Preparation for Lab Quiz #2

What does IMVic stand for?

Indole, Methylene Red / VP, citrate.

How do you set up these three slant tests?

Inoculate slants (zigzag pattern) with loop. Remember: for the TSI slant, you have to zigzag and stab!! Incubate at 37 degrees for 24-48 hours. Note: TSI slant results cannot be read after 48 hours. For citrate slants, color change can take up to 7 days when testing Bacillus species.

What is a bacterial lawn?

It is a solid layer of growth that can be seen on an agar plate if you use a culture that has not been diluted.

What is a candle jar?

It is an apparatus for incubating bacterial cultures that *provides a capnophilic environment (low O2, high CO2)*.

What is s Brewer's jar?

It is an apparatus for incubating bacterial cultures that *provides an anaerobic environment (no O2)*.

What kind of environment does this contraption provide?

It's a Brewer's jar, which provides an anaerobic environment.

What type of hemolysis is this? (greenish growth with empty space in the middle).

It's alpha hemolysis.

How would you characterize the oxygen requirements of this bacteria? (growth only on the top of the tube).

It's an obligate aerobe.

How would you characterize the oxygen requirements of this bacteria? (clear at the top of the reducing media tube and cloudy near the bottom).

It's an obligate anaerobe.

What do the normal flora in the family Enterobacteriaceae ferment?

Lactose. The pathogen of this family *do not* ferment lactose.

What kind of media plate (MSA or blood) would I grow Staphylococcus bacteria on to see if it is pahtogenic?

MSA.

Shake culture (thioglycollate agar)

Molten agar that is *allowed to harden* once inoculated. It contains a *reduced amount of oxygen allowing anaerobes to grow near the bottom* of the tube.

Remember

Not all pure cultures are clones!

How do you calculate the original concentration of bacteria?

Original concentration = (# of colonies) x 1/dilution factor

What is peptonization? What does this look like in the litmus milk test?

Peptonization means that proteins have been completely digested. It appears as a clear brown iced-tea color.

Is the Indole test based on carbohydrate or protein digestion?

Protein digestion; specifically, the breakdown of the amino acid tryptophan. If there is a pink ring at the top = positive for breakdown of tryptophan. If there is a yellow ring at the top = negative for breakdown of tryptophan.

What is the Triple Sugar Iron (TSI) test?

Red slant that tests for the fermentation of three sugars (glucose, lactose, sucrose) and the production of hydrogen sulfide gas (H2S). NOTE: This slant must be streaked and stabbed to inoculate.

What kind of bacteria is catalase negative?

Streptococcus.

The pathogenic and normal flora types of Streptococcus and Staphylococcus.

Streptococcus: S. pyogenes = pathogen (identified by *beta hemolysis*, complete clearing on blood agar) S. pneumoniae = pathogen (identified by alpha hemolysis, partial clearing on blood agar with greenish growth). Staphylococcus: S. aureus = pathogen (identified by *yellow halo on MSA* plate, which means it makes mannitol) S. epidermidis = normal flora (identified by *pink color on MSA* plate, which means it does not make mannitol).

Generation Time

The *goal of the spread plate* is to determine the generation time, or the *length of time necessary for a particular bacterial species to reproduce once*.

What is the Durham test?

The Durham test is a red broth that tests for the *fermentation of one carbohydrate*; identifiable by the inverted gas tube.

What is the Durham test used to test for? What type of media does this test use? Describe the results of this test.

The Durham test is used to test for carbohydrate fermentation. It is a broth. The broth starts out red and contains an inverted gas tube.

What are the two differences between the EMB plate and the MacConkey plate?

The EMB plate grows E. coli; MacConkey does not. The starting color of EMB is darker than MacConkey color.

What is the Eosin Methylene Blue (EMB) plate test for?

The EMB test is a burgundy colored plate that is selective for Gram negatives and differential for lactose fermentation.

As we previous learned, what is usually the first step in identifying a bacterium?

The Gram stain, which allows us to classify the organism based on morphology and Gram reaction. However, species identification is not possible using this stain.

What is the Indole test?

The Indole test is a clear broth that tests for the *breakdown of the amino acid tryptophan*; the waste product indole is used as an indicator.

What is the Methyl Red, Voges-Proskauer tests (MR-VP)?

The MR-VP tests are yellowish broths that test for the waste products of glucose fermentation. Methyl red - tests for acidic waste. VP - tests for neutral waste (acetoin).

What is the MacConkey plate test for?

The MacConkey test is a rose colored plate that is selective for Gram negatives and differential for lactose fermentation.

What is the TSA plate test for?

The TSA test is a beige colored plate that is enriched for growth of fastidious bacteria; used as a control for growth.

How do you choose a plate that will help you determine an estimate of bacteria per mL in the original culture?

The golden rule is to select the plate with *30-300 colonies*.

What are two isolation techniques we learned?

The spread plate and the streak plate.

Spread Plate

The spread plate involves *counting bacterial colonies to estimate the total number of bacteria per mL in a broth culture*.

By the way, if a bacterium is positive for catalase...

Then when you add the bacterium to hydrogen peroxide, it will produce bubbles.

What is the special ingredient in the reducing media broth?

Thioglycollate (removes O2)

What is this test called? What's does it test for? (The image shows a slant with a red top & yellow bottom, a completely yellow slant, a slant with a yellow top and a black bottom, and a slant with a pink top and a black bottom). Which tube(s) produced hydrogen sulfide? What does yellow slant/yellow butt indicate? What does the pink top/yellow butt indicate? What is the disadvantage of this test compared to the Durham tubes?

This is called the TSI test. It tests for the fermentation of carbohydrates as well as the production of H2S. The tubes with the black bottoms are positive for hydrogen sulfide (H2S) production. The yellow slant/yellow butt indicates the fermentation of glucose and sucrose and/or lactose; negative for H2S production. The pink top/yellow butt indicates the fermentation of glucose; negative for H2S production. In TSI, we don't know what the 2nd sugar is; however, in the Durham test, you would have separate tubes to test for each specific carb. It's more accurate.

What is the name of this plate? (Image shows a reddish/brown plate with 4 quadrants. In three of the quadrants, there is bacteria growing. One of the streaks is green, one is clear, and one is pink). Is this selective and/or differential? What is the name of the other plate that has similar functions? Which quadrants are Gram negative? What is the name of the bacteria in quadrant 1? (green). Is this bacteria pathogenic?

This is the EMB (Eosin Methylene Blue) test, which is selective for Gram negative and differential for lactose fermentation. The other plate that has a similar function is called MacConkey. The quadrants that have bacterial growth are Gram negative. The bacteria in quadrant 1 is E. coli. It is not pathogenic -- it's normal flora.

Dichotomous key

This key is combined with the results of the biochemical tests to help pinpoint the bacteria species. They key works by process of elimination for bacterial identification.

Serial Dilution

This technique is used in the process of creating a spread plate. You take a concentrated broth culture and put it through a series of dilutions.

(Image represents MR/VP test) A & B represent different bacterial species. If this is MR, which tube is positive? What color would we expect for bacterium B if we ran its VP test?

Tube A is positive. REMEMBER: A red color for either the MR or the VP = a positive result. You would expect a red color for bacterium B's VP test (because the MR result is yellow; thus the VP has to be red).

After adding reagents A and B in the nitrate test, we see no color change -- what could we add? After adding the second reagent, what color indicates a POSITIVE result?

We could add Zinc. After adding the second reagent, it would be clear to indicate a positive result (the bacteria is reducing nitrate).

How to find the dilution factor (cont'd...)

We then transferred the dilutions onto plates. There are two volumes that we can transfer to the plates-- 1mL or 0.1mL. If we transfer *1mL, it is the mathematical equivalent of 1/1*. There are no additional zeroes at the bottom of the fraction, so the dilution factor remains the same. If we transfer *0.1mL, however, this is equal to 1/10mL*. Now we have an extra zero at the bottom of the fraction, so we need to add 1 to the exponent for the dilution factor. Now you'll be able to determine the initial and final concentrations! (study the practice problems).

Colonies

When bacteria appear to grow as round clumps of growth on the surface of an agar plate. Each colony is composed of millions of bacterial cells that all originated from one parent cell.

The spread plate has a second part...

You basically just take the culture that you put back in the incubator for 2 hours and perform the same procedure. Create a 1st dilution, 2nd dilution, and 3rd dilution. Then you transfer samples of the diluted cultures onto 4 plates (T = 2 plates): *10^-4 plate*: transfer 1mL from 2nd dilution bottle. *10^-5 plate*: transfer 0.1mL from 2nd dilution bottle. *10^-6 plate*: transfer 1 mL from 3rd dilution bottle. *10^-7 plate*: transfer 0.1mL from 3rd dilution bottle. 6. Allow liquid to absorb into agar and place in incubator for 48 hours.

Clones

You can spread bacteria into a thin layer of single cells on a plate. Each single cell then begins to reproduce until there are so many bacteria that we can see the visible mass with our naked eye. Her definition: Population of cells all derived from a single parent cell.

How do you set up these three broth tests?

You have to aseptically inoculate the broths with your bacterial samples using the loop. Incubate for 24-48 hours and then check for growth by examining for turbidity. If growth is visible, analyze and record the results. If turbidity is not observed after 48 hours, reinoculate and reincubate. *For Durham tubes*: record any color changes and/or presence of gas in the Durham tube. *For Indole test*: add 4 drops of Kovac's reagent and observe the colored band at the top of the broth. *For MR-VP tests*: - Pour half of the turbid broth into an empty clean test tube. Label the original tube MR and the second tube VP. - For the MR tube: Add 5 drops of Methyl Red. - Fore the VP tube: Add 10 drops of Reagent A, followed immediately by 5 drops of Reagent B. - Observe broth tubes for color changes. NOTE: Although the MR will change color immediately, the VP can take up to 45 minutes to change color.

What are the possible results of the TSA plates?

You have to compare the amount of growth on the three plates for each organism. Rate growth using +++ (tons), ++ (some), + (little), and - (none). Facultative anaerobe should have + for Brewer's, +++ for Candle jar, and ++ for Incubator. In any case, it has +'s for each environment. Aerotolerant anaerobe should have one + for each environment. Obligate aerobe should have 0 for Brewer's jar, and +++ for other two environments. Obligate anaerobe should have ++ for Brewer's jar and 0 for other ++ environments.

Remember, for any of the TSI slants that have black butts...

You should assume that there is some yellow color underneath the black. If there is H2S production, then there is always glucose fermentation. So in other words, if you see a pink top/black butt, you would say that it's positive for glucose fermentation and positive for H2S production.

To remind you of the Nitrate process...

You start off by adding Reagents A and B, which will turn the culture pink if it is positive for reducing nitrate. If it's clear, then you can add Zinc. If it turns pink with the addition of Zinc, then the culture is negative for the reduction of nitrate. However, if it stays clear after the addition of Zinc, it is positive for nitrate reduction.

What is differential media?

Differential media allows us to distinguish between bacterial species based on the appearance of their growth on the plate.

How do you set up these plate tests?

Divide the plate into 4 quadrants. Inoculate plates by streaking the different bacteria on the appropriate quadrants. Incubate for 24-48 hours.

What is the name of these tubes? (The ones with gas tubes in them). What are they used to test? (image shows yellow tube w/ bubbles, red tube w/ no bubbles, and pink tube w/ no bubbles) Which tube fermented glucose? Did it produce gas? Which tube digested proteins? Is the pH high or low in these tubes?

Durham test. This test is used to test for carbohydrate fermentation. The yellow tube with bubbles is positive for carbohydrate fermentation because when carbs are fermented, they produce acidic waste, which turns the original phenol red pH indicator color to yellow, identifying a low pH. The pink tube w/out bubbles digested proteins. When proteins are digested, basic waste is created, which will turn the original phenol red indicator to pink, identifying a high pH.

How to find the dilution factor

During the first dilution step, we took 1mL containing millions of bacteria, and added it to a bottle containing 99mL of sterile saline. We have now spread out the bacterial cells into a final volume of 100mL (1mL culture + 99mL saline). In other words, we have created a 1:100 or 1/100 dilution. If you divide 1 by 100, you will find that it equals 0.01. Since we work in scientific notation, this would equate to 1 x 10^-2 or just 10^-2. Notice how the exponent in the dilution factor (-2) is the total number of zeros at the bottom of the fraction: 1/100.

Reducing media (thioglycollate broth)

Enriched liquid media in which the concentration of *oxygen is reduced allowing anaerobes to grow near the bottom* of the broth.

How to find the dilution factor (cont'd..)

Finally we performed a third dilution taking 1mL from bottle #2 and adding it to 99mL in bottle #3. Which is, of course, another 1/100 dilution. 1/10000 x 1/100 = 1/1000000 1/1000000 = 10^-6

How can you remember the above information?

For the Streptococci, both are pathogens! You just have to remember that S. pyogenes is beta hemolysis and S. pneumoniae is alpha hemolysis. For the Staphylocci, you can remember that S. aureus creates a yellow halo on the MSA plate because aureus sounds like an aura, which is like a halo :D But even though it looks like an angel, it's an evil pathogen! The sweet, innocent pink S. epidermidis is normal flora.. Of course epidermidis makes you think of epidermis, which means that it probably lives on the skin, and thus, is harmless.

How to find the dilution factor (cont'd)

For the second dilution step, we took 1mL from the first dilution and added it to a second bottle with 99mL of sterile saline. We have performed another 1:100 or 1/100 dilution. BUT this first bottle was already diluted when compared to our original culture. To calculate the total dilution thus far, we must multiply the dilution of bottle #1 by the dilution of bottle #2: 1/100 x 1/100 = 1/10000 1/10000 = 10^-4

What's the formula for generation time?

GT = Time / 3.3 x Log(B2/B1) In the GT equation, *time is always expressed in minutes*. B2 = final concentration of bacteria. B1 = initial concentration of bacteria. The number 3.3 is a constant necessary for the equation to work properly.

Remember, for the EMB test...

Green or pink = lactose fermentation = normal flora. White or cream = negative for lactose fermentation = pathogen.

What is the Citrate test?

Green slant used to test for the use of citrate as a *sole carbon and energy source*.

What are the results of the TSI test?

*All red/pink slant* = No sugars fermented. *Pink top/yellow butt* = Glucose fermented. *All yellow slant* = Positive for glucose fermentation and sucrose and/or lactose fermentation. *Black butt* = Positive for H2S production.

What are the results of the Citrate test?

*Green slant* = Negative for the use of citrate. *Blue slant* = Positive for the use of citrate.

What are the possible results of the reducing media and the shake culture?

*Growth throughout*: For the reducing media, it should look cloudy, and for the shake culture, it may be exploded (growth throughout with gas production)). This result indicates a facultative anaerobe. *Growth on top*: This indicates an obligate aerobe. *Growth on bottom*: This indicates an obligate anaerobe.

What are the results of the MacConkey plate?

*No growth* = Gram positive. *Growth* = Gram negative. *Pink growth* = Positive for lactose fermentation.

What are the results of the EMB plate?

*No growth* = Gram positive. *Growth* = Gram negative. *Pink growth* = Positive for lactose fermentation. *Metallic green growth* = E. coli (positive for lactose fermentation).

What are the results of the MR-VP test?

*Red MR / Yellow VP* = Positive for glucose fermentation. Acidic waste, pH < 7. *Yellow MR / Red VP* = Negative for glucose fermentation. Basic waste, pH > 7.

Note that both of the tests mentioned above contain thioglycollate. What is thioglycollate?

*Sodium thioglycollate chemically reacts with and eliminates any oxygen present in the media*. There will always be some amount of oxygen near the surface of the media that has diffused into solution from the atmosphere. This zone of oxygenation is visible as a pink/red band. Do not shake or mix these media to prevent the introduction of additional oxygen.

What are the results of the TSA plate?

*White ring around the line of inoculated bacteria* = Positive for the enzyme amylase (breaks down starch). *No white ring around the line of inoculated bacteria* = Negative for the enzyme amylase (doesn't break down starch).

What are the results of the Indole test?

*Yellow band* = Negative for the breakdown of the amino acid tryptophan. Acidic waste, pH < 7. *Pink band* = Positive for the breakdown of the amino acid tryptophan. Basic waste, pH > 7.

What are the results of the Urease test?

*Yellow slant* = Negative for the presence of urease. *Pink slant* = Positive for the presence of urease.

What are the results of the Durham test?

*Yellow* = Positive for carb fermentation. Acidic waste is produced, pH < 7. *Pink* = Negative for carb fermentation. Basic waste is produced, pH > 7. *Gas bubbles* = Positive for carb fermentation. *Turbid Orange w/ bubbles* = So many bacteria in tube, that they used up all the carbs and then began using up the protein, which takes it from the acid yellow color back to a more neutral orange color.

The following are the answers to the Lab Quiz #2 Practice Questions we went over in class...

...

What are the 3 traits that all species in the family Enterobacteriaceae have?

1. Gram negative bacilli. 2. Oxidase negative. 3. Glucose fermenters.

How do you set up these anaerobic bacteria tests?

1. Inoculate the reducing medium broths, inoculate and incubate for 24-48 hours. 2. Obtain a shake culture agar from the warm water bath. Inoculate with the appropriate culture, and roll between your hands for a few seconds. Place in an ice-water bath until agar is hardened. Incubate for 24-48 hours. Remember: Perform shake culture inoculations one at a time! 3. Observe your tubes after the 24-48 hours.

Describe the process of creating a spread plate.

1. To create the *first dilution*, you transfer 1mL of the pure bacterial culture to a glass bottle with 99mL of sterile saline. Close lid and shake for 30 sec. 2. Return the concentrated culture back to the incubator so that it can incubate for an additional 2 hours. 3. For the *second dilution*, transfer 1mL from the first dilution bottle to the second bottle with 99mL of sterile saline. Shake well. 4. For the *third dilution*, transfer 1mL from second dilution bottle to third saline bottle. Shake. 5. Transfer samples of the diluted cultures to your 4 labeled nutrient agar plates (T = 0 plates). *10^-4 plate*: transfer 1mL from 2nd dilution bottle *10^-5 plate*: transfer 0.1 mL from 2nd dilution bottle. *10^-6 plate*: transfer 1mL from 3rd dilution bottle. *10^-7 plate*: transfer 0.1mL from 3rd dilution bottle. 6. Allow liquid to absorb into agar and place in incubator for 48 hours.

Describe the process of creating a streak plate.

1. Use your sterile loop to transfer a small volume of bacteria from your assigned culture. Zigzag about 1/4 of the plate with the bacteria on the loop. Only dip the loop into hour broth culture once. 2. Flame the loop. 3. Rotate the plate by 90 degrees. Pass the loop over the first streak 2-3 times to pick up bacterial cells. Spread the bacteria on the loop into the next quarter of the plate using the same zigzag pattern. 4. Flame the loop. 5. Rotate the plate by 90 degrees. Overlap the 2nd streak 2-3 times to pick up bacteria and generate the third streak. Flame the loop. 6. Rotate the plate by 90 degrees. Overlap the 3rd streak and spread the bacteria in a wide zigzag through the center of the plate. Be careful not to pass over the previous streaks. 7. Incubate plates agar side up in the incubator for 48 hours.

How do you test for anaerobic growth on plates?

1. You obtain 3 TSA plates (1 for the Brewer's jar, 1 for the candle jar, and 1 for the incubator), and you draw 4 separate quadrants on them. 2. Inoculate all plates in the same order with the provided cultures and place in the appropriate locations. 3. Observe all plates after 24-48 hours to analyze oxygen requirements.

What is a capnophilic environment? What classification of anaerobe will grow in such an environment? How did we create a capnophilc environment?

A capnophilic environment is low in O2 and high in CO2. Facultative Anaerobes, Aerotolerant Anarobes, and Obligate Aerobes can grow in a candle jar. We create a capnophilic environment with the candle jar.

What is a culture?

A culture refers to the growth of microorganisms in or on media.

Streak Plate

A streak plate is used to *isolate pure cultures in the form of single colonies*. If you had a broth with multiple bacterial species, it would be extremely difficult to separate the organisms without looking under the microscope. With the streak plate technique, *we use a loop to take a small volume of the mixed culture and systematically spread it along the surface of the agar plate to distinguish the different appearances of the colonies*.

These are bacteria grown on starch plates. What enzyme do bacteria have to hydrolyze starch? Is it an endoenzyme or exoenzyme? What do we add to test for starch hydrolysis? Which bacteria (A: no halo, B: white halo) produces amylase?

Amylase. It's an exoenzyme (works outside of the bacteria). We add iodine to look for starch hydrolysis. Bacteria B, with the white halo, produces amylase.

What is an incubator?

An apparatus for incubating bacterial cultures that provides an environment with the same levels of oxygen as the atmosphere.

What is the Urease test?

Beige slant used to test for the *presence of the enzyme urease*, which converts urea to ammonia (NH3).

What is the purpose of biochemical testing?

Biochemical testing provides additional information that a Gram stain cannot provide, including which enzymes the bacterium might possess and its ability to metabolize various proteins and carbohydrates.

What kind of media plate (MSA or blood agar) would I grow Streptococcus on to see if it is pathogenic?

Blood agar.

Pure Culture

Culture can be composed of multiple species, or it can represent only *one species* in which case it is known as a *pure culture*.