Lab 12: Identification of Enteric Gram Negative Rods Flashcards
name some microorganisms in human intestinal tract (flora) that are shed in stool
- gram-positive bacteria
- family enterobacteriaceae
- clostridium (spore forming)
- candida (yeast)
- protozoa
- bacteroides (anaerobic bacteria)
- up to 50% most fecal matter is bacteroids fragilis cells
- What bacteria are not considered part of the normal flora?
- What can they cause?
- What family are they related to? Why is this important?
- Dalmonella & Shigella
- food poisoning, doarrhea & bacterial dysentery
- Family Enterobacteriaceae, very similar (morphologically) to nonpathogenic bacteriain flora
What kind of tests are used to distinguish harmful and nonharmful bacteria?
biochemical tests
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?
- 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)
Why live in large intestine?
-vast nutrients (carbs via fermentation)
what is exploited in lab that helps identify gram (-) organisms isolated from stool?
-which carbohydrates, and how many carbs an organism can ferment
- what two media are used to characterize these bacteria?
- what else is used?
- selective & differential media that contain carbohydrates
- also contain a pH indicator
- All Enterobacteriaceae can ferment what?
- not all posses enzymes to break down what?
- glucose
- lactose (disaccaride; eventually breaks into 2 glucose)
What two enzymes do these bacteria need to use lactose as a sole carbon source?
galactosidase & lactose permease
how can lactose be used in this experiment?
testing for the ability to ferment lactose can help with identification
organisms that can ferment lactose are termed?
coliforms
organisms that can’t ferment lactose are termed?
paracolons
types of agar mediums used in this lab
- MacConkey
- Eosin Methylene Blue Levine (EMB)
- Salmonella-Shigella (SS)
MacConkey agar
- media type
- what it contains and what it does
- changes expected in media?
- 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)
Eosin Methylene Blue Levine (EMB)
- media type
- what is it used for
- what it contains and what it does
- changes expected in media?
- 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
Salmonella-Shigeela (SS)
- media type
- what is it used for
- what it contains
- changes expected in media?
- 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
- 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?
- 4
- lactose fermentation properties (coliforms vs paracolons)
- gram-negative enterics
1st lab procedure
- streak bacteria onto three of the different media plates
- incubate
Triple sugar iron (TSI) agar allows for differentiation between bacteria how?
Generally used for which bacteria family?
poured how?
what does the medium contain?
- 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
bacteria prefer to break down glucose first. Why?
when glucose lacks it will continue to break down what?
what indicator added? Why?
-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)
based on color of slant & butt, what can be observed?
-fermentation patterns
what can be tested for in TSI slant?
- 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
TSI similarities to SS agar
- contains sodium thiosulfate & ferrous sulfate used to test for hydrogen sulfide (H2S) gas.
- black precipitate present if able to produce H2S
