Gram Negative Media and Testing 1
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What are gram negative rods that can ferment glucose?
What are coliforms?
Enterobacteriaceae that can also ferment lactose (lactose positive)
What are non-coliforms?
Enterobacteriaceae that can not ferment lactose (lactose negative)
What do lactose fermentation tests do?
help to distinguish coliform from non-coliform Enterobacteriaceae
Which Enterobacteriaceae are coliform (lactose positive)?
- Escherichia coli
- Klebsiella pneumoniae
- Enterobacter aerogenes
What are the three gram negative media and testing?
- eosin methylene blue agar (EMB)
- MacConkey agar (MAC)
- triple sugar iron (TSI)
What is the eosin methylene blue agar (EMB)?
- selective media for gram negatives
- the dye in the agar is bacteriostatic to gram positives
What does EMB contain?
lactose and pH indicators
What does the uninoculated color of EMB look like?
What does non-lactose-fermenters look like on EMB?
- colonies appear light colored, transparent
- lactose negative
What does lactose fermenters look like on EMB?
- take up some of the dye from the media
- colonies appear purple red
- lactose positive
What will Escherichia coli look like on EMB?
will have a metalic/green sheen
What is MacConkey agar (MAC)?
- selective media for growing gram negatives
- growth or no growth indicator of gram negative bacteria
What is MAC a preliminary test for?
What does uninoculated MAC look like?
clear light red
What nutrients are in the MAC?
What is the bile salt for in MAC?
selective for Enterobacteriaceae and salt tolerant gram negatives - suppresses gram positives
What is crystal violet for in MAC?
suppresses growth of gram positives
What are the indicators in MAC?
- lactose: differentiates between lactose and non-lactose fermenters
- neutral red indicator: colorless at pH above 6.8, red at pH less than 6.8
What does lactose fermentation produce in MAC?
What do lactose fermenters look like in MAC?
- grow as pinkish, purple-red, brick-red colonies
- turn MAC agar red
What do non-lactose fermenters do in MAC?
attack the peptone in the agar
What kind of reaction does non-lactose fermenters have in MAC?
What do non-lactose fermenters look like in MAC?
light pink, white, transparent colonies
What are clinical specimens usually cultured on?
both blood agar and MacConkey agar
What does no growth on MAC and a good growth on blood agar mean?
strongly suggests that the pathogen is gram positive
What is the TSI agar for?
initial differentiation of enteric bacteria
What nutrients are in the TSI agar?
- glucose, lactose, sucrose
- phenol red pH indicator
What does the TSI agar demonstrate the ability for the organisms to do?
- to ferment sugars
- to produce gas by fermentation of sugars
- to produce hydrogen sulfide
What does an uninoculated TSI agar look like?
What does sugar fermentation in the TSI agar do?
- causes production of acid products
- acid: yellow
- alkaline: red/pink
What may be produced in the agar with fermentation of sugar?
TSI agar detects the ability of the organism to reduce _____ to _____.
What reacts with the hydrogen sulfide to produce iron sulfide and what color does it produce in the agar?
What do we do an inoculation slant in a TSI agar?
- stab inoculation with needle
- use only one colony
- when withdrawing the wire, streak the surface of the agar
- leave cap slightly loose
- incubate for 16 hours and read
How do we read the TSI agar?
- check slant and butt for...
- acid or alkaline reaction
- hydrogen sulfide production
- gas production
What happens early in incubation with the TSI agar and why is this important for us to know?
- early in incubation the butt and slant will turn yellow due to glucose metabolism
- important for use to know so that we don't read it to early and think it is an acidic reaction
What happens if we leave a TSI agar in an anaerobic condition?
- the slant will stay yellow
- always loosen the cap of the slant
What would you like to do?
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