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Microbio Lab > Lab 12: Identification of Enteric Gram Negative Rods > Flashcards

Lab 12: Identification of Enteric Gram Negative Rods Flashcards

1
Q

name some microorganisms in human intestinal tract (flora) that are shed in stool

A
  • gram-positive bacteria
  • family enterobacteriaceae
  • clostridium (spore forming)
  • candida (yeast)
  • protozoa
  • bacteroides (anaerobic bacteria)
  • up to 50% most fecal matter is bacteroids fragilis cells
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2
Q
  • What bacteria are not considered part of the normal flora?
  • What can they cause?
  • What family are they related to? Why is this important?
A
  • Dalmonella & Shigella
  • food poisoning, doarrhea & bacterial dysentery
  • Family Enterobacteriaceae, very similar (morphologically) to nonpathogenic bacteriain flora
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3
Q

What kind of tests are used to distinguish harmful and nonharmful bacteria?

A

biochemical tests

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4
Q

Enterobacteriaceae

  • what kind of rods?
  • usually referred to as?
  • what kind of oxygen user?
  • motile or non-motile?
  • inhabit large or small array of environments?
  • fastidious or non fastidious?
  • opportunistic or non opportunistic?
  • small or large amount of species involved in food poisoning outbreaks?
A
  • gram-negative
  • enterics
  • facultative anaerobe, either respiring or fermenting
  • motile flagella
  • large (soil, animal, fruits, vegetables, humans)
  • nonfastidious (grow in most simple bacterial media)
  • opportunistic
  • large (ground beef, peanuts)
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5
Q

Why live in large intestine?

A

-vast nutrients (carbs via fermentation)

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6
Q

what is exploited in lab that helps identify gram (-) organisms isolated from stool?

A

-which carbohydrates, and how many carbs an organism can ferment

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7
Q
  • what two media are used to characterize these bacteria?

- what else is used?

A
  • selective & differential media that contain carbohydrates
  • also contain a pH indicator
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8
Q
  • All Enterobacteriaceae can ferment what?

- not all posses enzymes to break down what?

A
  • glucose

- lactose (disaccaride; eventually breaks into 2 glucose)

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9
Q

What two enzymes do these bacteria need to use lactose as a sole carbon source?

A

galactosidase & lactose permease

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10
Q

how can lactose be used in this experiment?

A

testing for the ability to ferment lactose can help with identification

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11
Q

organisms that can ferment lactose are termed?

A

coliforms

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12
Q

organisms that can’t ferment lactose are termed?

A

paracolons

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13
Q

types of agar mediums used in this lab

A
  • MacConkey
  • Eosin Methylene Blue Levine (EMB)
  • Salmonella-Shigella (SS)
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14
Q

MacConkey agar

  • media type
  • what it contains and what it does
  • changes expected in media?
A
  • selective & differential medium
  • bile salts (detergents) & crystal violet (select gram (-) bacteria and inhibit gram (+) bacteria)
  • lactose and pH indicator (neutral red) (colorless at pH 6.8 or higher and appears bright pink at pH less than 6.8)
  • when lactose fermented, pH is lowered causing coliforms to turn red
  • paracolons will be colorless b/c they use the peptides in the medium rather than ferment lactose (no pH change)
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15
Q

Eosin Methylene Blue Levine (EMB)

  • media type
  • what is it used for
  • what it contains and what it does
  • changes expected in media?
A
  • selective & differential medium
  • used for differentiation of enteric lactose fermentors & non-lactose fermentors

-contains lactose & dyes eosin & methylene blue, (partially inhibitory for gram (+) bacteria

  • lactose fermentors appear deep purple or blue-black with metallic green sheen depending on degree of acid
  • non lactose fermentors produce colonies, sometimes appear light pink/purple b/c color of the media
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16
Q

Salmonella-Shigeela (SS)

  • media type
  • what is it used for
  • what it contains
  • changes expected in media?
A
  • selective & differential medium
  • isolate Salmonella & Shigella species from other enteric bacteria
  • bile salts & brilliant green dye (select against gram (+) and gram (-)
  • pH indicator neutral red & lactose
  • coliforms able to grow appear red from neutral red
  • paracolons will be colorless
  • sodium thiosulfate & source of iron
  • some enteric bacteria can produce colorless hydrogen sulfide gas (H2S) from sodium thiosulfate
  • sodium thiosulfate act as sulfur source in H2S gas production
  • ferric citrate (H2S indicator forming insoluble black precipitate when H2S gas combines with it
  • bacteria that can produce H2S will have black center in colonies
17
Q
  • in this lab how many species of family enterobacteriaceae will be investigated?
  • what properties will they be divided by?
  • what type of gram bacteria are they?
A
  • 4
  • lactose fermentation properties (coliforms vs paracolons)
  • gram-negative enterics
18
Q

1st lab procedure

A
  • streak bacteria onto three of the different media plates

- incubate

19
Q

Triple sugar iron (TSI) agar allows for differentiation between bacteria how?

Generally used for which bacteria family?

poured how?

what does the medium contain?

A
  • ability to ferment glucose, lactose & or sucrose
  • produce gas from fermentation
  • reduce sulfur to hydrogen sulfide

-Enterobacteriaceae

  • agar slant (anaerobic butt = bottom, aerobic slant = top)
  • 0.1% glucose, 1% lactose, 1% sucrose (decipher carb fermentation patterns
20
Q

bacteria prefer to break down glucose first. Why?

when glucose lacks it will continue to break down what?

what indicator added? Why?

A

-lactose has to be broken down to glucose first

  • lactose or sucrose if they can
  • if not they use peptones present in media

-phenol red, detect sugar fermentation (yellow at acidic pH, pink at alkaline pH)

21
Q

based on color of slant & butt, what can be observed?

A

-fermentation patterns

22
Q

what can be tested for in TSI slant?

A
  • gas (CO2 or H2) end product of fermentation

- gas bubble or break in butt of slant indicates it produced CO2 or H2 gas during carb fermentation

23
Q

TSI similarities to SS agar

A
  • contains sodium thiosulfate & ferrous sulfate used to test for hydrogen sulfide (H2S) gas.
  • black precipitate present if able to produce H2S

Microbio Lab (6 decks)

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