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Bacte LAB > ISOLATION OF ENTERIC BACTERIA > Flashcards

ISOLATION OF ENTERIC BACTERIA Flashcards

1
Q

PART I
Materials: known enteric cultures and small spatulas, MAC, EMB, tubes of sterile saline, Gram stain reagents.

  1. Obtain MacConkey agar and EMB agar, and label them on the bottorn side with your name, the name of the medium, the date, and the designation
    “_____sample.” Obtain a loopful of the inoculum from cultures of enteric organisms and streak each plate for isolated colonies. Invert the plates, and incubate them at _____ (temp) for _____(hours)
  2. Observe the MacConkey agar for evidence of_______, which will appear as___ colonies. Escherichia coli will usually be_____, while Enterobacter, Klebsiella, and other lactose fermenters will be_____ with a____ texture.
    Colonies of non-lactose fermenters will appear______. Enter a labeled representation of the plate in the Results section. Gram stains should be prepared to confirm the presence of______
  3. Inspect the EMB plate and take note of pigmented colonies that appear
    ________when viewed in reflected light. These are probably colonies of Escherichia coli. Other pigmented colonies may be convex and mucoid with____ centers and ____borders. These may be Enterobacter aerogenes.
    Non-lactose fermenters will yield_____ colonies. A labeled representation of the plate should be made in the Results section. Gram stains should be prepared and observed for_____, and diagrams should be placed in the appropriate spaces.
A

fecal; 35°C for 24 to 48 hours

lactose-fermenting bacteria; red; brick red; pink to red; mucoid

colorless and transparent; gram-negative rods

blue-black with a green metallic sheen

dark; creamy pink

colorless

gram-negative rods

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2
Q

PART I
Materials: known enteric cultures and small spatulas, MAC, EMB, tubes of sterile saline, Gram stain reagents.

  1. Obtain MacConkey agar and EMB agar, and label them on the bottorn side with your name, the name of the medium, the date, and the designation
    “fecal sample.” Obtain a loopful of the inoculum from cultures of enteric organisms and streak each plate for isolated colonies. Invert the plates, and incubate them at 35°C for 24 to 48 hours.
  2. Observe the MacConkey agar for evidence of______, which will appear as red colonies. ______will usually be brick red, while ______(3) will be pink to red with a mucoid texture. Colonies of_____ will appear colorless and transparent.

Enter a labeled representation of the plate in the Results section. Gram stains should be prepared to confirm the presence of gram-negative rods.

  1. Inspect the EMB plate and take note of pigmented colonies that appear
    blue-black with a green metallic sheen when viewed in reflected light. These are probably colonies of______. Other pigmented colonies may be convex and mucoid with dark centers and creamy pink borders. These may be______.

______ will yield colorless colonies. A labeled representation of the plate should be made in the Results section. Gram stains should be prepared and observed for gram-negative rods, and diagrams should be placed in the appropriate spaces.

A

lactose-fermenting bacteria

Escherichia coli

Enterobacter, Klebsiella, and other lactose fermenters

non-lactose fermenters

Escherichia coli

Enterobacter aerogenes

Non-lactose fermenters

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3
Q

PART II
Materials: TSI agar slant, IMViC tube series, LIA, urea agar slant, isolated enteric bacteria from Part I.

  1. Using the plates from Part I, choose a lactose-fermenting colony of bacteria and/or a non-lactose fermenter. Label all tubes appropriately.
  2. TSI: Using a sterile inoculating needle, touch the top of a well-isolated colony. Inoculate TSI by first stabbing at least____ the depth of the TSI medium and then streaking the surface of the agar slant.

An uninoculated control tube should be included in the experiment.

Be certain the cap is loose, and incubate the tubes at_____. the tubes should be examined after___ hours of incubation or, if that is not possible, they should be____.

A

¾

35°C

24

refrigerated

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4
Q

TSI

Expected results:
• Alkaline slant/no change in the butt (K/NC) =

• Alkaline slant/acid butt (K/A) =

• Acid slant/acid butt: (A/A) =

Note: A black precipitate in the butt indicates production of______ and ______.

Bubbles or cracks in the tube indicate the production of_____.

Drawing a circle around the A for the acid butt, usually indicates the organism ferments glucose and sucrose, glucose and lactose, or glucose, sucrose, and lactose, with the production of gas.

Gram stains may also be prepared to confirm the presence of gram-negative rods.

A

glucose, lactose, and sucrose non-fermenter; this may also be recorded as K/K (alkaline slant/alkaline butt)

glucose fermentation only

glucose, sucrose, and/or lactose fermenter

ferrous sulfide and H2S gas

CO2 or H2

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5
Q
  1. LIA: With a straight inoculating needle, inoculate LIA by __________.

Cap the tube tightly and incubate at___ (temp) in ambient air for_____(hours)

A

twice stabbing through the center of the medium until almost at the bottom of the tube and then streaking the slant

35C

18-24 hours

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6
Q

• Red slant/acid butt (R/A)

LIA

Expected results:
• Alkaline slant/alkaline butt (K/K)
=

• Alkaline slant/acid butt (K/A) =

Note: Patterns can be accompanied by a black precipitate of_______, which indicates production of_____.

A

lysine decarboxylation and no fermentation of glucose

glucose fermentation

lysine deamination and glucose fermentation

ferrous sulfide (FeS)

H2S

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7
Q
  1. Citrate: Inoculate ______agar lightly on the____ by touching the tip of a needle to the same colony.

Note: There is no need to stab into the___ of the tube. Do not inoculate from a broth culture, because the inoculums will be too heavy. Incubate at____ for up to____. Observe for development of____ color, denoting alkalization.

A

Simmons citrate ; butt

35-37; 7 days; blue

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8
Q

Citrate

Positive:_____________

The color change of the indicator is due to acid or alkali production by the test organism as it grows on the medium.

Growth usually results in the______ indicator, turning from______.

Negative:______

A

Growth on the medium, with or without change in the color of indicator.

bromthymol blue; green to blue

Absence of growth.

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9
Q

Urea: Streak the_____ of a ______ with a portion of a well-isolated colony. Leave the cap on loosely and incubate tube at____ in ambient air for_____-_____

A

surface; urea agar slant

35C

48 hours to 7 days.

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10
Q

Urea

Positive:

Negative:

A

Change in color of slant from light orange to magenta

No color change (agar slant and butt remain light orange)

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11
Q
  1. SIM (or MIO): Touch a sterile inoculating needle to the same colony of a young (18 to 24-hour) culture growing on either MAC or EMB.

Stab once to a depth of only _____inch in the middle of the tube. Incubate at___\ for _____. Examine and record results.
Examine for motility:

A

1/3 to ½

35 C for 24 hours

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12
Q

SIM

Positive=

Negative =

Examine for Sulfide production:
Positive =______ in the medium; negative =_____

A

motile organisms will spread out into the medium from the site of inoculation.

nonmotile organisms remain at the site of inoculation.

blackening; no blackening

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13
Q

SIM

Examine for indole production:

While wearing disposable gloves, add_______reagent to the SIM tube, and shake the tube gently.

A _____develops in the presence of indole. Negative reactions remain______ or _____.

A

0.5 ml (about 10 drops) of Kovac’s

deep red

colorless or light yellow

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14
Q

Methyl Red–Voges Proskauer (MR-VP) Test

Label the MRVP broth tubes to be inoculated (1ST MR, 2ND VP).

Using aseptic technique, inoculate each tube with the unknown bacterium (same isolated colony) by means of a loop/needle. Incubate tubes at 35°C
for______.

For slow fermenters, it may take_____.

A

24 to 48 hours; 4 to 5 days

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15
Q

MRVP

To the MR culture tube while wearing disposable gloves, add 5 drops of ______indicator. Examine the color of the culture.
A positive reaction is indicated by a distinct____ color, showing the presence of____.
A negative reaction is indicated by a_____ color.

A

methyl red

red; acid

yellow

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16
Q

MRVP

To the VP tube,

add 0.6 ml (12-15 drops) of________in _____ and

0.2 ml (4-5 drops) of_____

Examine the color of the VP culture.

A pink color indicates the formation of_____.

No change in the color of the medium indicates a negative reaction.

Record your results in the Results section. Illustrate or take pictures of the biochemical results.

A

5% alpha-naphthol in absolute ethyl alcohol

40% potassium hydroxide

Pink= acetyl methyl carbinol

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17
Q

is both selective and differential.

It is designed to isolate and differentiate enteric bacteria, especially members of the Enterobacteriaceae family, based on their ability to ferment lactose.

A

MacConkey agar

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18
Q

MAC

Selective
Differential

A

S: Bile salts, Crystal violet
D: Lactose, Neutral red

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19
Q

MAC selective

inhibit the growth of non-enteric bacteria, which are often sensitive to the_____ found in the intestinal environment.

By preventing the growth of non-enteric organisms, it help to ensure that primarily enteric bacteria (those adapted to survive in the gut) are able to grow on the agar.

A

Bile salts; bile acids

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20
Q

MAC selective

further selects against gram-positive bacteria, which have a more sensitive cell wall structure that is disrupted by this dye.

This enhances the growth of gram-negative bacteria, which are more likely to include enteric pathogens and normal flora.

A

Crystal violet

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21
Q

MAC differential

is a fermentable carbohydrate that provides a way to differentiate between lactose-fermenting and non-lactose-fermenting bacteria.

Bacteria that ferment lactose produce acidic byproducts.

A

Lactose

22
Q

MAC differential

is an indicator dye that changes color based on pH. When lactose is fermented, acids are produced, causing the pH around the bacterial colony to drop.

At a lower pH, neutral red is taken up by the bacteria, causing lactose-fermenting colonies to appear pink or red.

Non-lactose fermenters do not produce acid, so their colonies remain colorless or transparent.

A

Neutral red

23
Q

MAC

Lactose-Fermenting Bacteria: These bacteria (e.g.,_______) appear as pink or red colonies because they produce acid that lowers the pH, leading to the uptake of the neutral red dye.

Non-Lactose-Fermenting Bacteria: These bacteria (e.g.,_______) do not ferment lactose, so their colonies remain colorless, indicating a lack of acid production.

A

Escherichia coli

Salmonella, Shigella

24
Q

EMB

Selective
Differential

A

S: Eosin and Methylene
D: Lactose

25
Q

EMB

act as selective agents by inhibiting the growth of most gram-positive bacteria. Additionally, they help differentiate lactose fermenters based on pigmentation.

A

Eosin and Methylene Blue

26
Q

EMB

Strong Lactose Fermenters (e.g.,______): These bacteria produce high levels of acid, leading to colonies that appear blue-black with a green metallic sheen when viewed in reflected light.

Weak Lactose Fermenters (e.g.,_______): These colonies often appear as mucoid with dark centers and a creamy pink border due to moderate acid production.

Non-Lactose Fermenters: These bacteria do not produce acid, so their colonies remain colorless or take on the natural color of the agar.

A

Escherichia coli

Enterobacter aerogenes

27
Q

is a differential medium primarily used to detect the decarboxylation or deamination of lysine and glucose fermentation in bacteria, particularly enteric bacteria.

A

Lysine Iron Agar (LIA)

28
Q

Lysine Iron Agar (LIA)

Inoculation Procedure:

Prepare the Inoculating Needle: Use a straight inoculating needle to transfer the bacterial sample.

Inoculate the Butt: Start by_______ the center of the LIA medium deeply with the needle until almost at the bottom of the tube. This step ensures the bacteria are exposed to____ conditions in the deeper part of the medium.

Inoculate the Slant: After the stab, ______of the medium with the same needle. This exposes the bacteria to_____ conditions on the slanted surface.

Seal and Incubate: Cap the tube_____ to maintain the anaerobic conditions in the butt and incubate at____ for 18-24 hours. This temperature is optimal for the growth of enteric bacteria.

A

stabbing; anaerobic

streak the surface (slant) ; aerobic

tightly

35°C

29
Q

LIA

Reaction: Both the slant and the butt of the tube remain red (alkaline) throughout the incubation period.

Interpretation: This result indicates that the organism has decarboxylated lysine, but no fermentation of glucose occurred.

Lysine decarboxylation produces_____ byproducts, which raise the pH of the medium, turning it red. The butt stays alkaline because no fermentation of glucose (which would produce acid) occurred.

A

Alkaline Slant/Alkaline Butt (K/K)

alkaline

30
Q

LIA

Reaction: The slant remains red (alkaline), but the butt turns yellow (acidic).

Interpretation: This result suggests that the organism has fermented_____, producing acid that turns the butt yellow, but did not decarboxylate lysine.

The slant remains alkaline because the bacteria in the aerobic slant area exhaust the glucose and metabolize peptones, which produces alkaline byproducts, turning the slant red.

A

Alkaline Slant/Acid Butt (K/A)

glucose

31
Q

LIA

Reaction: The slant turns red (alkaline) while the butt turns yellow (acidic).

Interpretation: This result indicates that the organism has________(removed the amino group), producing an alkaline environment in the slant and fermented glucose (producing acid in the butt). The red slant suggests______, which generates alkaline byproducts, while the yellow butt indicates_______, producing acid in the anaerobic conditions of the butt.

A

Red Slant/Acid Butt (R/A)

deaminated lysine

lysine deamination

glucose fermentation

32
Q

The Citrate Utilization Test is used to determine whether a bacterium can use citrate as its sole carbon source for growth.

________contains citrate as the only available carbon source and a pH indicator,________, to detect changes in pH as the bacteria metabolize citrate.

A

Simmons Citrate Agar

bromthymol blue

33
Q

CITRATE

Inoculation Procedure:

Prepare the Inoculating Needle: Use a sterile needle to obtain a small amount of the bacterial colony.

Inoculate the Slant:_______ of the Simmons citrate agar slant with the needle. Only touch the tip of the needle to the colony and streak across the slant surface—do not stab into the butt of the tube.

Avoid Heavy Inoculum: Do not use a broth culture to inoculate the medium, as this would provide too heavy of an inoculum, which can lead to inaccurate results.

Incubate: Incubate the inoculated tube at _____for up to 7 days.

A

Lightly streak the surface

35-37°C

34
Q

Citrate

Positive Result:

Growth: Growth on the medium indicates that the bacterium can utilize citrate as its sole carbon source.

Color Change: The pH indicator,_____, changes color from______ in the presence of alkaline byproducts. The alkaline pH results from the metabolism of citrate, producing alkaline compounds (like ammonia) that raise the pH.

Interpretation: A positive result indicates that the bacterium can utilize citrate, and as it grows, it increases the pH of the medium, turning the indicator blue.

A

bromthymol blue; green to blue

35
Q

Citrate

Negative Result:
: Absence of growth indicates the bacterium cannot utilize citrate as a carbon source.

: The medium remains green if there is no growth, as the pH stays neutral.

Interpretation: A negative result indicates that the bacterium cannot utilize citrate for growth, and thus no color change occurs in the medium.

A

No Growth

No Color Change

36
Q

The_______ is used to determine whether an organism produces the enzyme urease, which breaks down urea into ammonia and carbon dioxide. The urea agar medium contains urea and a pH indicator,_____, which changes color in response to the pH shift caused by ammonia productio

A

Urea Hydrolysis Test

phenol red

37
Q

Urea

Inoculation Procedure:

Prepare the Inoculating Loop: Use a sterile loop to obtain a portion of a well-isolated bacterial colony.

Inoculate: ______with the inoculum, spreading it gently across the surface.

Incubate: Leave the cap of the tube____ on to allow for gas exchange and incubate at_____ for 48 hours to 7 days.

A

Streak the surface of the urea agar slant

loosely

35°C

38
Q

Urea

Positive Result:
Color Change: A positive result is indicated by a color change of the agar slant from_____

Interpretation: The color change occurs due to the production of______ by the bacterium as it breaks down urea.

Ammonia raises the pH of the medium, causing the_____ indicator to shift from orange to magenta.

A

light orange to magenta (fuchsia).

ammonia

phenol red

39
Q

Urea

Negative Result:

No Color Change: If there is no color change and the agar slant and butt remain_______, the result is negative.

Interpretation: This indicates that the bacterium did not produce_____ or did not hydrolyze urea during the incubation period.

A

light orange

urease

40
Q

SIM

Inoculation Procedure:

Prepare the Inoculating Needle: Use a sterile inoculating needle to obtain a portion of a young (18 to 24-hour) culture grown on either (2)

Inoculate the Medium: Touch the needle to the colony, then stab once into the center of the SIM medium to a depth of approximately______

Incubate: Cap the tube_____ and incubate at_____ for 24 hours.

A

MacConkey (MAC) or Eosin Methylene Blue (EMB) agar

1/3 to ½ inch

loosely

35°C

41
Q

SIM

Motility:

Positive: Motile organisms will spread from the site of inoculation, causing the entire medium to appear_____

Negative: Nonmotile organisms will only grow along the stab line, leaving the surrounding medium_____.

Interpretation: This indicates the organism’s ability to move within the medium, which is characteristic of motile bacteria like______

A

turbid (cloudy) around and beyond the stab line

clear

Escherichia coli

42
Q

SIM

Sulfide Production (H₂S):

Positive: The medium will turn____ due to the production of _____by the bacteria, which reacts with iron salts in the medium to form______.

Negative: There will be no blackening in the medium.

Interpretation: Blackening indicates that the organism can reduce sulfur compounds, a feature commonly observed in bacteria like______.

A

black; hydrogen sulfide (H₂S); iron sulfide (FeS).

Salmonella

43
Q

SIM

Indole Production:

Procedure: After incubation, add_____ (about 10 drops) of______ to the SIM tube.

Positive: A _____color forms at the top of the medium within a few seconds, indicating the presence of indole.

Negative: If no color change occurs, the reaction remains_______

Interpretation: The production of indole indicates the organism’s ability to degrade______, a common trait of Escherichia coli and other tryptophan-degrading bacteria.

A

0.5 ml; Kovac’s reagent

deep red

colorless or light yellow

tryptophan

44
Q

The_____ is a set of two separate tests performed on the same broth culture to assess an organism’s ability to ferment glucose and produce different metabolic byproducts.

These tests help differentiate between enteric bacteria, particularly in their fermentation pathways.

A

MRVP Test (Methyl Red and Voges-Proskauer Test)

45
Q

MRVP

Inoculation Procedure:

Label the Tubes: Label two separate broth tubes as 1st___ and 2nd _____

Inoculate the Tubes:
• Using aseptic technique, inoculate both the MR and VP tubes with the same isolated colony of the unknown bacterium.
• Inoculate each tube by means of a sterile loop or needle.

Incubation: Incubate both tubes at_____ for 24 to 48 hours. For slow fermenters, incubation may need to be extended to 4-5 days.

A

1ST MR and 2ND VP.

35°C

46
Q

Methyl Red Test (MR):

•	Procedure:
•	After incubation, add\_\_\_\_\_ of \_\_\_\_\_to the MR tube while wearing disposable gloves.
A

5 drops

methyl red indicator

47
Q

MR

• : Indicates the organism ferments glucose to produce strong acids (e.g., Escherichia coli).

• : Indicates the organism produces neutral or weakly acidic byproducts (e.g., Enterobacter species).

A

Red color (positive)

Yellow color (negative)

48
Q

Voges-Proskauer Test (VP):

Procedure:
• To the VP tube, add_____ (12-15 drops) of______ and

______(4-5 drops) of _____

A

0.6 ml of 5% alpha-naphthol in absolute ethyl alcohol

0.2 ml of 40% potassium hydroxide.

49
Q

VP

• : Indicates the production of acetoin, commonly seen in bacteria like Enterobacter or Klebsiella.

• : Indicates the absence of acetoin production (e.g., Escherichia coli).

A

Pink color (positive)

No color change (negative)

50
Q

MR VP

• MR Positive / VP Negative: The organism ferments glucose to produce acidic byproducts (e.g., Escherichia coli).

• MR Negative / VP Positive: The organism ferments glucose and produces acetoin via the butanediol fermentation pathway (e.g., Enterobacter species).

• MR Negative / VP Negative: The organism does not ferment glucose in a significant way to produce acid or acetoin (e.g., Proteus species).

A

Bacte LAB (38 decks)

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