MIC 230 Lab Exam 2

Bacterial growth is affected by…

genetics (species), water and nutrient availability, carbon source, oxygen, temperature, pH, osmolarity, and competition

What are the direct measurement methods?

Cell count under a microscope, and viable plate count

What are the indirect measurement methods?

optical density (turbidity), dry weight, and molecular content

How is optical density measured?

spectrophotometer

What is the max reliable OD of a culture?

0\.5

What happens once the OD hits 0.5?

We must dilute the culture

Dilution =

volume culture / (volume of culture + volume of media)

Dilute 2 ml of culture into 4 ml of broth…

2/(2+4) = 1/3 dilution

Correct OD =

OD of diluted culture / dilution

If the OD of 1/3 diluted culture is .250 what is the correct OD?

.250 x 3 = .750

Metabolic reactions carried out by a cell are mediated by…

enzymes

Metabolic diversity is…

the major chritiera used to identify bacteria

Exoenzymes

Synthesized inside the cell and then secreted outside the cell where they catalyze reactions in the external environment

Why do some cells need exoenzymes?

Some substrates are too large to be brought into the cell so they exoenzymes work outside of the cell to produce a product which is small enough to be brought into the cell

What are hydrolytic reactions

reactions where exoenzymes degrade macromolecules by splitting chemical bonds between the polymeric subunits by adding water

Organisms that produce exoenzymes are able to use what as their nutrient source?

Macromolecules

What are hemolysins

exotoxins that are able to destroy RBCs

How can bacteria be differentiated on a sheep blood agar plate?

By their ability to hemolyze RBCs

Hemolysis that results in complete destruction of RBCs is called…

ß-hemolysis

Hemolysis that results in partial or incomplete lysis of RBCs is called…

a-hemolysis

No hemolysis is referred to as…

y-hemolysis

What is the inoculation method and time for starch agar, milk agar, and DNase test agar

Spot inoculation (48 hours)

Substrate and product for starch agar

starch and glucose

Substrate and product for milk agar

casein and small polypeptides

substrate and product for DNase test agar

DNA and nucleotides

Substrate and product for Gelitain deeps

gelatin and small polypeptides

Substrate and product for blood agar

RBC and hemolysin

Enzyme or extracellular protein for starch agar

amylase

Enzyme or extracellular protein for milk agar

caseinase

Enzyme or extracellular protein for DNase test agar

DNase

Enzyme or extracellular protein for gelatin deeps

gelatinase

Enzyme or extracellular protein for blood agar

hemolysis

Indicator/or observation method + results for starch agar

Add iodine and look around growth

\+ clearing

\- black

Indicator/or observation method + results for milk agar

Look for clearing

\+ zone of clearing

\- no clearing

Indicator/or observation method + results for DNase test agar

Methyl green

\+ zone of clearing (no white)

\- no clearing

Indicator/or observation method + results for gelatin deeps

After cooling, observe for liquefication

\+ any liquefication

\- no liquefication

Indicator/or observation method + results for blood agar

ß = growth is completely clear

a = around growth is green

y = no change

What are two medias used in sugar fermentation tests

Phenol red sugar broth and MacConkey agar

Inoculation method and time for phenol red sugar broth

loop and 48 hours

Inoculation method and time for MacConkey agar

streak and 24-48 hours

Substrate and product for phenol red sugar broth

Lactose, glucose, sucrose, or mannitol

Acid (sometimes gas too)

What is MacConkey selective for

gram negative

What is MacConkey differential for

lactose fermentation

Indicator and/or observation method for phenol red sugar broth

Change in color, phenol red = pH indicator

A = acid (yellow)

AG = acid and gas (yellow + gas in vial_

NC = no change

Indicator and/or observation method for MacConkey agar

PH indicator = neutral red

No growth = gram +

growth = gram -

pink colonies = lactose fermented

white colonies = no fermented

Triple sugar iron agar is used to determine what?

If sugar fermentation has occurred and if cysteine has been degraded

Inoculation method and incubation for TSI slant

Stab and streak and 18-24 hours

Substrate and products for TSI agar

Substrates: glucose, lactose, and sucrose (cycteine)

Product: acid (H2S gas)

Enzyme or process for TSI slant (glucose, lactose, and sucrose)

Fermentation

Enzyme or process for TSI slant (cysteine)

Degradation

Indicator and/or observation method for TSI agar slant (glucose, lactose, and sucrose)

Phenol red indicator and cracks/bubbles

Acid = yellow (A)

alkaline (no acid) = red (K)

gas = bubbles

What are the three options for TSI agar results

K/K

K/A

A/A

indicator and/or observation method for TSI agar slant (cysteine)

Ferric iron indicator

Color change (black = H2S)

Amino acid degradation is the ability to

degrade certain of the amino acids

Deamination reactions result in

the release of ammonia

Decarboxylation reactions result in

release of CO2

Lysine decarboxylase broth is used to show

decarboxylase activity

Phenylalaine malonate broth is used to

test for phenylalaine deaminase activity

Inoculation method for lysine decarboxylase and phenylalanine malonate

loop

Incubation time for lysine decarboxylase

1-4 days

Incubation time for phenylalanine malonate broth and urea broth

24-48 hours

Substrate and product for lysine decarboxylase

Lysine → CO2 (cadaverine)

Substrate and product for phenylalanine malonate

Malonate → CO2

Phenylalanine → phenylpyruvic ammonia (NH3)

Enzymes capable of using urea form

ammonia and CO2

Urea broth inoculation method

needle

Substrate and product for urea broth

nitrogen → ammonia

Enzyme or process for urea broth

urease

Indicator and/or observation method for lysine decarboxylase

Bromocresol purple added as pH media

\+ purple (cadaverine raises pH)

\- yellow

Indicator and/or observation method for phenylalanine malonate (malonate part)

Color change indicates pH change

\+ blue (uses malonate)

\- green

Indicator and/or observation method for phenylalanine malonate (phenylalanine part)

add FeCl3 to the reagent

\+ green precipitate

\- yellow precipitate

Indicator and/or observation method for urea broth

Phenol red is added as a pH indicator

\+ pink (ammonia present = increase pH)

\- no change

Indole tests examines the ability of bacteria to

produce indole from the amino acid tryptophan

Inoculation method for tryptone broth and MRVP broth

loop

Incubation for tryptone broth MRVP, and Simmons citrate

48 hours

Inoculation for Simmons citrate

stab and streak

Substrate and product for tryptone broth

Tryptone → indole

Enzyme or process for tryptone broth

tryptophanase

Indicator and/or observation method for tryptone broth

kovac’s reagent - look for layer on top

\+ pink layer

\- yellow/brown layer

Substrate and product for MR of the MRVP broth

glucose → acids

Substrate and product for VP of the MRVP broth

glucose → alcohols

Enzyme or process for MRVP

fermentation

Indicator and/or observation method for MR

Methyl red

\+ red

\- yellow

Indicator and/or observation method for VP

Testing for intermediate, add KOH and an a-naphthol, mix and wait 20 min

\+ red

\- yellow

Substrate and product for Simmons citrate

Citrate → citrate utilization

Enzyme or process for Simmons citrate

citrate permease

Indicator and/or observation method for Simmons citrate

can bacteria bring citrate into cell and use it

\+ blue

\- green

Inoculation method and incubation time for oxidase and catalase

plate and 24-48 hours

Substrate and product for oxidase test

cytochromes → O2 + H2O

Substrate and product for catalase test

Hydrogen peroxide → O2 + H2O

Enzyme for oxidase reaction

cytochrome oxidase

Enzyme for catalase reaction

catalse

Indicator and/or observation method for oxidase

Oxidase reagent

\+ purple - blue

\- colorless

Indicator and/or observation method for catalase

hydrogen peroxide

\+ bubble

\- no bubble

Nitrate broth inoculation method and incubation time

1 loopful and 48-96 hours

Substrate and produce for nitrate broth

Nitrate → nitrite or N2 gass

Process of nitrate broth

reduction

Indicator and/or observation method for nitrate broth (1)

Add Nitrate reagent A and B

\+ red (nitrate was reduced to nitrite)

\- colorless

Indicator and/or observation method for nitrate broth (2)

Add Zn dust

\+ no color (nitrate got all the way to N2 gas)

\- red

What is selection?

conditions that only cells with “desired” phenotype grow

Frequency

how often events occur among all potential chances