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: (1434-1435-2013-2014)

Classification of Bacteria

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 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 )

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

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

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

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

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 Pink colonies Lactose non feremters Salmonella, Shigella, Proteus Lactose feremters E. coli, Citrobacter Klebsiella, Enterobacter Uninoculated plate

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

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