Micro Lab Pratical

PPE

• Lab coat

• Gloves 

• Glasses

• Proper clothes 

• Closed toe shoes

Aseptic Technique

• Sterilizing loop between uses, not cross contaminating

• Negative control should have no grow

• Positive control should have growth

Magnification:

An increase in the apparent size of an image to resolve smaller separations between objects

Resolution:

the smallest distance by which two objects can be separated and still be distinguished

Contrast:

the difference in luminance (lightness/darkness) or color that makes an object, text, or element distinguishable from its surroundings

Coarse focus:

moves the entire platform, up and down to compensate for the different focal lengths of the lenses

Fine focus:

sharpens image focus, AFTER use of coarse focus

Phase contrast

utilized to produce high-contrast images of transparent/colorless specimens, such as living cells (usually in culture), microorganisms, thin tissue slides, etc.

Oil immersion:

 used to enhance the resolution of the specimen observed. The increased refractive index of the oil increases the numerical aperture of the microscope giving a better image. Used only for high magnification (100x)

• Wet mount:

• live cells, unstained, just a simple observation of a liquid under the microscope. Uses coverslips (10x and 40x magnification and can use phase contrast)

Basic dyes:

Crystal violet and Safranin are positively charged (cataionic) dyes in solution which bind to negatively charged cell components like the peptidoglycan in cell membranes

Synthetic media:

made of raw materials yeast extract from brewing industry, and milk protein (whey protein) from cheese making

• Forms of media:

• Solid, liquid, semi-soil, broth, plates, slant, deep

• Enrichment media:

complex media to which specific components are added

• Selective media:

favors growth of one organism over another

• Differential media:

exploits differences between two species that grow equally well

• 3 phase streaking:

• streak plate to isolate single colonies of bacteria (reduce density), sterilize loop, dip in culture, streak in quad 1, sterilize loop, streak from quad 1 into quad 2, sterilize loop, streak from quad 2 into quad 3 (can do 3 or 4 quads)

• Pasteurization in water bath:

• hot water bath to kill the bacteria

• Endospores are formed due to:

• starvation and extreme environmental stressors in gram positive bacteria

•  Endospore staining:

• mix a colony and water on a slide, heat the fix the slide to open up the spore to allow for staining, over steam flood plate with malachi green every 2 minutes for 5-7 minutes, rinse with water, counter stain with Safranin (30 sec) (10 and 100x magnification)

• Motility:

• not random , but directional movement of a cell (often slow) 

• Brownian motion:

• this random and erratic movement of cells in the droplet fluid caused by bombardment with other particles 

Water movement:

bacterial cells moving in the drop all at once and in the same direction

Drop slide:

drop culture on coverslip, apply vaseline to corners, flip coverslip onto drop slide (10x and 40x and phase contrast)

Azotobacter

Mobile, gram negative, nitrogen-fixing bacteria that live freely in soil

water quality testing parameters

one of the parameterds to assess water quality is testing total coliform bacteria

EMB agar

methylene blue is dye added to the agar that inhibits the growth of gram (+) bacteria, thereby selecting for only gram (-) bacteria

eosin responds to changes in pH changing from colorless to black under acidic conditions

also contains lactose and sucrose, as energy sources

water filter experiment steps

assemble vacuum filter, insert filter membrane, pour water sample, turn on vacuum, transfer filter paper onto agar make sure bacteria us facing upwards use both EMB and NA agar plates

fungi- mycelium

mycelium are formed by complicated networks of hyphae, hair like fibers

fungi spore baring structures

reproduction of fungi is facilitated by spores

sporangiospores- (asexual) produced inside a sporangium from a zygospore

Ascospores- (sexual) produced in sac-like structures. ascus can be surrounded by a protective cover (ascocarp)

Basidiospores- (sexual) produced on the surface of a basidium. mushrooms have a protective cover (basidiocarp) that hand basidia from the gills

Fungi: Sporangium

disperse spores that germinate and form their own mycelium

Fungi: Conidia

produced on conidiophores and release conidia to allow for asexual reproduction

Phage experiment protocol

4 test tubes, add 990 microliters of TS broth to test tubes 1 and 2, add 900 microliters of TS broth into tube 3 and 4

add 10 microliters phage stock into tube 1, mix then serial dilute 1 into 2, 2 inot 3, 3 into 4 (10^(-6))

pipette 100 microliters of E. coli into top agar tube 1 then pipette 900 microliters from tube 2 into top agar too, vortex

pour mixture onto agar plate, repeat for tubes 3 and 4

bacteriophage

a virus that infects bacteria, forms a plaque of lysed cells on a lawn of bacteria

bacterial growth curve stages

lag phase, log phase, stationary phase, death phase

optical density (OD) and spectrophotometer

the spectrophotometer will measure the intensity of light different wavelengths

higher OD= more light scattering, low OD= less light scattering

growth curve procedure

take 1 mL of sample into cuvette take spectra

3 sets of dilutions (1 each hour), set up 900 mL of sterile stock in 7 tubes

take 100 mL from cuvette into tube 1 containing 900 mL sterile broth, do again for all 7 tubes, plate 0.1 mL from tubes 4, 5, 6 on agar for the first plating use triangle to spread

repeat every hour plat tubes 5, 6, 7 for next two platings

sample titer

= # of plaques/ (cumulative dilution x phage volume plated)

doubling time

first calculate growth rate: =(ln (OD2)- ln (OD1))/(t2-t1)

then calculate doubling time td= ln(2)/growth rate

effects of different antibiotics on bacteria

CAM= prevents ribosomes synthesis cells are still intact, growth is inhibited OD remains the same

AMP= targets the cell wall cells are lysed OD drops

brief steps in the epidemiology experiment

cover glove in culture, shake hands with 2 people , rub saline on glove then plate q-tip, then shake 2 more hand then swab and plate again (NA+CAM plate)

Handwashing experiment brief steps

rinse hands in pre-scrub bowl, wash hands with antibiotic soap, erinse hands in post scrub bowl, plate 100 microliters of pre scrub water onto NA plate then again on EMB plate, then same for post scrub water

Azotobacter BLAST steps

we amplified conserved region of 16s rRNA ~1500 bp,

Mueller-Hinton plate

loose agar- easily diffuses antibiotics into agar

rich media- contain vitamins not found in NA

more defined than NA to ensure our conditions are controlled

Zone of inhibition

the area around the antibiotic disc that bacteria growth is inhibited in

can be effected by: the concentration of the antibiotic disc, the bacteria and/or strain plated - bacterial resistance, time allowed for the antibiotic to diffuse on the plate, temperature bacteria are grown at, rate bacteria are grown at

the larger the zone the more sensitive the bacteria is to that antibiotic

Antibiotics steps (Kirby-baurer test)

plate 100 microliters of stock culture on MH plate to create a lawn of bacteria, drop 3 antibiotic discs from the set on 1 plate

Enteric ID protocol

grab a colony from streak plate combine with 2 mL sterile water, transfer into panel rock forward to mix mixture with dried medias

after a couple hours add one drop of reagent into 3 wells

what makes soap antibacterial

chloroxylenol (softcide- which kills fungi, gram positive bacteria, and some viruses), benalkonium chloride (pink antibacterial soap), and/or benzethonium chloride