1. University of Tabuk
Faculty of Applied Medical Science
Department of Medical Laboratory Technology
Medical Microbiology II (p) Identification of Gram Negative Bacilli Culture Characteristics
Mr.AYMAN.S.YOUSIF
M.SC IN Microbiology &IMMUNOLOGY
Academic Year: ( )

2. Classification of Bacteria

3. 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).

4. 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.

5. General Procedure of Bacteriological Diagnosis
specimens
Microscopy & Staining
Cultivation in suitable types of media
Serological Test
Sub culture in the special types of media for confirmation
Biochemical tests ( Identification and Isolation )
Morphologic Identification
Susceptibility Testing ( to select the suitable antibiotics for treatment the pathogenic isolated bacteria from the specimen )

6. Identification of Enterobacteriaceae
Gram stain
All Enterobacteriaceae are Gram-negative rods
Arranged in single

7. Classification of Enterobacteriaceae
Klebsiella, Enterobacter
E. coli, Citrobacter,
Lactose fermenters
Non-lactose fermenter
Proteus, Yersinia
Salmonell, Shigella
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

8. Differentiation Between LF & NLF By Growth on Macconkey Agar
MacConkey agar is selective & differential medium for Enterobacteriaceae
MacConkey Agar
Contains
Bile salts
Crystal violet
Lactose
Neutral red
Inhibit growth of G+ve bacteria
pH indicator
Acidic: Pink
Cause of differential
Cause of selectivity
Lactose non fermenters
colorless colonies
Lactose feremnters
Pink colonies

9. Streak-plate technique
four-area streak plate technique I
1/10 II I
1/5
Rotate plate 90
Flame loop
Flame loop
Rotate 90
III
1/4 IV
Rotate 90

10. 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)

11. Growth of Enterobacteriaceae on MacConkey agar
Colorless colonies
Pink colonies
Lactose non feremters
Salmonella, Shigella,
Proteus
Lactose feremters
E. coli, Citrobacter
Klebsiella, Enterobacter
Uninoculated plate

12. Reaction on Salmonella Shigella (SS) agar
SS agar is a selective & differential medium used for isolation of 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 fermenter
Neutral red
Lactose
Acid
Pink colonies
H2S + Ferric ammonium citrate
Ferrous sulfide
Black precipitate

13. 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

14. 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).

15. 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

16. Growth of Enterobacteriaceae on EMB agar
colonies are very dark, almost black e.g. E. coli