Bio 315L Exam 2

Blood agar: You are given three plates with bacteria grown on sheep blood. On plate A the bacteria seems to have normal growth, the bacteria on plate B is clear and the bacteria on plate C is green. What do these results tell us?

This test tells us if a bacteria has the ability to lyse red blood cells (hemolysis). There’s three types of hemolysis. Beta hemolysis tells us that there’s complete destruction of red blood cells which would give us a clear plate. Alpha hemolysis indicates partial destruction which gives us a green color. Lastly, gamma hemolysis has no destruction which gives us natural growth.

You are given a salt agar plate divided into three quadrants. In quadrant A there is no growth or color change. Quadrant B has growth with no color change while quadrant C has growth and it is now yellow. What does each quadrant tell us about the bacteria that has grown?

These plates grown with NaCl and phenol red tell us if bacteria can grow in high salt and fermentation of mannitol. If there is growth then bacteria can grow in high salt, and a color change tells us if the fermentation of mannitol happened due to fermented mannitol decreasing the pH which gives a yellow color. Therefore, yellow is ++, growth with no color change is +-. And no growth or color change is - -. 

Nitrate Reduction: You are given 4 tubes, each with a different bacteria. Tube 1 has a gas bubble in the Durham before any reagents are added. Tube 2 turns red after reagents A and B were added. The other two tubes didn’t turn any color after the reagents were added. Zinc was added to tube 3 and 4 but only 3 turned red. What does this test tell us, and what does the result of each tube mean?

This test tells us if an organism can reduce nitrate by using the enzyme nitrate reductase. Step 0.5: if you see a gas bubble in the Durham tube = NO3 -> N2 gas

Step 1: add reagents A&B; if the media turns red = NO3 -> NO2

Step 2: add zinc; if the media turns red = negative for NO3 reduction; if media stayed clear = NO3 -> NO, N2O, or NH4+ (some nitrogenous compound) (positive for nitrate reduction)

Acidophiles

Deal with the low pH environments by altering their lipid profiles to limit protons from entering or extruding the excess protons out of the cells

Alkaliphiles

deal with the high pH environments by altering their cell walls with acidic polymers to balance the basic environment or with antiporters that pump out sodium to bring in protons

Neutrophiles

Na+/H+ antiporters, alterations to lipid composition

Halophiles

deal with osmotic stress by removing sodium with special pumps and replacing it with compatible ions such as potassium

You’re given a milk agar plate with two streaks of bacteria. One streak has a zone of clearing while the other one doesn’t. What does this mean?

This test identifies if there is the casease enzyme. If bacteria has it then it can break down the casein in milk into amino acids. If the bacteria has it, then there will be a zone of clearing. TCA helps to visualize. 

H2S: You are given two SIM tubes with two bacteria grown on them. Tube A is black while tube B has no color change. What do the results tell us? Explain what each

Tube A is black since since sulfide from cysteine was release, coupled to a hydrogen ion, and the formed H2S combines ferrous ammonium sulfate to make a black precipitate. This means that the bacteria has the enzyme cysteine desulfurase which allows it to break down cysteine. Since there aren't those enzymes in bacteria B, then it is negative and doesn't break cysteine to make H2S. 

Motility:

You are given two deeps of semi-solid agar. Tube A only has growth around the inoculum while the other deep is turbid. Explain what each result means.

This test is meant to see if a bacteria has flagella which allows it to move throughout the media. If a bacteria does have a flagella then we would expect sporadic growth which results in a turbid deep. If there isn’t growth away from the inoculum then the bacteria doesn’t have a flagella. 

Bile:

You are given two bile esculin slants with bacteria A and B. The slant with bacteria A is brown with no growth. The slant with bacteria B is black with bacteria growth. What do these two results imply? And what would it mean if a blank slant had no growth? 

Bile esculin plates test for two things, the first thing is growth on bile, and the other is the ability of esculinase to hydrolyze esculin. Slant A shows a positive result. Esclinase hydrolyzes esculin to esculetin which reacts with the ferric citrate in the media black. If the bacteria that don’t have it, it will not react with the ferric citrate staying brown. Therefore, bacteria A is negative for both bile and esculinase. 

You have two slides with bacteria. You put a drop of H2O2 on both and one bubbles. What is this test doing and what do the results mean?

This test tells us if a bacteria has the catalase enzyme that allows H2O2 to be converted to H2O and O2 bubbles. If there are bubbles then that means that O2 gas was.

You are given a plate with DNA in the agar. Two bacteria are put on there and one has a zone of clearing. What does this mean?

This test tells if a bacteria has DNase which breaks down DNA into factors. This is usually a form of a virulence factor that enhances the ability of a bacteria to cause disease. They’re grown on a plate with DNA and agar. You add HCl to precipitate DNA.  If there is a zone of clearing then that means that bacteria broke down DNA.

There is a vial with rabbit plasma. One has clotted while the other hasn’t.

One bacteria has coagulase which clots up rabbit plasma while the other doesn’t which lets it stay free flowing.

Fermentation:

There are three tubes with phenol red and durham tubes. The first tube has no bubbles and it is red, the next one is yellow and it has no bubbles and the third has a bubbles and it is yellow. 

What is a negative? What is a positive? What enzyme(s) are involved?

Fermentation tests tell us if a bacteria is able to use different substrates. We use phenol red as a pH indicator and the durham tube to capture gas. When the pH is lowered the media turns yellow and this indicates that the bacteria fermented a carbon source which made the media more acidic. A gas bubble also counts as a positive for fermentation.

You are given two tubes for an ONPG test. One is clear, while the other is yellow. What do these results tell us?

This test tells us if a bacteria has beta-galactosidase, which is an enzyme that breaks down lactose. ONPG is a substitute for lactose so it can also break it down. Bacteria that can break down the ONPG using beta gal will produce a yellow color. Bacteria without the enzyme cannot break down OBPG and remain colorless.

Indole:

You are given two tubes for an indole test. One has a pink ring and the other has an orange ring. What do these results need? What is a negative? What is a positive? What enzyme(s) are involved?

This test tells us if a bacteria can make indole by hydrolyzing tryptophan using tryptophanase. 

We use tryptophan broth with Kovac’s reagent and HCl. If indole reacts with kovac’s reagent then it’ll be pink but if there isn’t then it’ll be orange which is negative.

You have two tubes for a Methyl Red test. One is red and the other is yellow. What is a negative? What is a positive? What enzyme(s) are involved?

A methyl red test tells us if an organism can create an acidic end product via mixed acid fermentation. Methyl red is the pH indicator with it naturally being yellow but turns red as it get more acidic. If the MR broth has glucose that is fermented then that means that the bacteria has some kind of enzyme to do so.

You are given two tubes for a Voges-Proskaeur test. One is red and one is copper.

These tubes have VP or MR/VP broth with Barrit’s reagent A and B. This test determines if an organism can make acetoin by the degradation of glucose during 2,3-butanediol fermentation. The reagents turn acetoin red by oxidizing, but if it turns more copper, then that’s just the reaction of the reagents (neg).

You are given two slants for a citrate test. One is green and the other is blue. What is this test looking for and what do these results mean?

The media for this test is a simmons citrate agar slant, and it has bromethyl blue as a pH indicator. This test identifies if an organism has the ability to use citrate as its sole carbon source using the citrate permease enzyme. Bacteria without citrate permease enzymes can’t survive. Bacteria with it can convert ammonium phosphate to ammonium hydroxide which makes the media more basic which means it turns blue.

You’re given two phenylalanine slants. You grow bacteria on these slants and you add ferric chloride. One has a green shadow. What does that mean?

This test tells us if an organism can remove the alanine group (NH2) from phenylalanine using the phenylalanine deaminase enzyme. Bacteria that can deaminate phenylalanine using the enzyme will produce phenylpyruvic acid, which will react with ferric chloride creating a ‘green shadow’. Bacteria that cannot deaminate phenylalanine will not react with ferric chloride and cannot produce a color change.

You’re given two slants, one is pink and the other is orange. What does this mean?

This test for the enzyme urease which allows bacteria to hydrolyze urea to ammonia which increases the pH and turns Phenol red pink (more basic). Otherwise the color stays more acidic. 

What does an IMViC series tell us?

These tests differentiate bacteria in the family enterobacteriaceae, this divides them into lactose fermentors and non fermentors. Indole, Methyl Red, Voges, Citrate.

Lag Phase

period of slow/no growth as bacteria transition to acclimate to its new environment

Log Phase

cells begin to divide exponentially

Stationary Phase

cells can no longer grow due to lack of resources and overabundance of waste products

Death Phase

cells will either die off or enter a dormant state

μ = ??𝑡 & 𝑔 = ??

μ = [ln (𝑁2/𝑁1 )]/∆𝑡 & 𝑔 =ln2/μ

Obligate Aerobe

can only grow in oxygen

Obligate Anaerobes

can only grow without oxygen.

Facultative Anaerobes

Can grow with and without oxygen. Grows better with oxyfgen

Acidophiles

deal with the low pH environments by altering their lipid profiles to limit protons from entering or extruding the excess protons out of the cells

Alkaliphiles

deal with the high pH environments by altering their cell walls with acidic polymers to balance the basic environment or with antiporters that pump out sodium to bring in protons

How neutrophiles deal with pH tolerance

Na+/H+ antiporters, Alterations to lipid composition

Halophiles

deal with osmotic stress by removing sodium with special pumps and

replacing it with compatible ions such as potassium

You are given a starch plate with E. coli and S. epidermidis. You flood the plate with Iodine and see that S. epi has a zone of clearing whereas E. coli doesn’t. Explain what each bacteria did.

S. epidermdis was able to hydrolyze the starch in the media using either the a-amylase or oligo-1,6-glucosidase. The starch that was broke down did not react with iodine which gave us the zone of clearing. E. coli did not hydrolyze starch, so the whole starch reacted with iodine turning brown.