Microbiology Lab Midterm

Front 1

Bacillus subtilis

Back 1

Endospores, Gram Positive, Rod, Aerobic

Front 2

Citrobacter freundii

Back 2

Gram negative, Rod, Anaerobic, Positive for Fermentation

Front 3

Clostridium sporogenes

Back 3

Endospores, Gram positive, Rod, Anaerobic

Front 4

Staphylococcus aureus

Back 4

Gram Positive, Coccus, Catalase Positive, Facultative Anaerobes

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Mycobacterium

Back 5

Acid Fast, Rod, Aerobic

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Pseudomonas aeruginosa

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Gram negative, Rod, Aerobic, Negative for Fermentation

Front 7

Spirillum itersonii

Back 7

Endospore, Gram negative, Spirillum, Faculatative Anaerobic

Front 8

Enterococcus faecalis

Back 8

Gram positive, Coccus, Catalase negative, Anaerobic

Front 9

Escherichia coli

Back 9

Gram negative, Rod, Facultative anaerobic, Positive for Fermentation

Front 10

Streptococcus pyogenes

Back 10

Gram positive, coccus, catalase negative, facultative anaerobic

Front 11

Proteus vulgaris

Back 11

Gram positive, Rod, Facultative Anaerobe, Positive for fermentation

Front 12

Enterobacter aerogenes

Back 12

Gram negative, Rod, Facultative Anaerobic, Positive for Fermentation.

Front 13

Staphylococcus epidermidis

Back 13

Gram positive, Coccus, Catalase Positive, Facultative anaerobic

Front 14

Why is a dense or thick smear difficult to view under the microscope?

Back 14

It diminishes the amount of light able to pass through the organism making it difficult to distinguish features.

Front 15

Why do smears need to be air dried? What would happen if the smear was gently flamed to speed up the drying process?

Back 15

Overheating will denature and rupture cell walls. Excess water on the slides will boil during fixation. Aerosols could be formed and produce a hazardous contaminant.

Front 16

Why is it necessary to heat fix the smear?

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Heat-fixing kills the bacteria, makes them adhere to the slide, and coagulates cytoplasmic proteins to make them more visible.

Front 17

What would happen if you overheat the smear while heat-fixing?

Back 17

The sample’s cells would become distorted which could lead to an overall morphological change.

Front 18

Why could fingerprints on a glass slide result in a poor smear?

Back 18

Bacteria could adhere to the oils in our fingerprints and resist stainingIt could diminish the amount of microscopic light able to pass through making it more difficult to examine the sample

Front 19

Can simple staining identify more than the morphology and arrangement of bacteria?

Back 19

Morphology, size, and arrangement are the only things that a simple stain can determine.

Front 20

Why are basic dyes more effective than acidic dyes when staining bacteria?

Back 20

Basic dyes are attracted to the negative charges on the surface of most bacterial cells.

Front 21

What would you see under the microscope if you forgot to heat fix your smear before staining it?

Back 21

A live motile bacterial sample that is poorly stained.

Front 22

Why does a negative stain color the background and not the bacteria?

Back 22

The dye solution has a chromogen that is acidic and carries a negative charge. The negative charge of the bacteria then repels the basic dye and results in an unstained bacteria with a colored background.

Front 23

Why can’t methylene blue be used in negative staining?

Back 23

Methylene blue has a basic chromogen (i.e. positive basic attracts to negative bacteria)

Front 24

What is the advantage of negative staining? Why?

Back 24

To determine morphology and cellular arrangement in bacteria that are too delicate to withstand heat-fixing and/or an accurate size is needed to be determined. Heat-fixing causes distortion and can lead to improper assessment on morphologyNegative staining produces minimal cell shrinkage.

Front 25

What is a chemically defined media?

Back 25

composed of exact amounts of chemically pure, specifically identified organic or inorganic components. Examples include glucose salt broth or inorganic synthetic broth.

Front 26

Why is some media considered undefined or complex?

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composed of organic materials that are not chemically pure and not specifically identified chemical components. Examples include Nutrient Broth/Agar, Tryptic Soy Broth/Agar, and Blood agar.

Front 27

What nutrients do the following media ingredients supply?

Back 27

Beef Extract:A water-soluble substance which aids in bacterial growth (i.e. food)

Yeast Extract:A water-soluble substance which aids in bacterial growth by providing Vitamin B (i.e. food)

Peptone:An enzymatic digest of animal protein that is the principal source of organic nitrogen for the growing bacteria

Agar:An indigestible solidifying agent

Front 28

What is the advantage of performing a gram stain over performing just a simple stain?

Back 28

It is a differential stain that allows a microbiologist to detect differences between organisms with thicker peptidoglycan walls vs cell enveloped organisms

Front 29

List in order of the reagents used in the gram stain.

Back 29

Crystal Violet Mordant: IodineDecolorizer: alcohol or acetoneCounterstain: Safranin

Front 30

What color would the gram + cells stain with each step?

Back 30

Crystal Violet

Front 31

What color would the gram - cells stain with each step?

Back 31

Safranin

Front 32

Predict the effect on gram + and gram - cells if the following mistakes were made when performing the gram stain.

Back 32

Failure to add iodine.The crystal violet iodine complex would not form and the crystal violet would not adhere to the gram positive organisms. Failure to apply decolorizer.The Gram negative organisms would retain both of the dyes Failure to apply counterstain.The Gram negative organisms would appear clear

Front 33

What is the most critical step when doing a gram stain? Why?

Back 33

Decolorization because you can over-decolorize and under-decolorizeOver-decolorizing results in reddish Gram positive cells. Under-decolorizing results in purple Gram negative cells

Front 34

Why is it important to perform a gram stain with a fresh culture (24 hour or younger)?

Back 34

Old samples lose their ability to retain the crystal violet-iodine complex, especially Bacillus and Staphylococcus.

Front 35

What is the purpose of phenylethyl alcohol agar (PEA)?

Back 35

An undefined selective medium that inhibits the growth of Gram-negative (some Gram-positive) used to isolate staphylococci and streptococci. To screen out Escherichia coli and Proteus species

Front 36

Is PEA selective or differential or both? Why?

Back 36

Selective medium because it does not differentiate between the organisms that can grow on it.

Front 37

What is the purpose of B-phenylethyl alcohol in PEA?

Back 37

The selective agent and may be bacteriostatic or bactericidal, depending on the concentration.

Front 38

What is the purpose of Columbia CNA with 5% sheep’s blood agar?

Back 38

It is an undefined, differential, and selective medium that allows growth of Gram-positive organismsIt isolates staphylococci, streptococci, and enterococci, from clinical specimens.

Front 39

Is mannitol salt agar selective or differential or both?

Back 39

Both! Differential due to the Mannitol, which provides the substrate for fermentation. Selective due to the high salt concentration

Front 40

What is the purpose of the high salt concentration in the mannitol salt agar media?

Back 40

Sodium chloride makes the medium selective because the high concentration is high enough to dehydrate and kill most bacteria.

Front 41

Why mannitol agar contain mannitol and phenol red?

Back 41

Mannitol is a carb that can not be fermented by all staphylococci except S. aureus. Fermenters appear yellow. Nonfermenters that can grow will appear pin.Phenol red indicates whether fermentation with an acid end-product has taken place by changing color as the pH changes.

Front 42

Can you determine mannitol fermentation ability of bacteria that is not salt tolerant?

Back 42

No, an organism that is not salt tolerant would not be able to survive or ferment the mannitol

Front 43

MacConkey agar is selective, differential, or both?

Back 43

Both

Front 44

What is the purpose of crystal violet and bile salts in MacConkey agar?

Back 44

Inhibit the growth of Gram-positive bacteria

Front 45

What is the purpose of lactose and neutral red in MacConkey medium?

Back 45

Neutral red is a pH indicator that is colorless above a pH of 6.8 and red at a pH less than 6.8Lactose is fermented into acid end-products that lower the pH and result in colonies that turn a pink to red color.

Front 46

Nitrate broth contains 0.1% agar. Explain the function of the agar?

Back 46

It makes the media semisolid which selects for the organisms that are anaerobic which is required for nitrate reduction

Front 47

What is the purpose of Solutions A & B in Nitrate Reductions Tests?

Back 47

When there is no initial evidence of denitrification the solutions are added to test for nitrate reduction to nitrite. It is evidenced by a red color change

Front 48

What does it mean if there is no color change after adding Solutions A & B?

Back 48

No change to red means that the nitrate either was not reduced or was reduced to one of the other nitrogenous compounds.

Front 49

Why does the development of a red color after adding zinc indicate a negative test?

Back 49

The red color indicates that nitrate was not reduced by the organism because it may have catalyzed the reduction of any nitrate from KNO3 to nitrite.

Front 50

What is the purpose of having an uninoculated tube? Is it a positive or negative control?

Back 50

An uninoculated tube is needed as a negative control to compare all reactions to it. Negative control- no response is expectedPositive control- a response is expected

Front 51

What is fermentation?

Back 51

The metabolic process by which an organic molecule acts as an electron donor (becoming oxidized in the process) and one or more of its organic products acts as the final electron acceptor and becomes reduced.

Front 52

What is the purpose of using uninoculated controls?

Back 52

To have a basis to compare your results.

Front 53

Why does the medium contain phenol red?

Back 53

In order to detect acid and gas formation by observing a color change. It is yellow below a pH of 6.8, pink above 7.4, and red in-between. Alkaline reaction from the NH3 causes a pink color when no acid is produced.

Front 54

Why does the medium contain a Durham tube?

Back 54

As an indicator of gas production to indicate a bubble or pocket that displaces the broth formed by the organism.

Front 55

What is the purpose of the TSI test?

Back 55

The triple sugar iron agar is designed to differentiate bacteria (mostly Enterobacteriaceae) on the basis of sulfur reduction AND glucose, lactose, and sucrose fermentation.

Front 56

Why is it important to read the tubes no longer than 18-24 hours after incubation?

Back 56

Because the slants could go into reversion and produce an alkaline product thus allowing only identifying aerobic fermentation of glucose (and not sucrose or lactose like an earlier test would indicate).

Front 57

Why does TSI medium contain a lower concentration of glucose, than sucrose and lactose?

Back 57

Glucose causes acid products that lower the pH and turn the entire medium yellow. Therefore, as glucose diminishes ammonia will be produced causing the pH to rise and turn the medium red again. Process is called reversion and that is what we are observing.

Front 58

Why does TSI medium contain phenol red?

Back 58

It is the pH indicator that is yellow at a pH less than 6.8 and reddish above 7.4.

Front 59

Why does TSI medium contain thiosulfate

Back 59

It includes ferrous sulfate and sodium thiosulfate as sources of oxidized sulfur.

Front 60

If a black precipitate is produced in the butt, how do you know if the butt is alkaline or acidic?

Back 60

Ferrous sulfate reacts with the hydrogen sulfate to form a black precipitate and indicates that the sulfur reduction and glucose fermentation occurred. It is acidic since it is an acid condition from glucose fermentation.

Front 61

What is the significance of a methyl red test?

Back 61

It is designed to detect organisms capable of performing a mixed acid fermentation that is capable of overcoming the phosphate buffer in the medium and lowers the pH. Methyl red is red (positive) at pH 4.4 and yellow (negative) at pH 6.2.

Front 62

Why is bromothymol blue added to citrate medium?

Back 62

To indicate that citrate is utilized by the organism and that citrate permease is present.

Front 63

Why is it better to inoculate citrate slants with a needle

Back 63

To avoid confusion between actual growth and a heavy inoculum, which may be misinterpreted as growth.

Front 64

Is citrated medium a complex or chemically defined medium? Why?

Back 64

Chemically defined medium. Sodium citrate is the only carbon source in the medium and it provides the means for bacterial species that possess the enzyme citrate permease to transport citrate into the cell and perform citrate fermentation.

Front 65

How is the SIM medium used to detect motility

Back 65

The reduced agar concentration and the method of inoculation (single stab). Motile organisms are able to move about in the semisolid medium and their tracks can be detected.

Front 66

What is the function of ferrous ammonium sulfate in SIM medium?

Back 66

Reacts with the hydrogen sulfide gas to form ferric sulfide, which is a black precipitate that indicates a sulfur reduction and a positive test.

Front 67

What does Kovac’s reagent react with and what does a positive indole test indicate?

Back 67

Kovac’s reagent= DMABA DMABA reacts with any indole present and produces a quinoidal compound that turns the reagent layer red.A positive (red) indole test indicates the presence of tryptophanase.

Front 68

Why is heat necessary when applying the primary stain, but not the counterstain?

Back 68

Heat helps the primary stain penetrate the mycolic acid lipid membrane so the decolorizer will not wash out the carbolfuchsin

Front 69

Why is acid alcohol used as the decolorizing agent instead of ethyl alcohol?

Back 69

Mycolic acid fast cells resist this decolorization.

Front 70

What diseases can the acid fast stain help diagnose?

Back 70

Tuberculosis and leprosy

Front 71

How do you distinguish between Brownian movement and true motility?

Back 71

Brownian movement appears more like vibrations while true motility is more obvious.

Front 72

What are the advantages and disadvantages of the multi-test systems?

Back 72

Advantages: Minimal storage space, less media, speed of results, and computer identification.Disadvantages: Right amount of inoculation needed, compartment contamination, or improper age of inoculate

Front 73

Antibiotics

Back 73

Natural antimicrobial agents produced by microorganisms. Penicillium notatum produces penicillium

Front 74

Antimicrobials/antimicrobics

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Agents used to treat bacterial infections that are synthetic

Front 75

Zone of Inhibition

Back 75

The cleared area on an agar plate that indicates the organism is susceptible to the drug.

Front 76

Minimum inhibitory Concentration

Back 76

The junction of the zone of inhibition with growth where the concentration of anti-microbiotic has become too low to effectively stop growth.

Front 77

Bactericidal

Back 77

Drugs that kill the organism.

Front 78

Bacteriostatic

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Drugs that stop the bacteria from dividing but do not kill them.

Front 79

0.5 McFarland Turbidity Standard

Back 79

Corresponds to a cell density between 1 and 2 * 10^8 CFU/mL; this is the requirement necessary for bacterial suspensions so that the number of bacteria will be within a given range to standardize microbial testing.

Front 80

Resistance breakpoint

Back 80

The zone diameter below which all resistant strains are categorized; determined by the Clinical Laboratory Standards Institute

Front 81

Susceptibility breakpoint

Back 81

The zone diameter below which all susceptible strains are categorized; determined by the Clinical Laboratory Standards Institute.

Front 82

Antimicrobial Susceptibility Test

Back 82

used to standardize and measure the effectiveness of antibiotics and other chemotherapeutic agents on pathogenic microorganisms.

Front 83

Disinfectants

Back 83

An antimicrobial agent that kills the vegetative cells, but not the endospores, of disease-producing organisms. It can only be used on inanimate surface. It does not sterilize.

Front 84

Antiseptics

Back 84

Safe enough to apply to skin or mucous membrane (cannot be taken internally). It does not sterilize.

Front 85

Sterilize

Back 85

Completely destroy or remove all microorganisms.

Front 86

Use Dilution Test

Back 86

A standard procedure used to measure the effectiveness of disinfectants specifically against S. aureus, S. enterica, P. aeruginosa, and serovar Choleraesuis. Glass beads or stainless steel cylinders coated with living bacteria are exposed to varying concentrations (dilutions) of test germicides and then transferred to a growth medium. The solution must prevent microbial growth 95% of the time to be considered a suitable germicide.

Front 87

Indicator Bacteria

Back 87

Present in large numbers compared to the specific pathogen More likely to be constantly present rather than intermittentCan be monitored for just the one indicator rather than many different pathogensIndicator is safer to work with than the pathogens and it is easier to train lab workers to handle it.Can be used in the detection of deficiencies, such as insufficient chlorineCan detect a break in the distribution system by the presence of the indicator bacteria

Front 88

Coliforms

Back 88

Most used indicatorsGram-negative, facultative anaerobic rods, and ferment lactose to produce acid and gas in 24-48 hrs at 35oC. Typically Escherichia coli since it is present in the intestinal tract of warm-blooded animals. It’s presence in water indicates that fecal matter might be/have been presentIf E. coli is present, then enteric pathogens may be present.

Front 89

Enteric Pathogens

Back 89

Coliform groups that include Enterobacter aerogenes, Citrobacter freundii, & Klebsiella pneumoniae

Front 90

Most Probable Number

Back 90

The MPN procedure detects coliforms. Presumptive Test- lactose fermentation is detected; results are semiquantitative, allowing an estimation of the number of coliforms in the sampleConfirmed Test- Organisms that gave a positive presumptive test are grown on media that favor Gram-negative organisms and are differential for lactose-fermentation. Completed Test- Substantiates that the organism present is a coliform.

Front 91

Presence-Absence Test

Back 91

Performed instead of the multi-tube test, in which a larger volume of sample is employed and results are results are read as coliform positive or coliform absent.

Front 92

Membrane Filtration

Back 92

A non-turbid water sample is filtered onto a bacteriological membrane filter and incubated on selective/differential media. The number of coliforms in a given volume can be quantified.

Front 93

Does a positive Presumptive Test mean that the water is absolutely unsafe to drink?

Back 93

No, it is possible that non-coliforms can produce gas.

Front 94

What might explain the occurrence of false-positive presumptive results?

Back 94

2 or more non-coliforms working to perform sufficient gas to cause a false positive.

Front 95

What are the characteristics of an ideal indicator of fecal contamination?

Back 95

Gram-negative, facultative anaerobic rods, and ferment lactose to produce acid and gas in 24-48 hrs at 35oC.

Front 96

Why don’t health departments routinely test for pathogens instead of using a fecal contamination indicator?

Back 96

There are many more pathogens and indicator are easier to detect.

Front 97

Is E.coli always “just an indicator” or can it sometimes be a pathogen?

Back 97

Yes, because there are multiple strains or E.coli and some can be pathogenic.

Front 98

Aerosol

Back 98

Fine liquid droplets or solid particles that remain suspended in air

Front 99

Aseptic

Back 99

Free from pathogenic microorganisms

Front 100

Disinfect

Back 100

To reduce the number of pathogens on a material until they are no longer a hazard. (Some living microbes may remain)

Front 101

Escherichia Coli IMViC

Back 101

Indole (+), Methyl-Red (+), Voges-Proskauer (-), and Citrate (-)

Front 102

Enterobacter aerogenes IMViC

Back 102

Indole (-), Methyl-Red (-), Voges-Proskauer (+), and Citrate (+)