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Micro Practical 2


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what are the subunits that make up a protein?
amino acids
what do amino acids consist of?
carbon, hydrogen, oxygen, nitrogen and sometimes sulfur
how do amino acids bond together and what do these bonds form?
bond by peptide bonds

they form either a small chain called a peptide or a larger molecule called a polypeptide

what can bacteria do to peptides or polypeptides?
it can hydrolyze them to release amino acids
what do bacteria use the amino acids from peptides or polypeptides for?
they used them as carbon and energy sources when carbohydrates are not available
where are amino acids primarily used?
anabolic reaction
how are large protein molecules, such as gelatin hydrolyzed?
by exoenzymes
what happens to the smaller products of hydrolysis
they are transported into the cell
how can hydrolysis of gelatin be demonstrated?
by growing bacteria in nutrient gelatin
when does nutrient gelatin dissolve?
in warm water 50 degrees Celsius
what happens when an exoenzyme hydrolyzes gelatin?
it liquefies and does not solidify even when cooled below 20 degrees Celsius
a waste product of protein digestion in most vertebrates and is excreted in the urine
what does the presence of the enzyme urease help diagnose?
identify bacteria in urea
what does the enzyme urease do?
liberates ammonia from urea
what is in urea agar?
phenol red

what is the pH of urea agar?
what color is urea agar? why?
yellow because the pH turns the phenol red this color
during incubation, what happens to urea agar if there is bacteria possessing urease?
the urea possessing urease will produce ammonia which raises the pH of the medium, turning the indicator fuchsia or hot pink at a pH of 8.4
how is gelatin hydrolysis test conducted? how is hydrolysis indicated?
1. inoculate a tube of nutrient gelatin with a bacteria
2. incubate the tube at room temperature.
3. if the gelatin liquefied place it in a beaker of ice for a few minutes .

hydrolysis of the gelatin can be indicated by growth of the bacteria.
if it stayed liquefied even after the ice then hydrolysis occurred and it was a positive hydrolysis test

how is a urea test conducted? what are the possible results?
1.a urea agar slant is inoculated with a bacteria
2. it is incubated for 24-48 hrs

it is positive for the presence of urease is if the agar turns red and negative for urease if there is no color change and the agar remains yellow.

what color is phenol red at pH of 6.8 or below?
what color is phenol red at pH of 8.4 or above?
what were the results for psudomonas aeruginosa in the hydrolysis test?
it grew and was a positive hydrolysis test. the gelatin remained a liquid.
what were the results for the proteus vulgaris in the hydrolysis test?
there was no visible growth of the bacteria. negative hydrolysis
what were the results for pseudomonas aeruginosa in the urea agar test?
the agar liquefied. there was no growth. negative for urease / hydrolysis
what were the results for Proteus vulgaris in the urea agar test?
the agar liquefied and turned bright pink. positive urea / hydrolysis test.
what tests are part of protein catabolism part I?
urease test
gelatin hydrolysis test
what tests are part of protein catabolism part 2?
indole test
phenylalanin deaminase
hydrogen sulfide production

what happens once amino acids are taken into a bacterial cell?
various metabolic processes can occur using endoenzymes
what must happen before an amino acid can be used as a carbon and energy source?
the amino group must be removed
the removal of an amino group
how does deamination take place?
the amino group is converted to ammonia, which can be excreted from the cell
what results from deamination?
the formation of an organic acid
how is deamination of the amino acid phenyalalanine detected?
by forming a colored ferric ion complex with the resulting acid
the removal of carbon dioxide from an amino acid
what results from the presence of a specific decarboxylase enzyme?
the breakdown of the amino acid with the formation of the corresponding amine, liberation of carbon dioxide and a shift in pH to alkaline.
what media is used for decarboxylase reactions?
nutrient broth
a pH indicator
the desired amino acid

some names given to some amines such as putrescine or cadaverine indicate what?
how foul-smelling they are
which sulfur- containing amino acids do some bacteria liberate hydrogen sulfide from?

why is H2S commonly called rotten egg gas?
because of the copious amounts of hydrogen sulfide liberated when eggs decompose
how is H2S production detected?
a heavy- metal salt containing ferrous ion is added to a nutrient culture medium.

when H2S is produced the sulfide reacts with the metal salt to produce a visible black precipitate

what is the indole test used for?
the detection of bacteria to convert the amino acid tryptophan to indole or a blue compund called indigo
how is the indole test perfomred?
by inoculating a bacterium into tryptone broth and detecting indole by the addition of dimethylaminobenzaldehyde (Kovacs reagent)
what is motility indole ornithine agar used for -MIO?
test motility, indole production and ornithine decarboxylase activity
how is the Phenylalanine Deamination test conducted? what do the results mean?
1. a phenylalanine slant is heavily streaked with a bactium

2. it is incubated

3. observe for growth

4. add 4-5 drops of ferric chloride reagent to the top of the slant, allowing it to run through the growth on the slant

a positive test gives a dark green color

how is a hydrogen sulfide production test conducted? what do the results mean?
1. stab on peptone iron deep with a bacteria

2. incubate

3. observe for the presence of growth

blackening in the butt of the tube indicates a positive test

how is a MIO test conducted and what do the results mean?
1. stab and MIO deep with bacteria

2. incubate

3. compare the inoculated tube with an uninoculated tube

4. oberve for the presence of growth

motility is demonstrated by growth diffusing out from the stab inoculation line or by clouding of the medium

how is the ornithine decarboxylation reaction indicated? what about if it is negative?
by a purple color
and yellow it if is negative

when Kovacs reagent is mixed to the MIO what color indicates a positive test?
cherry red
electron acceptors
molecules that combine with electrons liberated during metabolic processes
when do electron acceptors become reduced?
when they gain electrons
how are electrons formed?
from the ionization of a hydrogen atom
what act as electron acceptors in fermentative metabolism?
organic molecules
what serve as electron acceptors in oxidative metabolism or respiration?
inorganic molecules
what is the electron acceptor in aerobic respiration?
molecular oxygen
how is the nitrate reduction test conducted? what do the results mean?
1. inoculate tubes of nitrate broth with bacterium
how is the nitrate reduction test conducted?
1. inoculate tubes of nitrate broth with bacterium
how is an oxidase test conducted and what do the results mean?
1. divide a petri plate containing trypticase soy agar in half streaking Escherichia on one side and Pseudomonas on the other

2. incubate the plate

3. to test for cytochrome oxidase drop oxidase reagent on the colonies and observe for a color change from pink within 1 minute then blue to blacl

oxidase-negative colonies will not change color

how is the catalase test conducted and what do the results mean?
1. divide a soy agar plate in half, streaking one half with Lactococcus and the other with a spot of Bacillus

2. incubate for 24 hrs.

3. to test for catalase drop hydrogen peroxide on the colonies and observe for bubbles

if bubbles appear - catalse positive, if not then negative

obligate aerobic bacteria
require oxygen for growth
anaerobic bacteria
no not use oxygen for growth
why cant abligate anaerobes tolerate the presence of oxygen?
they lack catalse, and the resultant accumulation of hydrogen peroxide is lethal
aerotolerant anaerobes
cannot use oxygen but tolerate it fairly well
grow best in an atmosphere with increased carbon dioxide and lower concentrations of oxygen
how are microaerophiles cultured on petri plates and nonreducing media?
they are placed in a candle jar with a lighted candle. after the lid is placed on the jar, the candle will extinguish when the concentration of oxygen decreases and the concentration of carbon dioxide will thus be raised.
faculative anaerobes
bacteria capable of living with or without oxygen
what five genera of bacteria lack catalase? what is their classifications?

clostridium are obligate anaerobes but the other four are aerotolerant anaerobes and do not need catalase

how are the tests of Aerobic and anaerobic bacterium conducted?
1. inoculate 4 tubes of thiglycollate broth with Clostridum, Enterococcus, Alcaligenes and Escherichia

2. incubate

3. record the appearance of growth

4. divide 2 nutrient agar plates in four sections and streak each of the bacteria on both plates

5. incubate the aerobic plate and place the anaerobic plate in the brewer jar then incubate the jar

6. record growth on both plates

7. perform the catalase test on the different colonies

how was the staphlococci lab conducted?
1. streak 3 nutrient agar plates with with a different organism using isolation technique

2. place staph aureus on a mannitol salt agar by itself in a straight line streak. split another plate and straight line streak one side with staph epidermidis and the other staph saprophyticus

3. inoculate a DNase plate with S. aureus in a straight line. streak s. epidermidis and s. saprophyticus on another dish in sraight lines

4. using isolation tech. streak the organisms on their own blood agar plates. using sterile forceps place a novobiocin disc in the area of heaviest inoculation

5. add two loops of each oranism to their own coagulate tubes.

6. incubate all the media

what were the results of the staph lab on the nutrient agar plates?
all were round, raised, white clusters
what were the results of the catalase reaction in the staph. lab?
staph. aureus- positive

staph. epidermidis -positive

staph. saprophyticus - positive

what were the results of the manital salt agar in the staph lab?
staph. aureus - area around streak was yellow - positive

staph epidermidis - area still red - negative

staph. saprophyticus - area yellow - positive

what were the results of the DNASE test reaction in the staph lab?
s. aureus - clear zone - positive

s. epidermidis - still green - negative

s. saprophyticus - still green - negative

what were the results of the blood agar plates / hemolysis in the staph lab?
s. aureus - clear zone - beta hemolysis - blood cells lysed

s. epidermidis - green zone - alpha hemolysis - partial lysis

s. saprophyticus - no change - gamma reaction

what were the results of the novobiocin reaction in the staph lab?
s. aureus - clearing zone - sensitive

s. epidermidis - clearing zone - sensitive

s. saprophyticus - growth next to disc - resistant

what were the results of the coagulase test in the staph lab?
s. aureus - clump - positive

s. epidermidis - free flow- negative

s. saprphyticus - free flow - negative

how was the streptococci lab conducted?
1. streak all three organisms for isolation on tryptic soy agar plates

2. streak organisms on blood agar plate then with sterile forceps place an SXT disc onto area of heaviest inoculum

3. streak organisms into Bile Esculin media tubes (BEM) using straight line streak

4. inoculate one 6. 5% NaCl broth test tube and nutrient broth (control) with two loops of organisms.

5. inoculate sodium hippurate broth tubes with organisms

6. incubate

what were the colony characteristics on TSA plates in strep. lab?
enterococcus faecalis - small circular

strept. agalactiae - circular

stret. equi - circular raised

what were the results of the catalse reaction in the strep lab?
e. faecalis - negative

strept. agalactiae- negative

strept equi - positive

what were the results of the hemolysis on the blood agar plates in the strep lab?
enterococcus faecalis - alpha hemolysis

strept agalactiae - beta hemolysis

strept. equi - alpha hemolysis

what were the results to the reaction of the SXT discs in the strept. lab?
enterococcus faecalis - resistant

strept. agalactiae - resistant

strept. equi- resistant

what were the reactions to the BEM plates in the strept. lab?
enterococcus faecalis - blackening of agar - positive

strept. agalactiae - blackening of agar - positive

strept. equi - blackening of agar - positive

what were the results of the 6.5% NaCl broth in the strept lab?
enterococcus faecalis - cloudy - positive

strept agalactiae - no growth - negative

strept equi - cloudy - positive

what were the results of the hippurate hydrolysis reaction in the strep lab ?
enterococcus faecalis - ninhydrin reagent didnt change tube - negative

strept. agalactiae - ninhydrin reagent didnt change tube - negative

strept. equi - ninhydrin reagent didnt change tube - negative

what were the results of the demo plate camp tests in the strep lab?
strept agalactia - arrow head shaped hemolytic zone - positive

strept, equi - no arrow head shape - negative

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