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 waterb Sterile 0.85% saline, packaged 99 ml per bottle
Bacterial loops 25% acetone in
isopropyl alcohol
MaterialsTDC
culture medium for Azotobocter
Thioglycollote
medium
India ink
70% ethanol
Microscope
slides and coverslipsTest substances, such as antibiotics,
metal salts, or
the kingdom Monera
Livingcultures2.
To illustrate some of the techniques in determinative
Anabaenamicrobiology
Bacillus megaterium
Pseudomones fluorescensrelated organisms. The largest of these categories is the
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.
.
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.