Micro Lab 2

• simple fats 
• glycerol and three fatty acid chains

fat derivatives in which one fatty acid has been replaced by a phosphate group and one of several nitrogen-containing molecules.

enzymes that hydrolyze lipids

lipolysis (lipid hydrolysis)

rancidity testing determines the level of oxidation in a sample.

• sim medium used to determine 3 bacterial activities:
• 1. Sulfur reduction
• 2. Indole production from tryptophan,
• 3. Motility

• 1.anaerobic fermentation where the products are a complex mixture of acids, particularly lactate, acetate, succinate and formate as well as ethanol and equal amounts of H2 and CO2. It is characteristic for members of the Enterobacteriaceae family.
• 2. MR test detects organisms capable of this

family of extracellular enzymes produced and secreted  by some microorganisms to hydrolyze gelatin

turns grey , black , or blue

agar would be broken, cracked or pushed up in tube

complex strand, branches off at multiple spots

break down large molecules in presence of water

break down big starch molecules

• distinguishes bacteria 
• sum of total reactions in bodies

breaking down large molecules due to release in energy

energy producing biochemical reactions usin electron donors from organic compounds

gentler indicator, phenol red, used for living cells, becomes bright yellow

traps any gas produced, can tell whether or not its fermenting

consume energy by building large molecules

• 1. put 3 different sugars into each tube with a bacteria
• 2. Definition: hydrolysis of disaccharides prior to the fermentation reaction
• 3. Goal: specific byproducts from different hydrolysis reactions indicates type of bacteria

• 1. tests to see if B-galactosidase is produced by bacteria, which in turn indicates fermentation of lactose
• 2. Definition: B-galactosidase is an enzyme that is produced to ferment lactose
• 3. Test Result: 
• a. use ONPG disc as lactose source, place in bacteria sample which goes in 37 C room.
• b. will turn yellow due to a byproduct

• 1. Purpose: to differentiate bacteria based on glucose, lactose or sucrose fermentation, or sulfur reduction
• 2. whole tube will turn yellow if bacteria uses multiple sugars
• 3. only yellow butt =glucose fermentation but not other sugars
• 4. yellow = acidic 
• 5. pink = alkalinic , or did not use a sugar but peptone and amino acids to alkalinize medium 
• 6. H2S production: turns medium black, grey or blue
• 7. gas production: agar would be broken, cracked, or pushed up in tube

• 1. tests if bacteria has enzyme alpha amylase, which breaks down big starch molecules
• 2. test itself:
• a. put straight line of bacteria on a starch agar plate.
• b. sat in 37 C room for at least 24 hours
• c. after 24 hours, apply iodine onto starch agar.
• d.positive for glucose production, of alpha amylase present is the "halo effect"

• P.A.: w/ oil, green color; w/out oil, yellow color
• a. means it is an obligate aerobe (needs O2)
• E.coli: both w/ and w/out were yellow (doesn't need O2)

• 1. Purpose: test for lactose fermentation, reduction of litmus, casein coagulation and casein hydrolysis
• 2. Result: lavendar to white = lactose fermentation

• Purpose: to test for lipase productivity, an enzyme that hydrolyzes lipids
• S. Aureus: colonies stay dark, halo of light indicates lipase activity (+ test)
• Proteus mirabilis: colonies spread

• Indole
• Methyl Red Test
• Vogues-Proskauer Test
• Citrate utilization (Simmons Citrate)

• Purpose: to see if organism can convert tryptophan, an amino acid, into indole
• tryptophanase: an enzyme, breaks down tryptophan to make indole and pyruvic acid
• Positive test: use indole reagent, pink ring = indole production

• Purpose: testing for mixed acid fermentation
• Positive: pink/red color

• Purpose: test for 2,3 butanadiol fermentation
• since an alcohol, can't use an indicator
• use methyl red and  acetoin instead (the product from fermentation)
• Positive: pink red color due to acetoin production

• Purpose: test to see if bacteria can use citrate as its only C source, end product is alkyl
• Positive: if medium turns blue, positive for the products (pyruvic acid, CO2, and oxaloacetic acid)
• ex/ that is positive: Salmonella typhimurium
• tip: make sure lid is loose to allow CO2 to escape

• 1. Positive for motility and indole production: dark color
• ex/ P.V. is motile
• PM: grew everywhere like clouds
• KO: single stabline w/ bugs

• Purpose: looking for bacteria that can produce gelatinase
• Gelatinase: enzyme that breaks down gelatin
• gelatin: protein fibers w/ matrix of water inside
• Positive: if gelatin is liqueified, flowing freer than normal, proteus vulgaris is positive
• TIP: will get FALSE + in 37 C
• takes 10 days - 2 weeks

• Purpose: to test for catalase present
• Positive: if H2O2 put on bacteria and bubbles, sign for the products water and free oxygen 
• (means protection from red blood cells)
• S. aureus is Positive!

• Purpose: identify bacteria w/ enzyme cytochrome c oxidase
• cytochrome oxidase = found in electron transport chain
• Positive: happens rapidly, will turn blue due to production of oxidized c cytochrome

• REAction: ammonia + phenol red + urease    --> ammonia, water and CO2
• Purpose: to detect urease 
• Positive: deep pink color from ammonia and phenol red
• 24 hr test

• 1. tests bacteria's ability to make enzyme deaminase
• 2. phenyl alanine + phenylalanine deaminase --> phenylpyruvic acid + ammonium ion + H2O
• 3. phenylpyruvic acid + ferric Cl -> green complex
• 4. Positive test: this green color NOT YELLOW-GREEN

• Purpose: to test the ability that a bacteria can reduce nitrate (NO3) to nitrite (NO2) using the enzyme nitrate reductase. 
• 2. if  red, positive for nitrate reduction

• 1. put on square
• 2. anitibody solution, 1 drop. 
• 3. when antibodies are matched with their antigen 
• 4. positive when beads form ( or clumping)

• (the too expensive test)
• 2. put known antibodies around unknown antibody. 
• 3. if match, makes division sign

round, spherical

straight rod

curved rods

very curvy rods

long and slender bacteria

looks like thin crackers

found in water

found in water

variable, indistinct

pairs

irregular clusters

in chains

groups of 4

3D

parallel

thin walls, red or pink

thick walls, blue or violet

adjustment period, no cell division initially

growth rate takes off, exponential increase

reproduction rate is equal to the death rate

decline of organisms, more are dying than surviving- cannot do a viable plate count because you cannot tell which cells are living and which are dead

Time it takes the population to double

The number of generations produced per hour

• 1) Make your own blank- using distilled water- in a cuvetteKeep this refrigerated between uses
• 2) Fill another cuvette ¾ full of the broth (this is your bug)
• 3) Run the % trans and record result Keep this in the shaker bath between uses
• 4) Do your serial dilutions and plate to find the countable CFU
• 5) Repeat every hour, on the hour

the temp the bug will best grow at- classifications are made using this

“cold lovers” live at 0-11 degrees C

“middle lovers” live at 12-44 degrees C

“heat lovers” live at 45 degrees and up

live way above, or way below the limits

don’t like high temps, but they can endure them

grow best at pH between 0-5.5

- 5.6-8.5

- 8.6-14

resist change in pH

pressure created when water goes from high to low concentration (usuallythrough a membrane)

dissolves

amount of water available for metabolism

there is more water outside of the cell than there is inside

more water inside of the cell than there is outside

water moves from inside the cell to the outside of the cell so quickly it pulls awayand kills the cell

having the same amount of water both inside and outside of cell – same osmotic pressure

like high salt concentrations

like high sugar concentrations

how bacteria survive extreme environments

love low water concentrations

require oxygen to live

grow better when oxygen is present but don’t need it to survive

presence of oxygen will kill them

don’t require oxygen to live- presence of oxygen doesn’t help or inhibit

require a very small amount of oxygen to live

media that lacks oxygen

wisk away oxygen

limitations of UV light

use energy from energy from visable light to repair what UV light killed

use chemical reactions to repair damage from UV light

can be used on nonliving surfaces

gentle enough to be used on living surface

kills bugs

inhibits further growth but doesn’t kill the living

• relative effectiveness
• <1 doesn’t kill as well as phenol
• >1 kills better than phenol

• 1) Add 0.1 ml bug to sterile saline
• 2) Make a 0.1 ml spread plate
• 3) Add 0.1 ml of selected disinfectant- This is the mix tube
• 4) Add 0.1 ml to TBA at 0 min, 1 min, 5 min, and 10 min- after each TBA tube is made spread plate immediately!!
• 5) Incubate at 37 degrees C

relative effectiveness of antibiotic- but doesn’t tell you how much youneed

if there is one the antibiotic is effective

what’s the lowest amount of the antibiotic that will do thejob- easy experiment- serial dilutions will tell you where to start your prescription