Gram Negative Algorithm Flashcards by Ann M

What are the Gram-negative diplococci?

Neisseria meningitidis

Neisseria gonorrhoeae

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What lab test can you use to differentiate between the Gram-negative diplococci?

Neisseria meningitidis ferments maltose (and glucose). Neisseria gonorrhoeae does not ferment maltose (it can only ferment glucose).

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What bacteria is:
pink on Gram-stain
diplococci
oxidase +, catalase +
motile, capsule
maltose fermenter, glucose +

Neisseria meningitidis

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What bacteria is: 
pink on Gram stain
diplococci
oxidase +, catalase + 
motile (twitching), no capsule 
maltose -, glucose +

Neisseria gonorrhoeae

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What bacteria is: 
pink on Gram stain
diplococci
oxidase +, catalase +
non motile, capsule variable
maltose +, glucose +, lactose +, sucrose + 
DNase + 
Pinkish brown hockey puck colonies on CHOC

Moraxella catarrhalis

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Which diplococci has a distinctive feature of pinkish brown hockey puck colonies on CHOC?

Moraxella catarrhalis

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What are 2 key lab tests to distinguish among the Gram-negative rods?

1) if it ferments lactose
2) if it ferments lactose fast or slow (among lactose fermenters)
3) if it has oxidase (among non-lactose fermenters)

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Differentiating between fast lactose fermenters

IMViC
- > E coli: IMViC ++–
- > Klebsiella IMViC –++
- > Enterobacter IMViC –++
Motility
- > E. coli: Motile
- > Klebsiella: Non-motile
- > Enterobacter: Motile
Urease
- > E. coli: Urease -
- > Klebsiella: Urease +
- > Enterobacter: Urease -

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Growth on EMB Agar for E. coli vs Klebsiella

E coli: black colonies with metallic sheen

Klebsiella: purple colonies

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Which gram -ve fast lactose fermenter bacilli has distinctive feature of a capsular halo on gram stain?

Klebsiella

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Name the bacteria:

Glucose +, Maltose +
IMViC ++ - - (Indole +, Methyl Red +, Voges-Proskauer -, Citrate Utilization -)
Urease -
Motile (flagella)
Growth on Eosin Methylene Blue (EMB) Agar = black colonies with metallic sheen
can cause disease in anyone
TSIA: A/A (Acid slant/Acid butt) gas +, H2S -

E coli

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Name the bacteria:

Glucose +, Maltose +, Sucrose +
IMViC - - ++ (Indole-, MR -, VP+, Citrate+)
Urease +
Non motile
Growth on Eosin Methylene Blue (EMB) Agar = purple colonies
Capsule, capsular halo on gram stain
can cause disease in anyone
TSIA: A/A (Acid slant/Acid butt) gas +, H2S -

Klebsiella

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Name the bacteria:

Glucose + , Maltose +, Sucrose +
Oxidase -, catalase +
IMViC - - + + (Indole -, MR -, VP+, Citrate +)
Urease -
Motile, non-capsulated
opportunistic pathogen
TSIA: A/A (Acid slant/Acid butt) gas +, H2S -

Enterobacter

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Ways to differentiate slow lactose fermenters

Citrobacter
IMViC - + - +
No red pigment
TSIA: A/A +, +/-

Serratia
IMviC - - + +
Red pigmented colonies (Prodigiosin)
TSIA: K/A - -

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Name the bacteria

IMViC - + - + (Indole -, MR +, VP -, Citrate +)
Motile
No red pigment
TSIA: A/A +, +/- (Acid slant/Acid butt, gas+, H2S +/-)

Citrobacter

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Name the bacteria

IMViC - - + + (Indole -, MR -, VP +, Citrate +)
Motile
Red pigmented colonies due to Prodigiosin (anti-cancer, anti-microbial, anti-oxidant and immunosuppressant)
TSIA: K/A - - (Alkaline slant/ Acid butt, gas -, H2S -)
opportunistic pathogen

Serratia

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Which gram negative slow lactose fermenter has distinct red pigmented colonies?

Serratia

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IMViC for Citrobacter

- - +

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IMViC for Serratia

- - +

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Name fast lactose fermenting gram negative bacteria

E. coli, Klebsiella, Enterobacter

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Name slow lactose fermenting gram negative bacteria

Citrobacter, Serratia

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Non lactose fermenters are colourless on MacConkey (T/F)

True

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How can we separate NLFs (Non Lactose Fermenters)?

Split into oxidase + and -

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Oxidase + NLF

Pseudomonas

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Two distinct features of Pseudomonas

Blue green pigment | Grape odour

Name the bacteria - IMViC - - - + (Indole -, MR -, VP -, Citrate +) - Motile - Urease + - TSIA: K/K + + (Alkaline slant/Alkaline butt, gas+, H2S+) - Blue-green pigment - Grape odour - P. aeruginosa grows on cetrimide selective media

Pseudomonas

Give IMViC for Pseudomonas

- - - +

Pseudomonas is non motile (T/F)

False

Oxidase - NLF

Shigella, Salmonella, Proteus

Differentiating between the gram negative, oxidase -ve, NLFs

``` - Growth on Salmonella Shigella Agar Shigella: green colonies Salmonella: black colonies - IMViC Shigella: - + - - Salmonella: - + - - Proteus: - + - + - TSIA Shigella: K/A - - Salmonella: K/A + + Proteus: K/A + + - Motility Shigella: Non-motile Salmonella: Motile Proteus: Swarming motility ```

Name the bacteria: - Catalase +, Oxidase - - IMViC - + - - (Indole -, MR +, VP -, Citrate -) - Non-motile - TSIA: K/A - - (Alkaline slant/ Acid butt, gas -, H2S -) - Green colonies on Haktoen/Salmonella Shigella Agar

Shigella

Which of the gram negative, oxidase -ve, NLFs is non motile?

Shigella

Give IMViC for Shigella

- + - -

Give TSIA for Shigella

K/A - -

Name the bacteria: - Catalase +, Oxidase - - IMViC - + - - (Indole -, MR +, VP -, Citrate -) - Motile - TSIA: K/A + + (Alkaline slant/Acid butt, gas +, H2S +) - Black colonies on Haktoen/Salmonella Shigella Agar and XLD Agar

Salmonella

Which of the gram negative, oxidase -ve, NLFs shows black colonies on XLD Agar?

Salmonella

Give IMViC for Salmonella

- + - -

Give TSIA for Salmonella

K/A + +

Name the bacteria: - Catalase +, Oxidase - - IMViC - + - + (Indole -, MR +, VP -, Citrate +) - Motile (swarming on BA= thin layer arranged circular) - Fishy odour - TSIA: K/A ++ (Alkaline slant/ Acid butt, gas +, H2S +) - opportunistic pathogen

Proteus

Which of the gram negative, oxidase -ve, NLFs has swarming motility on BA?

Proteus

Give IMViC for Proteus

- + - +

Give TSIA for Proteus

K/A + +

Curved rods

Growth V. cholera: hemodigestion in areas of confluent growth on BA but non hemolytic Growth on TCBS (yellow colonies)/peptone water C. jejuni: Swarming growth on moist agar Grey, convex, glistening, non-hemolytic on BA H. pylori More difficult to grow on BA Growth on Skirrow's medium, transparent colonies ``` Flagella V. cholerae - Single flagellum C. jejuni - Capsule, Single flagellum H. pylori - 6-8 flagella ``` String test V. cholerae + C. jejuni - H pylori - ``` Urease test V cholera - Urease - C. jejuni - Urease - H. pylori - Urease ++ ```

Name the Curved gram -ve rods and how we can differentiate them

V. cholerae, C. jejuni, H. pylori Growth V. cholera: hemodigestion in areas of confluent growth on BA but non hemolytic Growth on TCBS (yellow colonies)/peptone water C. jejuni: Swarming growth on moist agar Grey, convex, glistening, non-hemolytic on BA H. pylori More difficult to grow on BA Growth on Skirrow's medium, transparent colonies ``` Flagella V. cholerae - Single flagellum C. jejuni - Capsule, Single flagellum H. pylori - 6-8 flagella ``` String test V. cholerae + C. jejuni - H pylori - ``` Urease test V cholera - Urease - C. jejuni - Urease - H. pylori - Urease ++ ```

Name the gram -ve, curved bacteria: - IMViC + - v + (Indole +, MR -, VP variable, Citrate +) - +ve string test - Non hemolytic (only take observations from isolated colonies due to hemodigestion in areas of confluent growth) - Urease - - Motile - Single flagellum - Growth on Thiosulphate Citrate Bile Salt (TCBS) (yellow colonies due to sucrose fermentation)/ alkaline peptone water

Vibrio cholera

Which gram -ve, curved bacterium has yellow colonies on TCBS due to sucrose fermentation?

V. cholerae

Which gram -ve, curved bacterium has a +ve string test?

V. cholerae

Which gram -ve, curved bacterium requires salt supplementation for optimal growth?

V. cholerae

Coalesce - define

come together to form one mass or whole.

Preliminary identification of Campylobacter species from primary culture is by ?

Colonial appearance, Gram stain, Growth in oxygen, Oxidase test, Hippurate test

Name the bacterium: - Catalase +, Oxidase + - Urease - - Motile - Capsulated, single flagellum - Non-hemolytic, grey, convex, glistening - Colonies coalesce on moist agar and do exhibit swarming growth - Hippurate hyrolysis + (only spp of Campylobacter to hydrolyze hippurate) - RAPID test (stool/body tissue)

Campylobacter jejuni

Which gram -ve, curved bacterium has swarming growth on BA?

C. jejuni

Which gram -ve, curved bacteria have a single flagella?

V. cholerae, C. jejuni

Which gram -ve, curved bacterium grows on TCBS/peptone water?

V. cholerae

In order to optimize the diagnosis of H. pylori, it is usually recommended that several tests be used together (T/F)

True

In order to optimize the diagnosis of H. pylori, it is usually recommended that several tests be used together, what are these tests?

1. Serology: measure pylori IgG antibodies via fecal antigen, urea breath test. 2. Microscopy (gold std): 3. Culture: - Growth on Skirrow's medium 4. Rapid Urease Testing (RUT): Urease ++

Name the bacterium: 1. Serology: measure pylori IgG antibodies to see if person is infection via fecal antigen, urea breath test. 2. Microscopy (gold std): - Curved, gram -ve rods, 6-8 flagella 3. Culture: - Growth on Skirrow's medium = transparent colonies 4. Rapid Urease Testing (RUT) indicated by change in pH indicator = Urease ++ - Catalase +, Oxidase + - Motile

Helicobacter pylori

Which gram -ve, curved bacterium grows on Skirrow's medium as transparent colonies?

H. pylori

Which gram -ve, curved bacterium has 6-8 flagella?

H. pylori

Which gram -ve, curved bacterium is urease ++ ?

H. pylori

Which gram -ve, curved bacterium is more difficult to grow on BA?

H. pylori

Which gram -ve, curved bacterium is grey, convex, glistening, non-hemolytic on BA

C. jejuni

Match the bacteria based on growth on BA: 1. Grey, convex, glistening, non-hemolytic on BA 2. Hemodigestion in areas of confluent growth on BA but non hemolytic 3. More difficult to grow on BA V. cholerae, C. jejuni, H. pylori

1. C. jejuni, 2. V. cholerae, 3. H. pylori

Skirrows medium: has _ Vancoymcin, _ Polymyxin, _ Trimethroprim

Skirrows medium: has 1 Vancoymcin, 2 Polymyxin, 3 Trimethroprim

Name the gram-ve coccobacilli and how we can differentiate them

H. influenzae, Bordetella pertussis, Brucella, Francisella tularensis, Pasteurella multicoda

What gram-ve coccobacilli requires factors V and X for growth?

Haemophilus influenzae

No growth on BA, gray, opaque, large colonies on CHOC, this describes which gram -ve coccobacilli?

Haemophilus influenzae

What gram-ve coccobacilli is oxidase + with +ve satellite test?

Haemophilus influenzae

Name the bacteria: - Coccobacilli - Oxidase +, Catalase + - No growth on BA, growth on CHOC = gray, opaque, large colonies - Growth factor requirement test (factor V and X) / Satellite test - Slide agglutination serotyping (determines capsular serotype)

Haemophilus influenzae

Bordet-Gengou agar contains blood, _______ extract, glycerol with _______/penicillin

Bordet-Gengou agar contains blood, potato extract, glycerol with cephalexin/penicillin

Name the gram-ve, coccobacilli: - Bordet-Gengou agar gives dome-shaped, small, pearl-like, opaque colonies - Detected via fluorescent antibody/PCR

Bordetella pertussis

Pearl-like, opaque colonies on Bordet-Gengou agar can be seen in what gram -ve bacterium?

Bordetella pertussis

What are other tests used to ID Bordetella pertussis?

Fluorescent antibody/PCR

Brucella easily mistaken for Haemophilus, what is a test used to differentiate the two?

Satellite test Brucella = no satellitism H influenzae = satellitism

If Brucella is Urease + what is the next test done?

Phenylalanine deaminase test (PDA)

- BA, CHOC, Brucella agar Brucella grows on both Blood and Chocolate agar without satelliting around staphylococcus. - Catalase +, Oxidase + - Rapid urease test + - If urease + do Phenylalanine deaminase test (PDA)

Brucella

Culture and isolation of Francisella tularensis is easy (T/F)

False; Culture and isolation of Francisella tularensis is difficult

The medium of choice for F. tularensis is ________ _______ blood agar Good growth has also been achieved on modified ______-______ broth, ______ agar supplemented with IsoVitale X and modified ______ yeast extract agar.

The medium of choice for F. tularensis is cysteine glucose blood agar Good growth has also been achieved on modified Mueller-Hinton broth, chocolate agar supplemented with IsoVitale X and modified charcoal yeast extract agar.

Biochemical tests aren't particularly used for F. tularensis (T/F)

True

Blue-gray, round, smooth and slightly mucoid colonies - which gram -ve coccobacilli?

Francisella tularensis

Name the gram -ve bacterium - Growth on: - - Cysteine glucose BA - - Modified Mueller-Hinton broth - - CHOC with IsoVitale X - - Modified charcoal yeast extract agar - Colonies are blue-gray, round, smooth and slightly mucoid. - On BA, a small zone of alpha-hemolysis surrounds colony. - Slide agglutination tests / direct fluorescent antibody using commercially-available antisera for ID

Francisella tularensis

Tularemia is not often confirmed by serology (T/F)

False; Tularemia is most frequently confirmed by serology

Pasteurella multicoda can be contracted from a dog bite (T/F)

True

Name the bacterium: - Oxidase + - Capsulated - Non-motile - Bipolar staining with Giesma/ methylene blue - No growth on MAC - 'Safety pin' appearance - Grows well on CBA (Columbia Blood Agar), CHOC - Ferments glucose, sucrose with little/no gas - Weak acid production in TSIA - Colonies grey and viscous but rough irregular colonies occur frequently. - Strong mucinous odour resembling H. influenzae - On CHOC - round, greyish/ yellowish colonies Butterfly colonies may also occur. - No haemolysis on BA - butterfly colonies

Pasteurella multicoda - dog bite

We differentiate 3 subspecies of Pasteurella by ?

Sorbitol and dulcitol fermentation

Differentiation of Pasteurella strains (multicoda, septica, gallicida)

multocida strains - ferment sorbitol but do not ferment dulcitol; septica strains - negative rxns for sorbitol and dulcitol fermentation, gallicida strains - ferment both sorbitol and dulcitol.

Which gram -ve coccobacilli has a safety pin appearance

Pasteurella multicoda

Which gram -ve coccobacilli has butterfly colonies on BA/CHOC?

Pasteurella multicoda

Which gram -ve coccobacilli has weak acid production in TSIA?

Pasteurella multicoda

Which gram -ve coccobacilli has strong odour resembling H. influenzae?

Pasteurella multicoda

Pasteurella multicoda - dog bite

Non haemolytic, grey, butterfly colonies on BA and non motile, which gram - coccobacilli?

Pasteurella multicoda