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Microbiology Lab > Lab Exam 3 Part 2 > Flashcards

Lab Exam 3 Part 2 Flashcards

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1
Q

How can you determine whether a particular carbohydrate is fermented when using a fermentation medium? What are the possible indicators?

A

Fermentation releases acid causing a decrease in pH, can be shown by a pH indicator. Sometimes fermentation causes gas release which can be indicated by the Durham tube.

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2
Q

How does the fermentation of monosaccharides differ from that of disaccharides? Answer in terms of enzymes needed.

A

Each use different enzymes and the rate of reaction differs. The monosaccharide (glucose) acted as an aid when it came to the rate of fermentation, while lactose (disaccharide) hindered the rate of fermentation

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3
Q

Name the four components of fermentation tube and explain the function of each.

A

Single chemically defined carbohydrate- what you are testing the bacteria against.
pH indicator- indicate acid increase as a result fermentation.
Durham tube- Gas indicator
Nutrient broth-media for the bacteria

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4
Q

Is nutrient broth synthetic or non-synthetic? Explain.

A

Non-synthetic aka complex media

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5
Q

Is the single sugar included in each type of fermentation broth a synthetic or non-synthetic ingredient? Explain.

A

Synthetic aka defined.

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6
Q

How is the ability to ferment a particular carbohydrate related to an organism’s genotype and phenotype? In your explanation consider the ability of an organism to produce the necessary enzymes needed to ferment a particular sugar.

A

Enzymes are substrate specific. So the ability of a organism o ferment a particular carbohydrate is determined by the enzyme it produces which is determined by it genes.

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7
Q

If a carbohydrate broth does not change color after it has been inoculated and incubated, how can you tell whether the unchanged color is due to failure of the organism to grow or failure to ferment the carbohydrate?

A

Look at the Duran tube to see if any gas has been produced, gas means bacteria had fermented and thus grown. Also look for any visual signs of growth in the broth.

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8
Q

A particular organism is able to ferment glucose but not sucrose (a disaccharide composed of glucose and fructose). What are some possible explanations?

A

The organism does not have the enzyme needed to ferment sucrose.

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9
Q

Sugar Fermentation Procedure:

A
  1. Depending on the test gather phenol red-dextrose broth, phenol red-sucrose broth, and or phenol red-lactose broth.
  2. Inspect each tube and make sure Duram tube contains no air. Ocassionally air can be come trapped due to agitation. If air is trapped invert the entire fermentation tube to release the air form the Durham tube.
  3. Inoculate each tube and place all tubes in test tube rack.
  4. Incubate tubes overnight at 37C.
  5. Observe results. Set of inoculated fermentation tubes will act as controls.
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10
Q

Define pathways used,final electron acceptor, and ~ATP generated. Aerobic Respiration, Anaerobic respiration, and fermentation.

A

Aerobic respiration: oxygen is the final electron acceptor, uses electron transport chain/krebs/glycolysis, produces 36-28 ATP.

Anaerobic respiration: uses other inorganic molecules as final electron acceptor, uses electron transport chain, yields <36 ATP.

Fermentation: only involves glycolysis, final electron acceptors lactic acid/ethanol, 2 ATP.

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11
Q

Synthetic vs. non-synthetic media

A

Synthetic= defined
Non-synthetic= complex

Synthetic media contain pure organic and inorganic compounds that are chemically defined (i.e. known molecular formula). Complex or undefined media contain ingredients that are not chemically defined or pure (i.e. animal extracts).

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12
Q

Sugar Fermentation results of the following bacteria agents PR-gucose, PR-sucrose, and PR-lactose:
Escherichia coli
Staphylococcus aureus
Bacillus subtilis
Alcaligenes faecalis

A

Escherichia coli- Ferment all 3 producing acid, but no gas with sucrose.
Staphylococcus aureus- Ferment all 3, only acid no gas.
Bacillus subtilis- Acid only with glucose and sucrose, NG for lactose
Alcaligenes faecalis- NG for any.

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13
Q

Media options for sugar fermentation testing?

A

Phenol red-dextrose (glucose) broth
Phenol red-sucrose broth
Phenol red-lactose broth

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14
Q

Obligate aerobe

A

require the presence of oxygen

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15
Q

Obligate anaerobe

A

oxygen is toxic, only survive in the absence of oxygen

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16
Q

Faculative anaerobe

A

organisms which can survive in both oxygenated as well as the deoxygenated environment

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17
Q

Microaerophile

A

Requires low levels of oxygen

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18
Q

Refer to the catalase lab. Which detoxifying enzymes would obligate aerobe, obligate anaerobe, faculative anaerobe, and microaeropile likely synthesize?

A

obligate anaerobe, none as oxygen is toxic. The rest likly synthesize catalase, peroxidase, and or superoxide dismutase.

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19
Q

Why boil agar deeps prior to use?

A

The boiling drives out any oxygen in the agar so that after hardening the top 1/2 is aerobic but the deep becomes increasingly anaerobic as you go deeper into the agar.

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20
Q

Why is it important that the deeps be colled to “baby bottle” temp before inoculation?

A

If its too hot it will kill the bacteria.

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21
Q

Oxygen Requirements Lab Procedure:

A
  1. Gather cultures, Tryptic Soy +5% glucose agar deeps that have been boiled for at least 10 minutes and cooled to 50C, Tryptic Soy agar slants, and sterile swabs.
  2. Gently agitate broth culture, saturate sterile swab with culture wringing out the swab on the side of the tube.
  3. Inoculate agar deep by swishing the swab in the melted, cooled agar and mix by rolling the tube between your hands.
  4. Place tube on rack to harden.
  5. Inoculate slant with bacteria.
  6. Lossen caps 1/4 turn and incubate at room temp for 48 hours.
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22
Q

Capnophiles

A

organisms that require increased concentrations of carbon dioxide for growth.

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23
Q

For the oxygen requirement lab why is it so important to incubate at room temp?

A

Some organisms grow so vigorously at warmer temps that gas splits the agar.

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24
Q

In the oxygen requirement lab would you expect the anaerobe to grow on a slant incubated aerobically? Why?

A

No, basically bacteria that are obligate anaerobe cannot grow on any slant media or plate media unless the enviroment are treated anaerobically, obligate anaerobe bacteria will die in presence of oxygen.

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25
Q

Which organisms could grow aerobically on slant? Why?

Escherichia coli
Micrococcus luteus
Clostridum sporogenes

A

Escherichia coli and micrococcus luteus, because they are able to grow in the presence of oxygen.

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26
Q

Identify the oxygen requirements of the following bacteria:

Escherichia coli
Micrococcus luteus
Clostridum sporogenes

A

Escherichia coli=faculative anaerobe
Micrococcus luteus=aerobe
Clostridum sporogenes=anaerobe

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27
Q

Oxygen Requirement lab results for:

Escherichia coli
Micrococcus luteus
Clostridum sporogenes

A

Escherichia coli, grows throughout tube but better at top, grows on slant.

Micrococcus luteus, grows only near top of tube, grows on slant.

Clostridum sporogenes, no growth near top of tube, no growth on slant.

28
Q

Why in a oxygen requirement lab did we boil the agar deeps longer than it took to merely melt the agar.

A

You boil the agar deep 10 minutes so that you can drive out all the oxygen

29
Q

If air can diffuse into agar and broth, how were the anaerobes grown in the broth for the class?

A

Using an anaerobic jar or reduced broth.

30
Q

How does synthetic (defined) media differ from non-synthetic (complex ) media?

A

Syntactic media are produced using precise amounts of pure chemicals . Non-synthetic media contains at least one ingredient with an imperfectly characterized nutrient content.

Complex media are rich in minerals and nutrients and are often used for growing a wide variety of microorganisms, including fastidious ones. Defined media, on the other hand, consist of ingredients whose chemical formulas are known. These pure substances are carefully measured and dissolved in double-distilled water.

31
Q

Which characteristics defines a selective medium?

A

Support the growth of some types of microorganisms wile purposefully suppressing the growth of other types of microorganisms.

32
Q

What characteristics define a differential medium?

A

Allow one to visually differentiate between multiple species growing on the medium. Bacterial colonies may display a unique appearance such as a particular color, or they may produce viable changes in the media itself.

33
Q

What is a growth factor?

A

Growth factors are secreted proteins that regulate a variety of biological processes, including cell proliferation, differentiation, survival, and migration. Required by the organism.

34
Q

What is a pH indicator and how is it useful?

A

a chemical compound which is added to a solution to visually determine the pH (acidity or basicity) of the solution. The characteristic color changes indicate the acidic, basic, or neutral character of the substance being tested.

35
Q

Which of the 4 organisms below can grow in Simmon’s citrate agar?

Bacillus subtilis
Escherichia coli
Serratia marceeescens
Staphylococcus aureus

A

Bacillus subtillis and Serratia marcescens

36
Q

What is possessed by the species that can grow on the surface of Simmons’ citrate agar? Do they require growth factors?

A

The ability to use citrate as sole carbon source and use ammonium ions as sole nitrogen source.

37
Q

Why might a bacterial species be unable to grow on Simmons’ citrate media?

A

They are unable to use citrate for carbon and ammonium for nitrogen.

38
Q

A student performs a citrate utilization test on a bacterium that should be citrate positive but observes no color change after a 48-hour incubation. What simple mistake in the procedure might have caused this false negative results?

A

Student might have failed to keep the citrate slants cap looses during incubation. Resulting in a false negative test.

39
Q

A student performs a citrate utilization test on a bacterium that should be citrate negative, but the media turns blue after a 48-hour incubation. What simple mistake in the procedure might have caused the aberrant results.

A

They might have used bacteria form a broth culture which risks the transfer of nutrients into the nitrate media causing a false positive result.

40
Q

Citrate Utilization Lab Procedure:

A
  1. Label Simmons’ citrate agar slants.
  2. Inoculate the surface of theses four slants with the four bacterial species, using a light inoculate from the solid media.
  3. Loosen each slant’s cap 1/4 to 1/2 turn to allow gas exchange.
  4. Incubate at 37C for 24-48 hours. If media has not turned deep blue in 24 hours, incubate for the full 48 hours.
  5. Examine the growth and color change. Blue=poss Green=neg
41
Q

What makes up Simmons’ citrate agar?

A

Synthetic (defined), both selective and differential. Consists of citrate, few inorganic salts ( including ammonium ion), bromothymol blue, agar, and water.

Citrate serves as carbon and energy source. Ammonium ions serve as nitrogen source.

Bromethymol blue is pH indicator. Green with pH of 6.9 and below and blue at 7.6 and above.

42
Q

Reading citrate test result:

A

Blue= positive
Greeen=negative

43
Q

Why do animals produce urea?

A

animals must detoxify ammonia by converting it into a relatively nontoxic form such as urea or uric acid. Because they secrete urea as the primary nitrogenous waste product.

44
Q

What is a virulence factor?

A

Virulence is described as an ability of an organism to infect the host and cause a disease. Virulence factors are the molecules that assist the bacterium colonizing the host at the cellular level.

45
Q

Urease

A

Enzyme that hydrolyzes urea into carbon dioxide and ammonia, resulting in a significant increase in the area pH.

46
Q

Christensen’s Urea Agar

A

Complex and differential media containing urea, peptone, glucose, a few salts, an phenol red.

47
Q

Urea Hydrolysis Lab Procedure

A
  1. Obtain Christensen’s Urea agar slants.
  2. Inoculate each slant surface with bacteria.
  3. Loosen slant cap 1/4 to 1/2 turn for gas exchange.
  4. Incubate the slants for 43 hours. The observe slants for color change and record results.
  5. If no color change is noted after an overnight incubation, incubate for additional 6 days.
  6. observe slants for color change.
48
Q

Which of theses bacteria displayed unease activity?
Escherichia coli
Klebella pneumoniae
Proteus vulgaris
Staphylococcus aureus

A

Klebsiella pneumoniae and Proteus vulgaris

49
Q

In molecular terms, why do some bacteria produce the enzyme urease whereas other species do not.

A

Depends on if the enzyme produces urea or not.

50
Q

If a bacterium lacks the ability to hydrolyze urea, does this mean it does not have virulence factors? Explain.

A

No, other virulence factors present?

51
Q

What component of urea agar makes it a complex medium?

A

urea, pep-tone, glucose, salts

52
Q

Interpreting urea lab results

A

Pink= positive
Yellow=negative

Clear positive results after a 24 hour incubation indicate rapid urea hydrolysis. Weak positive results after a 24 hour incubation indicate substantially slower hydrolysis of urea. Delayed positive organisms may take a week to turn the entire media pink.

53
Q

Why to humans produce bile salts?

A

The bile helps break down and digest the fats present in food. Another primary function of bile that bile salts help with is the removal of toxins. Toxins (bacteria) are secreted into the bile and eliminated in feces. Antimicrobial nature.

54
Q

What is a selective medium?

A

A selective medium is a medium that allows the selection of one or more types of microorganisms.

55
Q

What is a differential medium?

A

contains specific ingredients or chemicals that allow the observer to visually distinguish which species possess and which species lack a specific biochemical process

56
Q

Which of theses bacteria were able to grow in bile esculin media?

E.coli
Enterococcus faecalis
Klebsiella pneumoniae
Staphylcoccus aureus

A

Enterococcus faecalis and Klebsiella pneumoniae

57
Q

Which of theses bacteria were able to hydrolyses esculin in the presence of bile salts?

E.coli
Enterococcus faecalis
Klebsiella pneumoniae
Staphylcoccus aureus

A

Enterococcus faecalis and Klebsiella pneumoniae

58
Q

How is bile esculin media selective, and what types of organisms does it inhibit? Are all bacteria of the type inhibited? Explain.

A

Selective due to present of bile slats. Selective for Gram-negative bacteria. Inhibits Gram-positive bacteria.

59
Q

How is esculin media differential, and how might its appearance differ with different bacteria?

A

Differential due to presence of ferric ions (Fe3+) which reacts with esculetin to produce an insoluble, black colored ion salt.

60
Q

What component or components of bile esculin agar makes it a complex medium?

A

Presence of bile salts, esculin, and ferric citrate.

61
Q

Where are bile salts found in the body?

A

Produced by the liver and stored in the gallbladder before being secreted into the small intestine.

62
Q

What does bile esculin test for?

A

The ability of a bacteria to hydrolize esculin in the presence of slats.

63
Q

What purpose is a bile esclin test normally used for?

A

Differentiate between Gram- positive enterococci and group D streptococci

64
Q

What do bile esclin test results look like?

A

Growth= Gram-negative
No growth = Gram-positive

Black color= esculin hydrolyzed
No color change= escuulin not hydrolyzed

65
Q

What is Esculin?

A

Glycoside, consisting of glucose contently bonded to a two-ringed aromatic compound. Can be hydrolyzed to from glucose and esculetin. Many bacteria can do this but no in the presebce of bile salts.

Microbiology Lab (7 decks)

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