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Bacillus

Bacillus is a genus of rod-shaped bacteria.

Gram-positive organism.

(looks like funky coral)

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Spirillum

Spirillum in microbiology refers to a bacterium with a cell body that twists like a spiral.

Spirillum is a genus of gram-negative bacteria.

(Neon sign, black background with glowing bacteria.)

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Staphylococcus

Staphylococcus is a genus of Gram-positive bacteria. Under the microscope they appear round (cocci), and form in grape-like clusters.

(Blue, looks like color-blind test.)

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Streptococcus

Streptococcus is a genus of spherical Gram-positive bacteria. They grow in chains or pairs, hence the name — from Greek στρεπτος streptos, meaning easily bent or twisted, like a chain (twisted chain). Contrast this with staphylococci, which divide along multiple axes and generate grape-like clusters of cells.

(Blue.)

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Clostridium Tetani

Clostridium tetani is a Gram positive rod-shaped bacterium.

Endospore

(Easter Grass, Swollen Ends)

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Amphitrichous Flagella

Amphitrichous bacteria have a single flagellum on each of two opposite ends (only one flagellum operates at a time, allowing the bacteria to reverse course rapidly by switching which flagellum is active).

(Brown snakes)

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Peritrichous Flagella

Peritrichous bacteria have flagella projecting in all directions.

(Brown furry)

Mycobacterium Tuberculosis

M. tuberculosis is characterized by caseating granulomas containing Langhans giant cells, which have a "horseshoe" pattern of nuclei. Organisms are identified by their red color on acid-fast staining.

(Acid-fast stain - red/purple)
(Non-acid stain - blue)

Blood red

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Capsule Stain

Capsule Stain

Stain background - Red

Capsule - Unstained

Ubiquity of Microbes - Microorganisms

They are everywhere.  They are free-living and do not need a host.

They are commensal/mutualistic - they benefit/we benefit.

They can also be opportunistic pathogens - if conditions are right they multiply and cause harm.

Germ Theory of Disease - theory that microorganisms cause disease.

Tryptic Soy Agar - (TSA) - Solid medium, food for bacteria.

Fomite - any inanimate object or substance capable of carrying infectious organisms (such as germs or parasites) and hence transferring them from one individual to another.

Colony - each dot

Only takes one cell on nutrient medium to start growing/multiplying.

Eventually you can see an area of growth on the medium = colony.

 Morphology - study of shape

Every colony has a specific set of characteristics:

Shape - circular, irregular, ounctiform (tiny)

Margin - entire(smooth), undulate/wavy, lobate, filamentous, rhizoid (branched)

Elevations - flat, raised, convex, pulvinate (very convex), umbonate (raised in the center)

Texture - moist, mucoid, dry

Pigment - opaque, translucent, shiny, dull

Stains are solutions consisting of a solvent and a dye.

Charged regions of the dye molecule that give color are termed chromophores.

Chromophores stain bacteria through bonds formed with the cell. Bacterial cell surfaces tend to have a - charge so a dye with a + chromophore will adhere to the cell.

Common stains - Methylene Blue, Crystal Violet, Safranin

Heat fixing has two important purposes: kills bacteria and coagulates cell proteins so they are more visible.

Cells are transparent and dyes make them visible.  We can then see cell morphology and arrangement.

Differential Stain - allows us to detect changes between organisms

2 stain process with de-colorization inbetween.
Primary stain - crystal violet

Iodine added as mordant.
Mordant enhances cv stain by forming cv-iodine complexes)

Decolor (alcohol wash).

Gram - will decolor.  Gram + - keep color.

Counterstain - safranin is applied and gram negative cells turn the color of this dye which is pink/red.

Gram - have more lipid content in cell wall.  Alcohol forms holes in wall releasing CV in decolorization.

Decolorization of gram + does not make them porous so stain remains in cell.

Decolorization is most imp process of the gram stain.

Poor technique and age can give false results.

Shapes of bacteria:

Bacillus - rod shaped

Coccus - spherical shaped

Spirillum - spiral shaped

Staphylo - cluster arrangement

Strepto - chain arrangement

Acid fast prganisms have mycolic acid in cell wall.

Mycolic acid is a waxy substance that prevents true decolorization of primary stain.

Two methods employed for acid fast bacteria - Ziehl-Neelsen and Kinyoun methods.
ZN uses heat as part of the process. K does not.

Primary stain in ZN method is Carbolfuschin (red).
This dye is lipid soluble and will penetrate waxy coating.  Steaming allows the dye to penetrate further.

Alcohol is used as a decolorizer in non-acid fast cells.  Acid fast cells will not decolorize.

After decolorizing, another stain is applied to those that lost the carbolfuschin stain.  Methylene blue is used.

Acid fast=red.  Non-acid fast=blue.

Capsules are composed of mucoid polysaccharides that repel most stains.
Capsule staining colors the background of the slide and leaves a "halo" around bacteria.

This area is hard to stain, so we stain everything else.

Congo red or nigrosin are commonly used to stain.

Endospore - a dormant form of bacterium.  Not all bacteria form endospores.

If a bacteria produces an endospore it means the conditions around it have deteriorated.

Spores resist heat as well as most chemicals.

Tough outer coating of a spore is made of keratin.

Scaheffer-Fulton Method - primary stain is Malachite green which is forced into spore by heating.

Counterstain is safranin.

Spores loacted according to the following: terminal, central, subterminal

Hanging Drop - wet mount that is inverted on a depression slide.
Used to view bacteria under low light and look for motility. 

Ring of petroleum jelly prevents evaporation of H2O and allows coverslip to adhere to depression slide.

Motility should not be confused with Brownian Motion.
Vibration or evaporation of H2O.

Ryu stain is used to see flagella on bacteria.  It allows you to see the various arrangements of the flagella.

Montrichous

Amphitrichous - both ends

Lophotrichous - tufts at both ends

Peritrichous - flagella all over

Gram Stain after Heat Fix

1-Cover smear with crystal violet stain for 1 minute.

2-Rinse with distilled H2O.

3-Cover Smear with iodine for 1 minute.

4-Rinse with distilled H2O.

5-Decolorize with alcohol until run-off is clear.

6-Rinse with distilled H2O.

7-Counterstain with safranin for 1 minute.

8-Rinse with distilled H2O.

9-Gently blot dry in biulous paper.

Observe under oil immersion.