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Clinical Bacteriology Lab > Gram-Negative Bacilli > Flashcards

Gram-Negative Bacilli Flashcards

1
Q

What specimen is used for laboratory diagnosis of Family Enterobacteriaceae and Related Enterobacteria ?

A

Urine, blood, pus, spinal fluid, sputum, or other material, as indicated by the localization of the disease process.

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2
Q

What is of unique significance in the gram staining of Klebsiella species ?

A

presence of large capsules, which can be observed as colorless to light pink halo around the bacilli

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3
Q

What is a useful alternative gram staining for Yersinia species ?

A

Wayson staining

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4
Q

What are the significant features shown by the Yersinia species under wayson staining ?

A

bipolar purple staining

central vacuole giving a characteristic “safety pin” appearance

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5
Q

What enrichment medium is best used in isolation of Salmonella ?

A

Selenite broth

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6
Q

What conditions must be met for Salmonella subcultures to be formed in selenite broth ?

A

8 - 12 hours

35°C

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7
Q

What is the reason for the specific conditions of Salmonella and selenite broth ?

A

coliforms or other intestinal flora may overgrow the pathogens within a few hours

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8
Q

What may happen if you overheat selenite broth ?

A

Overheating may produce a visible precipitate, making it unsatisfactory for use

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9
Q

What makes selenite broth a specific enrichment medium for Salmonella ?

A

It inhibits the growth of many species of gram-positive and gram-negative bacteria including the coliforms and many strains of Shigella

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10
Q

What is used as an enrichment medium for the recovery of Salmonella and Shigella from clinical and nonclinical specimens ?

A

GN (Hajna) broth

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11
Q

What conditions must be met for GN (Hajna) broth ?

A

4 - 6 hours

35°C

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12
Q

What is the reason for the specific conditions of the GN (Hajna) broth ?

A

Due to the relatively low concentration of desoxycholate, it is less inhibitory to E. coli and other coliforms

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13
Q

In the GN (Hajna) broth higher concentration of mannitol increases the growth of which Enterobacteriaceae species ?

A

Salmonella and Shigella

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14
Q

In the GN (Hajna) broth what does the sodium citrate and sodium desoxycholate do ?

A

inhibit gram-positive and some gram-negative bacteria

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15
Q

What broth is used as a selective enrichment for salmonella species ?

A

Tetrathionate broth with iodine-iodide solution

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16
Q

What makes tetrathionate broth a selective medium ?

A

The tetrathione which is formed in the medium by the addition of the iodine-iodide solution inhibits the normal intestinal flora of fecal specimens

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17
Q

What conditions must be met for the tetrathione broth ?

A

18 - 24 hours

35°C

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18
Q

What are things to take note of in preparation of tetrathione broth ?

A

After heating the broth base to boiling, add the iodine solution
No heating must be done after adding the iodine solution

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19
Q

What hemolytic signs are observed in E. coli on BAM ?

A

beta-hemolytic, many are shown from isolates of infections

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20
Q

What hemolytic signs are observed for Klebsiella pneumoniae on BAM ?

A

Non-hemolytic (gamma-hemolytic)

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21
Q

Significant characteristics of growth of Proteus on BAM ?

A

Swarming growth, distinct zonation

Culture usually gives off a “burnt gun powder” odor

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22
Q

What makes MAC agar a slightly selective medium for Enterobacteriaceae growth ?

A

The crystal violet dye and bile salts in the medium inhibit G+ bacteria and allow G- bacteria to grow

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23
Q

What change is there on MAC agar when a lactose fermenter is present ? and why ?

A

Bacteria that ferment lactose, the sole carbohydrate in the medium, produce acids, which, in the presence of neutral red as the pH indicator, results in the formation of pink to red colonies.

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24
Q

What is observed for non-lactose fermenters on MAC agar ?

A

Colourless colonies

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25
Q

What selective and differential medium is used for the detection of sorbitol-nonfermenting Escherichia coli serotype O157:H7 ?

A

MAC agar with sorbitol

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26
Q

What makes EMB agar a slgihtly selective medium for Enterobacteriaceae growth ?

A

Eosin Y and methylene blue inhibits growth of G+ bacteria

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27
Q

What is observed in lactose fermenting bacteria colonies on EMB agar ? and why ?

A

Lactose fermenting bacteria produce pink to purple colonies
due to the taking up of an eosin-methylene blue dye complex by the bacterial cells when the pH drops

Escherichia coli colonies show a characteristic green metallic sheen (GMS)
due to the rapid fermentation of lactose

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28
Q

What is observed in non-lactose fermenting bacteria colonies on EMB agar ?

A

Colourless or transparent

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29
Q

What makes SSA a moderately selective agar for Enterobacteriaceae growth ?

A

Bile salt, brilliant green, and citrates inhibit the growth of G+ bacteria

Detects lactose fermentation with the neutral red indicator forming pink or red colonies

Sodium thiosulfate is a source of sulfur. Any bacteria that produce hydrogen sulfide (H2S) are detected by the black precipitate formed with ferric citrate.

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30
Q

What are things to take note of in preparation of SSA ?

A

During preparation, heat to boiling to completely dissolve the agar. DO NOT OVERHEAT. DO NOT AUTOCLAVE.

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31
Q

What is observed in E. coli growth on SSA ?

A

pink to red colonies

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32
Q

What is observed in Salmonella or Proteus growth on SSA ?

A

colourless colonies with black centers

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33
Q

What is observed with Shigella growth on SSA ?

A

colourless colonies

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34
Q

What makes XLD agar a moderately selective agar for Enterobacteriaceae growth ?

A

Bile salt (sodium desoxycholate) inhibits G+ bacteria growth

Xylose, lactose, and sucrose are the fermentable carbohydrates, Xylose is fermented by practically all enterics except for the shigellae

Lysine decarboxylation by Salmonella, which initially produce yellow colonies due to xylose fermentation, results in delayed red colonies due to alkaline amines produced

Sodium thiosulfate and ferric ammonium citrate are the H2S indicator system. Formation of colonies with black centers.

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35
Q

What is observed with E. coli, Klebsiella, and Enterobacter growth on XLD agar ?

A

Yellow colonies and yellow conversion of the medium due to fermentation of carbohydrates

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36
Q

What is observed with salmonella growth on XLD agar ?

A

Red colonies surrounded by pink halo, the pink halo is due to the decarboxylation of lysine
There is also blackening of the colonies due to H2S production

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37
Q

What is observed with Proteus and Edwardsiella growth on XLD agar ?

A

Red colonies with black centers but no pink halo

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38
Q

What is observed with Shigella growth on XLD agar ?

A

Red colonies

39
Q

What makes HEA agar a moderately selective agar for Enterobacteriaceae growth ?

A

Bile salt inhibit G+ bacteria growth and retard growth of many coliforms

Lactose, sucrose, and salicin are available carbohydrates for fermentation; acid fuschin reacts with bromthymol blue creating a yellow colour

Sodium thiosulfate source of sulfur and H2S is detected by ferric ammonium citrate

40
Q

What is observed with E. coli growth on HEA agar ?

A

Yellow conversion of the medium

Yellow-orange to salmon-pink colonies

41
Q

What is observed with Salmonella on HEA agar ?

A

Greenish-blue colonies with black centers

42
Q

What is observed with Shigella on HEA agar ?

A

Faint green colourless colonies

43
Q

What makes BSA a highly selective agar for Enterobacteriaceae growth ?

A

Isolating Salmonella typhi

Bismuth sulfite and brilliant green inhibit G+ bacteria growth and coliforms allowing Salmonella to grow

Bismuth sulfite is the source of sulfue and H2S is detected by ferrous sulfate, forming brown to black metallic sheen

44
Q

What makes BGA a highly selective agar for Enterobacteriaceae growth ?

A

Isolation of Salmonella other than S. typhi/S. paratyphi

Brilliant green dye inhibits growth of G+ bacteria growth and most of G-

Phenol red is indicator for fermentation of lactose and/or sucrose

45
Q

What is observed with Salmonella species other than S. typhi and S. paratyphi growth on BGA ?

A

White to red colonies surrounded by red zones

46
Q

What is observed with S. typhi and S. paratyphi growth on BGA ?

A

No traces of growth

47
Q

In the inoculation and incubation of the specimen what is to be done if negative after incubation of 24 hours ?

A

Reincubate for an additional 24 hours and select suspicious colonies for definitive biochemical or serologic testing

48
Q

In preparation of Yersinia enterocolitica what is the enrichment process ?

A

Cold enrichment of feces by 4°C for 1-3 weeks in buffered saline to enhance Y. enterocolitica recovery

49
Q

What is observed with Y. enterocolitica growth on MAC ?

A

Lactose-negative colonies , flat, colourless, or pale pink

50
Q

What is observed with Y. enterocolitica growth on CIN agar ?

A

Fermentation of mannitol in the presence of neutral red results in a characteristic “bull’s-eye” colony, colorless with red center

51
Q

What makes CIN agar a good medium for detection of Y. enterocolitica ?

A

Crystal violet, sodium desoxycholate and Irgasan (triclosan) inhibits growth of G+ bacteria and G- bacteria

Supplementation with cefsulodin and novobiocin improves inhibition of normal enteric bacteria.

52
Q

What makes TSI nutritionally rich ?

A

incorporation of four protein derivatives—beef extract, yeast extract, peptone, and proteose peptone

53
Q

What fastidious bacteria specie is excluded in TSI growth ?

A

obligate anerobe

54
Q

What is the difference between the two chambers in the TSI tube ?

A

upper slant is aerobic

bottom slant is anaerobic

55
Q

What colour indicates an acid slant or butt in TSI agar ?

A

yellow

56
Q

In the result of an alkaline slant and acid butt what causes the change of the slant ?

A

the release of amines to counteract the small percentage of acid

57
Q

What showcases gas production in TSI ?

A

Presence of cracks in the medium or the “pulling away” of the medium from the walls of the test tube

58
Q

Where is hydrogen sulfide production first seen in the TSI tube ?

A

The butt because it becomes acidic with glucose fermentation

59
Q

How is inoculation done on TSI ?

A

First stab the butt 2-3 mm deep

Then stroke the slant from bottom to top in back forward motion

60
Q

Why must the screw caps be loosened slightly during incubation for TSI ?

A

to promote typical reactions

61
Q

How will alkaline slant, acid butt, with gas, and H2S production be presented if shown in the TSI ?

A

K/Ag + H2S

62
Q

What is the difference between KIA and TSI ?

A

KIA lacks sucrose and the H2S indicator is ferric ammonium citrate

63
Q

What does the IMViC reactions stand for ?

A

Indole test
Methyl red test
Voges-Proskauer test
Citrate utilization test

64
Q

What is the incubation period for MR-VP broth ?

A

2 - 4 days

65
Q

What must be taken note of when inoculating a series of tubes of differential culture media with an unknown organism for the IMViC reactions ?

A

the citrate medium be streaked first to prevent carryover of proteins or carbohydrates from the other media

66
Q

What must be taken note of when using the ehrlich reagent in the indole test ?

A

Step should be preceded with the addition of 1ml of xylene or chloroform, because indole is soluble in organic compounds

67
Q

What is the positive result of the indole test ?

A

bright fuschia red colour at interface of the reagent and the broth

68
Q

What is the negative result of the indole test ?

A

yellow colour or no change

69
Q

In the MR test, because other organisms may produce smaller quantities of acid from the test substrate, an intermediate orange color between yellow and red may develop. What is the result ?

A

This does not indicate a positive test

70
Q

What is the right order of addition of reagents in the VP test ?

A

alpha-naphthol and then 40% KOH

71
Q

What is the purpose of alpha-naphthol in the VP test ?

A

serves as a catalyst to bring out a red complex

72
Q

After shaking the tube for exposure of the VP test to oxygenic atmosphere how long must it be left undisturbed for ?

A

10 - 15 minutes

73
Q

How many drops of p-dimethylaminobenzaldehyde containing solutions must be added to the indole test ?

A

15 drops

74
Q

How many drops of methyl red indicator must be added to the MR test ?

A

5 drops

75
Q

Why must the VP test not be read for standing over an hour ?

A

negative VP cultures may produce a copper-like color, potentially resulting in a false-positive interpretation

76
Q

What is the positive and negative result for citrate utilization test ?

A 
\+ = prussian blue or deep colour blue
- = retention of green colour
77
Q

How is motility test possible with SIM agar ?

A

semisolid nature of the medium

78
Q

What is the correct order of observation of results in the SIM test ?

A

Motility first and then sulfide before addition of reagents for indole detection because reagent may diffuse into the medium making it hazy or cloudy which may result in false motility interpretation

79
Q

What other media is available for motility testing ?

A

Motilility Test Medium
Motility Indole Ornithine (MIO) Medium
Motility Indole Lysine Sulfide (MILS) Medium
MIL (Motility-Indole-Lysine) Medium

80
Q

Lysine decarboxylation by bacteria produces what alkaline reacting amine ?

A

cadaverine

81
Q

What is the positive and negative result for lysine decarboxylation ?

A 
\+ = purple butt
- = yellow butt
82
Q

What is the positive and negative result for lysine deamination ?

A 
\+ = red slant
- = purple slant
83
Q

What are the three amino acids routinely tested for the identification of Enterobacteriaceae ?

A

Lysine, ornithine, and arginine

84
Q

What is the positive and negative result for the urease test ?

A 
\+ = pink red 
- = yellow
85
Q

What is done after incubation in PAD tests ?

A

add 4 or 5 drops of the ferric chloride directly to the surface of the agar

As the reagent is added, the tube is rotated to dislodge the surface colonies

86
Q

What is the positive and negative result for the PAD test ?

A 
\+ = green
- = not green
87
Q

What does ONPG mean ?

A

o-nitrophyl-β-D-galactoyranoside

88
Q

What is the visual evidence of ONPG going through hydrolysis ?

A

ONPG is a colorless compound and after it cleaves into o-nitrophenol it becomes yellow

89
Q

What are the two enzymes required for the production of acid in the lactose fermentation test ?

A

lactose permease and β-galactosidase

90
Q

What enzyme of the lactose fermentation test is required for the lactose molecule to penetrate the bacterial cell ?

A

Lactose permease

91
Q

What enzyme of the lactose fermentation test is required to cleave the galactoside bond, producing glucose and galactose ?

A

β-galactosidase

92
Q

What considerations are there when reporting results for ONPG test ?

A

visible yellow color reaction within 5–10 minutes
Most tests are positive within 1 hour; however, reactions should not be interpreted as negative before 24 hours of incubation

93
Q

What enzyme is part of the MUG test ?

A

β-glucuronidase

94
Q

What is the result for MUG test of E. coli serotype O157:H7 ?

A

negative

Clinical Bacteriology Lab (2 decks)

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