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one bacterial cell that has DIVIDED into a group of hundreds of thousands of identical cells.
formula= (# of colonies)/(dilution factor)(volume plated)
existing or being everywhere
oxidize inorganic cmpds to yield energy and reduce CO2
3 parameters critical in obtaining sterility, and why
Temperature, Time, Pressure
-one of the only effective ways to kill spores
used for the sterilization of "dry" items AKA glassware/certain plastics
used to sterilization of liquids in order to reduce volume loss due to boil over
Uncontaminated Broken Glass
broken glass box at the back of the lab.
ex: heat-fixed slides!!!
pails on the discard cart at the back of the lab.
ex: petri plates
What characteristics are used to describe parameters?
form, elevation, margin, pigmentation, growth patterns, texture.
- ~Capsid/head with DNA inside
- ~tail fibers
Difference between Bacteriophage and Animal Virus
- -Bacteriophage requires a BACTERIA host and Animal requires a host that's a EUKARYOTIC CELL.
- -Bacteriophage contains DNA ;; Animal Virus contains RNA or DNA never both
- -Bacteriophage has a COMPLEX structure ;; Animal virus has SIMPLE structure
- -Bacteriophage lyse bacteria cells ;; Animal virus lyse euk. cells
Cycle for Bacteriophage
Attachment, Penetration, Synthesis, Assembly, Release
allow bacteriophage to attach to bacteria (set aside for 15 min.)
Why is soft agar used?
-Consistency is firm enough that bacteria can't move through it but FIRM enough to let bacteriophage move short distances so bacteriophage can infect neighboring bac. cells.
Parts of a microscope
- -ocular lens
- -objective lens(on nose piece)
- -stage clip
- -coarse focus
- -fine focus
- -lamp control
- -condenser (narrows beam of light that strike specimen)
- 4x: red (TOTAL magnification: 40x)
- 10x: yellow ("" 100x)
- 40x: blue ("" 400x)
- 100x: white ("" 1000x)
CANNOT see any characteristics under 100X! TOO SMALL.
Why do we use oil immersion?
Oil immersion allows light going up through the specimen to remain UNREFRACTIVE so it passes through the objective.
-WITHOUT OIL:: light-refracted away from objective and image=blurred
-under oil you'll see shape and pattern of bacteria easily and clearly visible.
more easily seen bc organism contains PHOTOSYNTHETIC pigments that give them a blue/green color.
Diatoms / Phytoplankton
over 100,000 species, cells incased in a cell wall made of silica.
-reproduce by budding
-bud scars: indicate site where new cell budded off
Yeast and Mold
- mold : looks fuzzy
- yeast : looks like bacterial colonies
-hyphae can be septate
(divided by cell walls) or nonseptate
- -primary mode of reproduce=ASEXUAL spore formation
-Zygospores=SEXUAL spore formation
-a DIFFERENTIAL stain : shows size, and identify characteristics to that bacterium
- 1. Fixation: let air-dry and heat-fix
- 2. Primary Stain: Crystal Violet
- 3. Mordant: Iodine
- 4. Decolorizer: Ethanol (dissolves lipids in outer membrane of gram(-) letting crystal violet & iodine escape from cell wall; but gram(+)'s thick layer of peptidoglycan retains purple color.)
- 5. Counterstain: Safranin (stains transparent bacteria PINK while gram(+) masks the pink and stays purple.)
Gram stain ERRORS
-using OLDER culture; cell walls lose integrity and can produce inaccurate results.
-OVER-decolorization: crystal violet/iodine complexes will be released from ALL cells making bacteria appear gram(-).
UNDER-decolorization: outer membrane will NOT be entirely dissolved so crystal violet/iodine complex will NOT be released from gram(-) bacteria.
Using thick bacterial smears can lead to uneven staining. Cell arrangements may be hard to distinguish.
- Coccus (Cocci)
- pairs and single
- Bacillus (rods)
- pairs and single
- flagellated Bacilli
Nocardia and Mycobacterium
these species have a unique
cell wall structure.
- Mycobacteria cell wall
- -mycolic acid (waxy lipid)
- -inner membrane
- Gram stain don't work on Nocardia and Mycobacteria bc...
- -dyes have difficulty penetrating the bacterium due to its waxy cell.
- -dyes that DO enter bacterium are difficult to move bc of bacterium's waxy cell.
Gram stain=inaccurate results!
-causes tuberculosis (TB) : has a waxy cell wall and is highly communicable.
- -1/3 of world's pop is infected with TB
- -almost 2-mill TB-related deaths each yr
- -TB leading killer of HIV infected individuals
- -in 2008, 4.2 cases/100,000 people in the US
-Acid-fast NON-pathogenic mycobacterium & nocardia species exist in the env.
- Specialized Stain
- -carbolfuchsin: a phenolic-based primary stain that is LIPID SOLUBLE (ex. it can more easily penetrate through the waxy cell wall)
2 ways: ZIEHL-NEELSON METHOD/KINYOUN METHOD
- Ziehl-Neelson Method
- -apply HEAT in the form of steam to drive the stain in for 5 minutes (all bacteria appear PINKISH/RED)
- -DECOLORIZE with ethanol; releases the carbolfuchsin stain from all bacteria except those with waxy cell walls
- -COUNTERSTAIN with methylene blue//brilliant green. Dye taken up by nonacid-fast cells but waxy cell wall does NOT allow dye to enter acid-fast bacteria.
- Kinyoun Method
- -bacteria stained with carbolfuchsin that contains a higher concentration of PHENOL (lipid soluble). The higher concentration of phenol in stain allows dye to penetrate into ALL bacteria (all bacteria appear PINKISH/RED).
- -DECOLORIZE with ethanol, extremely effective in releasing the carbolfuchsin stain from all bacteria EXCEPT those with a waxy cell wall.
- -COUNTERSTAIN with methylene blue/brilliant green. Dye taken up by nonacid-fast cells but waxy cell wall does NOT allow dye to enter acid-fast bacteria.
REMEMBER: only bacteria with WAXY CELL WALL will appear pinkish
after acid-fast staining.
- Flagella staining can provide info about a bacterium's flagella arrangement.
Motility media helps determine if a bacterium is motile.
- Motile Media:
- -semi-solid media has enough agar added to produce a media with consistency of "sloppy-jello" (4x less agar than media used for agar)
- -this media is THICK enough to allow motile bacteria to move thru BUT its too thick for non-motile bacteria to disperse even as they replicate.
- -inoculate needle the contains bacteria added ONCE STRAIGHT into middle of motility media 3/4 down and pull straight out.
- -motility can be seen as DIFFUSE growth radiating from stab line.
- -TETRAZOLIUM SALT (TTC) makes motility easy to read (red)
- (-)NEGATIVE: nonmotile growth only along stab line
- (+)POSITIVE: motile growth spread out from stab line (diffuse red color)
-must be careful not to jiggle, may give appearance of motility=FALSE POSITIVE
TTC (tetrazolium salt)
-when oxidized its colorless and soluble
-when reduced by metabolically active bacteria its red and INsoluble
-is an e- acceptor thats used to detect cellular respiration
-motility media TTC indicates location of metabolically ACTIVE/growing bacteria
Bacterial Motility: Flagella
-flagella provides a bacterium with the means to move towards more favorable conditions (ex: an area with more nutrients)
-NOT ALL bacteria have flagella so whether a bacterium is motile or not can help in identification.
-flagella may also contribute to the virulence of a bacterium since it can allow the bacterium to move to its preferred anatomical site to start the disease process
Types of Flagella
- Monotrichous: 1 flagella at 1 pole
- Lophotrichous: several flagella at 1 pole
- Amphitrichous: 1 flagella at each pole
- Peritrichous flagella ALL over
-flagella is EXTREMELY thin so have to use MORDANT
that will bind/stick flagella to thicken it so it can be seen under oil.
- CONS of flagella staining
- -very delicate and can easily break off during staining procedure
- -mordant is NON-SPECIFIC so it'll bind onto anything aka dust/dirt on slide (ex: oil on yo finger tips)
-this is a differential stain bc not all bacterias have capsules.
-capsules:a complex layer of sugars and proteins that tightly
surrounds a bacterium
can a capsule be stained? --NO bc its water soluble so the stains will not ADHERE/STICK.
-this stain is a combo of negative
- -a capsule stain is NOT heat-fix so its still alive...gotta throw it in the AMPHYL/DISINFECTANT BUCKET!
Role of a capsule
1. protecting the bacterium against harsh env. conditions and dehydration
2. helps bacterium ADHERE to surface of an object (aka wall of catheter tube)
3.helps PREVENT PHAGOCYTOSIS of bacteria by macrophages.
-In pathogenic bacterial species a CAPSULE is considered a VIRULENCE FACTOR.
-non-pathogenic bacteria can also possess capsules!
Capsule Stain (PROTOCOL)
- Step1: (-) stain w/ Congo Red. (should show up clear/white bacteria on a REDDISH bg)
- -Congo Red is an acidic dye (carries (-) charge)-surface of a bacterium also carries (-) charge
- -llke charges REPEL.
- with (-) staining remember....
- -bacteria CAN be cocci or a rod
- -it can be gram (+)/(-)
- -it may or may NOT have a capsule
- -in a (-) stain only the BG will be stained not the bacteria!
- Step2: air dry slide
- NEVER HEAT-FIX
- -bc it could SHRINK a cell resulting in a halo around the bacterium that could be mistaken for a capsule (FALSE (+))
-heating could destroy capsule so that a bacterium w/ a capsule would not appear to have had one (FALSE (-))
- Step3: simple staining with MANEVAL'S stain
- -maneval's stain is a basic dye (carries (+) charge)
- -surface of bacterium carries (-) charge
- -opposites attract
- Simple staining with Maneval's stain
- -bacteria CAN be cocci or a rod
- -it can be gram (+)/(-)
- -in a simple stain ALL bacteria will be stained
- -a stain w/(+) charge can penetrate capsule to bind to bacteria; however,stain CANNOT bind to capsule itself.
-this is a differential stain; size,shape, and location of endospore w/in a bac. can help identify the bacterium. (6 diff. spots)
-Some bacterias can survive in inhospitable env. conditions (aka too cold/too hot/too dry/not enough nutrients)
- -HOW? bc they grow endospores. Endospores are INDESTRUCTIBLE.
- -They withstand extreme temps, disinfectants, desiccation and UV radiation.
- CHARACTERISTICS OF ENDOSPORES-contains bac. DNA but is metabolically inactive(DORMANT)
-consists of a very tough outer cell COAT
, a CORTEX
made-up of peptidoglycan and an inner CORE
that contains the bacterium's DNA.
- Under favorable conditions:
- -VEGETATIVE cells (the bac. normally used & grown in labs)
- Under UNfavorable conditions:
- -undergo sporulation = formation of endospore
- -several stages gone thru to produce and endospore within bacterial cytoplasm.
When conditions become favorable the endospore will germinate bck into a vegetative cell.
Endospore Stain (Protocol)
- 1. Stain with Malachite Green using steam
- 2. Decolorize with H2O; removes color off of bacteria but NOT the endospore
- 3. Counterstain with Safranin; making bacteria pink/red. did not use STEAM so endospore IS NOT STAINED.
- The age of the bacterial culture can influence the endospore formation.
- -24 hr Bacillus culture=endospore STILL inside bacteria so you see pink/red and green inside.
- -5 day Bacillus culture= no more cell, endospores only so you see all GREEN.
Poly Beta-hydroxybutyrate (PHB) Staining
-to overcome the constant flux in nutrient concentration some bacteria have the capacity to produce INCLUSION BODIES
-can range in diameter from 0.2 to 0.7 um
-PHB inclusion are containing w/in a unique membrane that's composed of a single lipid layer.
-when nutrients are high in concentrations some bacterias can store it in inclusion w/in the cytoplasm.
-when nutrients in env. is scarce inclusions can by used by bac. to sustain its growth (reserve deposits
-if a bacterium is in a nutrient rich
env its likely to contain more/larger inclusions
-in depleted nutrients env. there are more likely to be fewer/small inclusions
since bac. may have utilized some of the store macromolecules.
- Why produce inclusion bodies?
- 1. nutrient reservoirs
- 2.since macromolecules are concentrated in inclusion bodies rather than dispersed throughout cytoplasm the microbe avoids an increase in osmotic pressure.
NOT ALL bacteria produce inclusion bodies so their presence can help ident. certain bacterial species.
Examples of PHB
Carboxysomes: inclusions that contain enzyme ribulose 1,5-diphosphate carboxylase
Sulfur Granules: inclusion contain sulfur/sulfur-containing cmpds
Polysaccharides Granules: inclusions that typically contain glycogen and/or starch as a source of energy
Metachromatic Granules: inclusions contain volutin (inorganic phosphates), can be used for synthesis of ATP.
Magnetosomes: inclusions contain iron oxide, which can act like tiny magnets.
Lipid Inclusions: inclusions contain lipid polymer poly B-hydroxybutyrate (PHB), which is used as both carbon and energy sources.
PHB (poly B-hydroxybutyrate) Staining Protocol
1. heat-fix a sample, flood it w/ SUDAN BLACK B
stain for 5 min.
- SUDAN BLACK B....
- -a lipid soluble stain, DOES NOT carry a charge (aka neutral stain)
- -stain can dissolve into lipid components of inclusion; but it does NOT associate w/any cellular components since its not charged.
2. SUDAN BLK B is removed and sample decolorized with an organic solvent.
- (when bacteria are stained w/ SBB and decolorized, only the INCLUSIONS will stain blk and rest of bac. cell will be transparent.)
- 3. Counterstain with Safranin.