Download 19 Micro lab

LAB 19
REVIEW FOR LAB PRACTICUM
DATA SHEETS DUE NEXT WEEK
3.1 Microscope
3.2 Ocular micrometer
1..1 Media preparation and plate exposure
2.1, 2.2, 3.4, 3.5 Colony morphology, simple and negative stains
3.3 Eukaryotic microbes
3.6 Gram stain
3.7 Acid fast stain
3.9 spore stain
4.1, 2.8, 3.10 morphologic microbes, anaerobic cultures, motility
4.7, 5.4, 5.21, 5.23, 5.29 MacConkey agar, MR-VP, TSI, Litmus milk, API 20-E
REVIEW OF API-20E TEST STRIPS
The purpose of this test strip is to test for 20 different enzymes to get a profile of
organisms to help identify them. The profile is based on the fact that different genes
produce different enzymes for these organisms.
One such enzyme is β-galactosidase, which breaks lactose (a milk sugar) into glucose and
galactose. If you don’t have this enzyme and you drink milk, you feed the bacteria in
your colon, and they break the lactose down and produce CO2 gas, and you also get
diarrhea. There was no lactose in the API strip, so we tested for it in litmus milk (fig 5-41
in lab manual). Actually, bacteria need the enzyme plus a product called a “permease” to
break it down: the permease in the API strip is ONPG (fig 5-42, page 132).
ONPG was colorless. If the bacteria had the enzyme, it turned yellow.
ADH (argentine dehydrolase) breaks down argentine, which is an amino acid. All amino
acids have a carboxyl group (with a free radical attached to the carbon) and an amino
group. (fig 5-45). When ADH breaks down argentine, it becomes ornithine, which is
more basic. A pH indicator (phenol red) is present in the well. The pH of phenol red is
6.8, which is close to neutral. If the pH goes down, it is acid (yellow), and if the pH goes
up, it is basic (red). Phenol red, in the presence of ornithine, gets redder, although in the
first 24 hours it may still be just orange. The substrate of the ADH test is arginine,
because that is what is broken down. The product is ornithine. Since ornithine is more
basic, the phenol red turns red.
Other pH indicators we used are brom thymol blue (in API strip), methyl red, and litmus
(not in API strip).
LDC: Lysine decarboxylase. The substrate is lysine, another amino acid. A
decarboxylase breaks off the carboxyl group. The result in this case is cadaverine. When
an animal dies, there is a lot of protein breakdown, called putrefaction, and give that stink
of “death”. Cadaverine contributes to that smell. Lysine breaks down into cadaverine,
which is more basic, so phenol red becomes redder.
ODC: Ornithine decarboxylase. Ornithine was the PRODUCT of the ADH well, but in
the ODC well, it is the SUBSTRATE. The product here is putricine, which is also a
product of putrefaction and has that death smell. It is more alkaline (basic), so phenol red
turns red. Therefore, in tubes 2, 3, and 4, we are looking for red as a positive result.
CIT: citrate (a salt of citric acid). Citric acid is a part of the Krebs’s cycle. Citrate is the
only API well with one carbon source: citrate. The break down of citric acid produces a
more basic medium because an acid is breaking down. In this case, the pH indicator is
brom thymol blue. Blue color after the test indicates a basic pH, which is a positive
result. Yellow indicates acid, and green is slightly acid.
H2S: Hydrogen sulfide gas. This is the result of the reduction of thiosulfate (the
substrate). Thiosulfate breaks down into hydrogen sulfide gas. Iron is added to the
medium, and if positive, it precipitates out and turns the medium black. The enzyme
involved is thiosulfate reductase (therefore, it is a reduction reaction). The gas has to
react with iron to detect it. This reaction can also occur in eggs, which have the amino
acid cysteine in them. The bacteria break down the amino acid into H2S, and you get a
rotten egg smell from the sulfur in the H2S. The substrate is sodium thiosulfate. You look
for the black color in the TSI slant. Remember, iron is needed for this test.
UREA: Urea is broken down into ammonia by the enzyme urease. Urea is made when
proteins are broken down. The kidneys are supposed to filter the urea and put it into the
urine for elimination. If the kidneys malfunction, urea can build up in the blood and be
very toxic. The smell of OLD urine is from ammonia. Bacteria break down urea into
ammonia (a base). Phenol red turns red for a positive test. Urea broth can also be used
(fig 5-49). The most basic tube in this photo is pink (positive test), and the least basic is
yellow (acid). Only a few of your unknowns were positive.
TDA: tryptophan deaminase (breaks off an amine group). The substrate is tryptophan (an
amino acid; also found in turkey. This amino acid might encourage sleep, which is why
you might feel sleepy after a Thanksgiving turkey meal. The TDA tests for the product:
endopyruvic acid. Reagent (ferric chloride: FeCl3) is added to the tube. A positive result
turns dark brown with a reddish hue. The same people who had a positive urea test will
have a positive TDA test.
INDOLE: This test also uses tryptophan. It breaks down more than the amino group, and
you end up with indole, which contributes to the smell of feces. Many of your unknowns
are positive for this test, including E. coli. To test for indole, add Kovac’s reagent, which
has alcohol in it. Alcohol is lighter than water, so when the test is positive and turns red,
the red ring floats to the top of the tube (fig 5-66 and 5-88). TDA and indole have the
same substrate.
VP: Voges-Proskauer (fig 5-8). Glycolysis forms pyruvic acid, which undergoes
fermentation, which produces acetoin, which then ends in the product 2, 3 butandiol. We
don’t have a test for the end product, but we can test for acetoin. This test uses Barret’s
reagent A (alpha napthol, a carcinogen!) and Barret’s reagent B (KOH, a very caustic
base, found in draino). When reagent A reacts with acetoin in a basic environment (takes
10 minutes), it turns red if positive and brown if negative (fig 5-11).
GEL: gelatin is broken down into amino acids in the presence of gelatinase (fig 5-53).
Gelatin comes from the hooves of horses, pigs, and cows. If the enzyme is present, it
breaks the gelatin down and liquefies it (fig 5-54). It is seen on the API strip (fig 5-88). If
the charcoal stays clumped, the test is negative. If it diffuses, it is positive. The same
people who had a positive TDA and urease will have a positive GEL test. This organism
makes several protein-breaking enzymes, and gets its name from these proteonacious
enzymes. It is the only organism that breaks down gelatin.
SUGAR WELL TESTS
These wells contain different carbohydrates and the same indicator: brom thymol blue.
They all start blue, and we are looking for the acid products (yellow) of fermentation.
Fermentation is anaerobic, so look for the color change at the bottom of the tube. Yellow
is positive. All Enterobacteraceae should be positive for glucose. None should be positive
for all of the sugar wells. If you see green, that is a weak acid, and is still positive.
NITRATE REDUCTION (fig 5-21)
This is a separate test, but it is performed in the glucose well because nitrate was added to
that well by the API manufacturer. Nitrate is reduced to nitrite, which can be further
reduced to nitrogen. All of you should have had a positive test. Add two reagents. If it
turns red, the test is positive for nitrate. If it does not turn red, add zinc powder to detect
for nitrogen, since reduces nitrate to nitrite. This time, if there is a color change, it means
there was nitrate present, so the organism did not reduce it, and the test is negative. If
there is NO color change, the organism already reduced the nitrate, so the test is positive
for nitrate reduction.
CATALASE TEST
In the presence of the enzyme catalase, H2O2  H20 + O2
Hydrogen peroxide was added to three of the sugar tubes. If catalase was present, there
will be bubbles. All of your unknowns should be positive.
LOG IN YOUR API NUMBERS
After you decide which of your tests on the API 20 E strip ar positive, add up the
numbers on every three tests that are positive. This will give you a 9-digit profile number.
Go to www.apiweb.com
Log in as gmorales
Password is micro1
Click on API 20 E
In the dark circles, type in your profile numbers (should be 0-7 for each set of three tests.
Then hit confirm.
Your organism will be listed under “significant taxa”.
MICRO REVIEW UNIT 1
Be able to match the parts of a microscope to a photo of a microscope.
How many grams of pure agar must be weighed out to make 600ml of nutrient agar
(1.5% pure agar)?
1.5% means 1.5 grams of agar per 100ml.
1.5
100
=
x
600
 100x = 600 (1.5)  x = 900  x = 9 grams
100
Properties of agar:
It is a seaweed from the ocean
Its purpose is to solidify
Its advantages over gelatin is that it does not melt in the autoclave and it is not a
nutrient
Dissolving point: 100° C
Melting point (becomes molten): 100° C
Pouring temp: 50° C
Solidifying temp: 42° C
Autoclave it at 121°C and 15 p.s.i.for at least 15 minutes (longer for larger
volumes)
Ingredients of nutrient agar: agar, water, nutrients (peptone, beef extract)
Agar slants are made by pouring them, then sterilizing, then inclining while
cooling
On a stage micrometer, the large lines are 0.1 mm apart; how many microns apart are
they?
Answer: 0.1 x 1000 = 100 microns
The smaller lines on a stage micrometer are 0.01 mm apart; how many microns?
An autoclave is better than an oven because the temperature necessary for sterilization is
lower in an autoclave than an oven.
When we “streaked for isolation”, the procedure is called a “streak plate”. The reason we
make a streak plate is to get isolation of organisms. If we are successful, we see just one
type of cell when we Gram stain.
When you flame a loop, make sure it is not wet or it will spatter, and the bacteria with it!
It will become an aerosol of bacteria.
……………………………………………………………………….
Know what a spirochete looks like, and that you can usually find one in your mouth.
Know terms describing types of elevation in colonies from page 31in your lab manual,
such as erose, umbonate, flat, convex, filamentous, rhizoid, and lobate. Be able to use
these words to describe the photos on pages 31-37 of the manual.
When you do a Gram stain:
What would happen if you forget to use Gram’s iodine? (cells would be pink)
Over-decolorize? (cells would be pink)
Use no alcohol? (cells would be purple)
Use an old culture? (Gram + cells would be purple and pink because some PG has broken down)
Why do endospores resist stain? Because of mycolic acid.
Why is heat necessary with SF technique?
Allows stain to penetrate resistant cells because mycolic acid is waxy.
Why is blotting paper used with heat steam? Keeps the stain from drying out.
What does a slide of ZN look like? Check your lab manual.
What does a slide of SF look like? Check your lab manual.
What diseases are diagnosed by a positive ZN? TB and Hansen’s disease (leprosy)
What does the pink layer mean in a Thioglycollate broth tube? (There is oxygen present)
What agent in it is a reducing medium? (Resazurin)
Know what a tube of Thioglycollate would look like when inoculated by the following
types of organisms: strict anaerobe, microaerophile, strict aerobe, facultative anaerobe.
API strips:
All Enterics are negative for the oxidase test.
All Enterics are positive for the Glucose test.
All members of the Enterobacteriaceae can reduce the inorganic substrate NO2
The product in ONPG is colored, whereas the substrate is colorless.
Tryptophan is the substrate for TDA and INDOLE tests.
Know which wells have what color of a positive test: Brown, Orange or Red, Blue,
Yellow, Diffused black pigment, Pink ring on top
Which test checks for the production of acetoin, a product of fermentation? VP
Which test has only one carbon source in the medium. Citrate
Which test uses a substitute for lactose as substrate. ONPG
Which test checks for the production of certain proteases UREASE
The products of which two tests are primary amines. Urease and Nitrate
Litmus Milk:
Know how to record the results of this test, and know what each of the following looks
like:
AR, ACR, AGCR,
TSI slant:
Know how to record the results of this test, and know what each of the following looks
like:
A/A,G, K/A,G K/A,G, H2S K/A, H2S A/A, H2S
A/A,G, H2S
Glucose has a 0.1% concentration in TSI.
Know what colors are positive and negative for an IMVC test
When the H2S tube is checked for color change, it indicaates FeS gas present
When bubbles appear due to nitrate reduction, it indicates nitrogen gas present.
When bubbles appear after the addition of hydrogen peroxide, oxygen gas present
After fermentation, glucose broth produces carbon dioxide gas
Which Methyl Red tube shows more acid: yellow or orange?
Phenol red is used for a lower pH than an MR.
Urea broth turns pink if it produced ammonia.
Urea broth turns yellow if it produced acid.
What product of nitrate reduction is in the second tube that turned red? presence of
nitrite
If the results of the above test was yellow, zinc powder is added to the tube to detect the
possible presence of nitrogen gas.
What would happen if you prepare TSI:
You forget to add ferrous sulfate: You will be unable to detect hydrogen sulfide gas
You forget to slant the media: You will not be able to differentiate glucose from lactose
or sucrose fermentation.
You make a very small butt. You will not be able to differentiate glucose from lactose or
sucrose fermentation.
You make the concentration of glucose 0.1%: This would have no effect
You make the concentration of lactose 1.0% : This would have no effect
What would happen if you are preparing a hanging drop:
You are observing a wet mount and the drop of broth culture starts to recede.
False positive results
You are observing a wet mount and you notice that the culture is too concentrated.
Motility could be observed without false results
You are observing a hanging drop and the drop falls into the well.
No motility could be observed
You are observing a hanging drop and have the iris opened wide.
No motility could be observed
MacConkey agar inhibits the growth of Gram positive organisms because it contains
crystal violet and bile salts. You need to keep Staph aureus out of your medium because
it can ferment all three sugars, so it will throw off your results. We use MacConkey’s
agar to differentiate Gram negative species according to their ability to ferment lactose.
Mac Conkey agar turns pink if lactose is fermented.