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.

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 References

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 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 Enterobacter cloacae on MacConkey Agar: growth with pink colonies Eschericia coli on MacConkey Agar: growth, with pink colonies Staphylococcus aureus

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. Neisseria Pseudomonas 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