterial colonies appear white, cream, or yellow in color, and fairly circular in shape
Yeasts
Yeast colonies generally look similar to bacterial colonies.
Some species, such as Candida, can grow as white patches with a glossy surface.
Round Yeasts
Candida albicans
Pink Yeasts
Molds
Molds are actually fungi, and they often appear whitish grey, with fuzzy edges.
They usually turn into a different color, from the center outwards.
Black Mold (Aspergillus nidulaus)
Green Mold (Trichoderma harzianum)
Other Fungi
Moss green colonies, a white cloud, or a ring of spores can be attributed to the growth of Aspergillus, which is common in such fungal infections as athlete's foot.
Aspergillus
Agar Slant
Agar butt
Growth only within the line of inoculation (non-motile)
Growth spread or not only within the line of inoculation (motile)
Agar broth
Amount (scanty, moderate, abundant)
Distribution and type of growth
Uniform (even turbid)
Scum or film (pellicle)
Sedimentary (granular)
Ring at the top of the rim
CULTURE MEDIA
Culture Media
A liquid or gel designed to support the growth of microorganisms or cells.
Culture medium- nutrients prepared for microbial growth
Inoculation- introduction of microbes into medium
Culture/Colony- microbes growing in/on culture medium