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University of Tabuk
Faculty of Applied Medical Science
Department of Medical Laboratory Technology
Mr.AYMAN.S.YOUSIF
M.SC IN Microbiology
&IMMUNOLOGY
Academic Year: (1434-1435-2013-2014)
Bacteria
Gram positive
Cocci
Bacilli
Gram negative
Cocci
Rods
Characters of Enterobacteriaceae
 All Enterobacteriaceae.
 Gram-negative rods.
 Ferment glucose with acid production.
 Reduce nitrates into nitrites.
 Oxidase negative.
 Facultative anaerobic.
 Motile except shigella and klebsiella .
 Non-capsulated except Klebsiella.
 Non-fastidious.
 Grow on bile containing media (MacConkey agar).
Enterobacteriaceae
 Some Enterobacteriaceae are true pathogens
 Salmonella spp.
 Shigella spp.
 Yersinia spp.
 Certain strains of E. coli (ETEC, EPEC, EIEC, EHEC)
 Most members of the Enterobacteriaceae are
opportunistic or cause secondary infections of wounds,
the urinary and respiratory tracts, and the circulatory
system e.g. E. coli.
General Procedure of Bacteriological Diagnosis
specimens
Cultivation in suitable types of media
Morphologic Identification
Microscopy & Staining
Biochemical tests ( Identification and Isolation )
Sub culture in the special types of media for confirmation
Serological Test
Susceptibility Testing ( to select the suitable antibiotics for
treatment the pathogenic isolated bacteria from the specimen ) 5
Identification of Enterobacteriaceae
 Gram stain
 All Enterobacteriaceae are Gram-negative rods
 Arranged in single
Classification of Enterobacteriaceae
Enterobacteriaceae
Lactose fermenters
E. coli, Citrobacter,
Klebsiella, Enterobacter
Non-lactose fermenter
Salmonell, Shigella
Proteus, Yersinia
There are several selective and differential media used to
isolate distinguishes between LF & LNF
The most important media are:
MacConkey agar
Eosin Methylene Blue (EMB) agar
Salmonella Shigella (SS) agar
In addition to Triple Sugar Iron (TSI) agar
Differentiation Between LF & NLF
By Growth on Macconkey Agar
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MacConkey agar is selective & differential medium for Enterobacteriaceae
MacConkey Agar
Contains
Bile salts
Crystal violet
Inhibit growth of G+ve bacteria
Cause of selectivity
Lactose feremnters
Pink colonies
Lactose
Cause of differential
Neutral red
pH indicator
Acidic: Pink
Lactose non fermenters
colorless colonies
Streak-plate technique
four-area streak plate technique
I
1/10
I
II
1/5
Rotate plate 90
Flame loop
Flame loop
Rotate 90
Rotate 90
III
1/4
IV
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Identification of Enterobacteriaceae
Differentiation between LF & NLF by Growth on MacConkey agar
 Method:
 MacConkey agar is inoculated with tested organism using
streak plate technique.
 Incubate the plate in incubator at 37 C/24 hrs
 Results :
 LF organism appears as pink colonies (e.g. E. coli)
 NLF organism appears as colorless colonies (e.g. Shigella)
Growth of Enterobacteriaceae on MacConkey agar
Colorless colonies
Uninoculated plate
Lactose non feremters
Salmonella, Shigella,
Proteus
Pink colonies
Lactose feremters
E. coli, Citrobacter
Klebsiella, Enterobacter
Reaction on Salmonella Shigella (SS) agar
 SS agar is a selective & differential medium used for isolation of
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Salmonella and Shigella
The selective agents are bile salts, and brilliant green dye, which inhibit
gram-positive organisms
The medium contains only lactose as a differential agent and thus
differentiates on the basis of lactose fermentation
The formation of acid on fermentation of lactose causes the neutral red
indicator to make pink colonies.
Non lactose fermenting organisms are colorless on the medium.
SS agar contains sodium thiosulfate and ferric ammonium citrate allows
the differentiation of organisms that produce H2S
 Lactose fermenters, such as E. coli, have colonies which are pink
 Shigella appears transparent or amber
 Salmonella appears transparent or amber with black centers due to H2S
production
Lactose
Lactose fermenter
Acid
H2S + Ferric ammonium citrate
Neutral red Pink colonies
Ferrous sulfide
Black precipitate
Growth of Enterobacteriaceae on SS agar
A .Klebsiella pneumoniae
B .Escherichia coli
C :Salmonella sp.
D :Proteus mirabilis
E :Ps. aeruginosa
.
Both are lactose fermenters
Both Salmonella sp. & Proteus product H2S
Pseudomonas colonies are nearly colorless
Growth of Enterobacteriaceae on EMB agar
 Constituents: Nutrients, lactose, neutral red, eosin and
methylene blue.
 Appearance: A clear red medium
 A selective stain for Gram-negative bacteria. It is a mix of
two stains, eosin and methylene blue in the ratio of 6:1.
 Acommon application of this stain is in the preparation
of EMB agar, a differential microbiological medium,
which inhibits the growth of Gram-positive bacteria and
provides a colour indicator distinguishing between
organisms that ferment lactose (e.g., E. coli) and those
that do not (e.g., Salmonella, Shigella).
Eosin Methylene Blue (EMB)
 Lactose fermentation produces acids, which
lower the pH. This encourages dye
absorption by the colonies, which are now
coloured purple-black.
 Lactose non-fermenters may increase the
pH by deamination of proteins. This ensures
that the dye is not absorbed.
 On EMB if E. coli is grown it will give a
distinctive metallic green sheen
Growth of Enterobacteriaceae on EMB agar
colonies are very dark, almost black e.g.
E. coli