CLED agar contd....

This is medium for the differentiation of urinary organisms. Its basic colour differs according to the manufacturer. Previously we had pale pink plates; now they are the prettiest green I've seen in a long time. Almost translucent green, like the sea on a clear summer day.

From the name, it contains cysteine, lactose and is electrolyte deficient. The electrolyte deficiency prevents swarming of .......?
LF are yellow
NLF are blue
but in addition to that.......
Pseudomanas is green with a rough colony edge
Staphylococcus aureus and Enterococcus faecalis are yellow (other Staphylococcus can be white)

A likely story......

the following relates to the orgs we have/are studying this semester.

Mr Brown is sick. He goes to the doctor who takes a specimen. Culture reveals an org that is citrate neg, urease neg, and does not produce hydrogen sulphide. It is a Gram negative bacillus.
What reaction would it give on Mac and CLED?
Why would it not grow on MSA?
If you inoculated a TSI and SIM, would it be incorrect to say that the org is non motile, indole pos and produces an alkaline slant? Why. (explain fully)
Name the organism.

identification of Staphylococcus and Streptococcus

these orgs are Gram positive cocci. Strept occur either in chains or as diplococci. Staph occur in grape like clusters.

to differentiate between Staph and Strept
catalase test (3% H2O2)
Staph is pos (immediate bubbling when org is added)
Strept is neg (no immediate bubbling when org is added)

to differentiate between S aureus and S epidermidis
DNAse test (stab inoculation)
S aureus is pos (clear zone around colonies after 1N HCl is added)
S epidermidis is neg (medium turns opaque around colonies after 1N HCl is added)

reagents tall and ugly

Kovacs reagent for indole production
p-dimethylamino benzaldehyde

nitrate A = sulfanilic acid
nitrate B = dimethyl alpha naphthylene

VP 1 (Barritts A) = 40% KOH
VP II (Barritts b) = alpha naphthol

10 FeCl2 for phenylalanine deaminase

1N HCl for DNase

I dont have a way to remember these. If you do, please share.

MR VP test

stands for methyl red and voges proskauer test
same medium ,sort of clear or colourless broth before inoculation
test is used to differentiate between E coli and Enterobacter
if E coli is positive in one test, then it will be negative in the other

add methyl red reagent to MR
look for red colour which is positive which is E coli
add VP1 and VP2 to voges proskauer
red colour is positive which is Enterobacter

methyl red
looking for breakdown of glucose to acidic end products at ph4
non acidic end products at ph 6 is negative

voges proskauer
non acidic end products at ph 6 is positive
acidic end products at ph 4 is negative

easy way to remember
acid 4 ME
(acidic end products ph 4 MR E coli)

orgs and Gram reactions

GPC
Staphylococcus aureus/epidermidis
Streptococcus pneumoniae/pyogenes
Enterococcus faecalis

GNB
Escherichia coli
Klebsiella pneumoniae/oxytoca
Proteus vulgaris/mirabilis
Shigella sonnei
Enterobacter aerogenes
Pseudomonas aeruginosa

GNC
Neisseria

GPB
Bacillus subtilis/cereus

SIM

used for H2S production, motility and indole production

H2S production
medium turns black

motility
organism grows outwards from line of inoculation

indole
add Kovacs reagent and look for cherry red layer at top of medium

H2S producers are motile

H2S organisms
Salmonella
Proteus

non motile
Klebsiella
Shigella

indole pos
E coli
Proteus vulgaris
Klebsiella oxytoca
Enterobacter agglomerans

reactions for coliforms (GNB)

UREA
pale peach to pink
Proteus is pos
Klebsiella pneumoniae is late pos
citrate
green to blue
Enterobacter aerogenes is pos
Salmonella typhimurium is pos
Proteus mirabilis is positive
phenylalanine deaminase
pale brown (like nutrient agar)
Proteus is pos (green colour after adding 10% FeCl2)
nutrient gelatin
remove after incubation and refridgerate for 20 min
Bacillus subtilis, Proteus is positive
indole
use SIM, add kovacs reagent
look for cherry red colour on top of medium
Escherichia coli, Enterobacter agglomerans, Klebsiella oxytoca are positive
motility
Klebsiella and Shigella are non motile