Transcript Continuation Lecture 1

CONTINUATION……..
Three domain
classification
system
How it is discovered??



The discovery of the three cell types was based on
the observations that ribosomes are not the same in
all cells.
Prokaryotic 70S ribosomes while eukaryotic 80S
ribosomes
The sequence of nucleotides in ribosomal RNA from
different kinds of cells shows that there are 3
distinctly different cell group.
Three Domains
Eukarya
 Bacteria
 Archaea

Carl R.Woese

He believed that even though Archae
and Bacteria have similar appearance
but they should form their own domain.
Domain Archae and Bacteria

-

Domain bacteria
includes all of the prokaryotes as well as many of
non-pathogenic prokaryotes found in soil and water.
Domain Archae
includes prokaryotes that do not have
peptidoglycan in their cell wall.
Domain of Eukarya




Animalia: Multicellular; no cell walls;
chemoheterotrophic
Plantae: Multicellular; cellulose cell walls; usually
photoautotrophic
Fungi: Chemoheterotrophic; unicellular or
multicellular; cell walls of chitin; develop from
spores or hyphal fragments
Protista: A catchall kingdom for eukaryotic
organisms that do not fit other kingdoms
 Grouped
into clades based on rRNA
Bergey’s Manual of Systematic
Bacteriology




Identify and classify
Served the community of microbiologist since 1923
In 5 volume
IJSEM
Official publication of record for taxonomy and
classification of prokaryotes.
References
International Journal of
Systematic and Evolutionary
Microbiology (IJSEM)
Articles with evidence of
new species or classification
Bergey’s Manual of Systematic
Bacteriology
Provides phylogenetic and
identification information on
bacteria and archaea
Approved Lists of Bacterial
Names
Lists species of known
prokaryotes
Based on published articles
Colony morphology
1. Colony shape and size: round, irregular, punctiform (tiny)
2. Margin (edge): entire (smooth), undulate (wavy), lobate (lobed)
3. Elevation: convex, umbonate, flat, raised
4. Color: color or pigment, plus opaque, translucent, shiny or dull
5. Texture: moist, mucoid, dry (or rough).
Bacillus
Proteus
Staphylococcus
http://www.sciencebuddies.org/
Streptococcus
Motility agar–
stab agar with
wire, not loop
Mannitol Salt Agar



Purpose Mannitol salt agar is both a selective and differential
growth medium. It is used to differentiate pathogenic
Staphylococcus species from nonpathogenic members of the
genus Micrococcus.
Principle Mannitol salt agar can help determine two
characteristics of bacteria, whether they are salt tolerant or not,
and whether they are able to ferment mannitol.
Salt Tolerance (Does it grow?). This medium contains 7.5%
salt and therefore "selects" for organisms that are able to
tolerate the presence of high levels of salt.If the organism
grows, it is salt tolerant.
If the organism does not grow, it is not salt tolerant.
Therefore, we say that MSA is "selective," as it will "select"
for salt-tolerant organism.
MSA continued
salt/mannitol fermentation
1. S. aureus
+/- (yellow)
2. S. epidermidis +/3. M. luteus
-/na
MacConkey Agar
Purpose MacConkey agar is a widely-used culture medium which is
both selective AND differential. The medium is primarily used to
differentiate between Gram negative bacteria while inhibiting the growth
of most Gram positive bacteria. The medium also differentiates between
lactose-fermenting coliforms and lactose nonfermenters, which include
potential pathogens.
Principle Addition to the nutrient agar base of bile salts and crystal violet
will inhibit the growth of most Gram positive bacteria, making
MacConkey agar selective. Lactose, a fermentable carbohydrate, and
neutral red, a pH indicator, are added to differentiate the lactose positive
coliforms from the potentially pathogenic lactose nonfermenters.
Additional Information When lactose is fermented, acid products lower
the pH below 6.8, with the resulting colonial growth turning pinkish-red. If
an organism is unable to ferment lactose, the colonies will be colorless.
Mac continued
Staphylococcus aureus
Enterobacter cloacae on
MacConkey Agar:
growth with pink colonies
Eschericia coli on MacConkey Agar:
growth, with pink colonies
DIFFERENTIAL STAINS – GRAM
STAINING cont..


Fresh reagents - of proper strength
Control cultures - for a known GP bacterium and
GN culture (S.aureus & E.coli)
The process includes the use of:
a primary stain (crystal violet)
a mordant (helper) iodine solution,
a decolorizer (95% ethanol),
a counterstain (safranin).
The Gram stain
Thin smear/heat fix
Gram stain:
a. Flood slide with crystal violet and let stain
for 1 minute.
b. Drain off crystal violet and rinse off
with distilled water; flood slide
with Gram's iodine for 1 minute.
c. Rinse off Gram's iodine with distilled water.
d. Hold the slide on an angle (preferably with
a clothes pin) and drop 95% ethyl alcohol
onto it until the alcohol leaving the slide no
longer has a purple tint; be sure to drop the
alcohol onto the upper portion of the slide
so that the smears are subjected to uniform
decolorization. Be careful not to
"decolorize" dye from the clothes pin!!
e. Rinse with distilled water and flood the
slide with safranin and let stain for 2-3
minutes.
f. Rinse with distilled water and blot dry with
bibulous paper.
Gram
positive
Gram
negative
Demos: Gram stained slides of
Neisseria, Streptococcus, Pseudomonas, Actinomyces species.
Pseudomonas
Neisseria
Streptococcus
WHAT ARE ENDOSPORES?

Two genera of bacteria, Clostridium and Bacillus,
produce endospores. Endospores are tough,
resistant structures that allow the bacteria to
essentially exist in ‘suspended
animation.’ Endospores do not metabolize and do
not reproduce but merely exist, much like plant
seeds, until exposed to environmental conditions
suitable for bacterial growth.
Example of Clostridium bacteria with characteristic drumstick-shaped
endospore-producing cells. The dark rod-shaped cells are vegetative,
actice cells. The clear ovals are endospores, and the objects consisting of
both dark rod and clear oval are vegetative cells producing endospores.
Variations in endospore morphology: (1, 4) central
endospore; (2, 3, 5) terminal endospore; (6) lateral
endospore.
ENDOSPORE SPORULATION




Endospores are made through a process known as sporulation
in response to extreme environmental conditions.
Extreme environments include high temperatures, drying out
(dessication), extremes in pH, bacteriocidal chemicals and lack
of food.
When environmental conditions are favorable, the endospore
germinates.
Upon germination, the cell returns to its normal metabolic state,
capable of reproduction.
STAINING ENDOSPORES


Normal staining techniques will not stain the resistant
endospores. Here are the steps that are required:
Malachite Green: The stain, malachite green, is forced
into the spore with heat much like the carbol fuschsin
was forced through the waxy mycolic acid layer of
Mycobacterium. After flooding the slide with
malachite green, it is suspended over a boiling water
bath for 5 minutes and then rinsed with water.
Staining Endospores cont..

Safranin: Once the endospore is stained, the counter
stain, Safranin, provides color for the vegetative (i.e.
metabolically active) cells.
WHAT VEGETATIVE CELLS AND
ENDOSPORES LOOK LIKE?

Below is a photo of Bacillus bacteria that have been
stained using the endospore stain. At the end of this
differential staining process the vegetative cells
(active, metabolizing cells) are pink and the
endospores, if present, are green.
BACILLUS SP. ENDOSPORES