Introduction

 Identification of enteric bacteria which may be responsible for some food and water-borne diseases
 Mostly from the family Enterobacteriaceae
o Short Gram (-) non-spore forming bacilli
 Selective and differential media is used to identify causative organism of enteric diseases
o Separation of intestinal pathogens from the other gram (-) bacilli that are part of the normal flora
o E proteus, Enterobaccter, Klebsiella And Pseudomonas
 Other media (ex. Biochemical activities) may be used as final identification of pathogens
 Medically important genera of enteric pathogens:
o Salmonella  typhoid fever
o Shigella  bacillary dysentery
 Gram (-) bacteria can be generally be classified into lactose or non-lactose fermenters
o Normal Intestinal bacteria: Escherichia and Enterobacter are lactose fermenters
o Pathogens: Salmonella and Shigella are non-lactose fermenters
o Klebsiella are lactose fermenters
o Proteus are non-lactose fermenters
 MacConkey agar
o Lactose fermenters = red
 Generally, all gram (-) bacilli (normal intestinal flora) are lactose fermenters
 Some intestinal organisms , Proteus and Pseudomonas, are easily confused with enteric pathogens
becase they are non-lactose fermenters
o Lactose non-fermenters = colorless
 Bacterial colonies producing H2S (enteric or not enterics) have a dark center on SSA
o Because of small amount of iron in the medium  ferrous sulfide formation

Culture Media for Enterics

 Selective Media
o Salmonella-shigella (SS) agar
o TCBS
 For isolation of vibrio
 Differential Media
o MacConkey Agar
o EMB
 Enrichment Media
o Selenite broth = allows the growth of pathogens
o Gram (-) broth (GN)
o Alkaline peptone broth (pH 8) = enhance growth of vibrio

Procedure
A. Collection of Specimen
• freshly voided stool should be collected in a covered sterile, dry bottle
• sent to the laboratory within 2 hours
• sterile swab impregnated with stool placed aseptically in a bottle or transport medium (may also be done)

•In case of blood mucoid stool, ensure to get material for proper isolation of the specific microbe

•Contamination of the stool with urine MUST be avoided

•Collection of stool specimen must be done preferably before antibiotic therapy has started

B. Microscopic Examination
a. Should be included ONLY if cause of GI infection is parasitic in nature
b. If Staphylococci enteritidis is suspected, do Gram staining
i. If due to normal flora, no need for Gram stain
C. Inoculation of Culture
 •1st swab, inoculate with the MacConkey agar (differential media) with rectal swab or freshly passed stool specimen for
primary isolation of the pathogen

•2nd swab, inoculate first enrichment media, selenite blue and alkaline peptone broth, to assure growth of fastidious
organisms
•NOTE: broth is designed to support the growth of enteric pathogen while suppressing the growth of other intestinal
bacteria

•turn plate upside down to prevent contamination by water condensation

•incubate all inoculated media (broth and plates) at 37°C for 18-24 hours

•streak SSA plate the inoculum from selenite broth

•Streak on TCBS agar from alkaline peptone broth
•NOTE: Both SSA and TCBS has lactose and a pH indicator that allows separation of lactose and non-lactose fermenting
enteric pathogens from intestinal flora that are also lactose fermenters (coliform group)

•Incubate both plates at 37°C for 18-24 hours

•Examine plates and do Gram stain on growth on the medium

•continue to identify isolates by doing biochemical and serological tests

D. Biochemical Tests

•Inoculate biochemical media with colonies of suspected pathogens

•Incubate all inoculated media at 37°C

•Interpret and record results

Medium Gram (+) Fermentable Indicator Colony Color Category

Bacteriostatic Carbohydrate Fermenter Nonfermenter
agent
EMB Eosin Y Lactose+ Eosin Y Red or black Colorless S,D
methylene methylene with sheen
blue blue
MacConkey Crystal violet Lactose Neutral red Red Colorless S,D
Bile salts
Xylose-lysine Bile salts Xylose Phenol red Yellow Red S,D
deoxycholate Lactose
(XLD) Sucrose
Hektoen Bile salts Salicin Bromothymol Yellow-orange Green S,D
enteric (HE) Lactose blue Blue-green
Sucrose
Salmonella- Bile salts Lactose Neutral red Red Colorless S
shigella (SS)
Bismuth Brilliant Glucose Bismuth ≠ ≠ S
sulfate (BS) green sulfite
TCBS Bile salts Sucrose Thymol blue Yellow Colorless S,D
 pH8.6 Bromthymol
blue
 D = differential, S = selective
 +
= Levine’s formulation
 ≠ = H2S producing salmonellae have black colonies
 TCBS used for isolation of vibrio

Gram (+) Bacteriostatic agent = inhibitor, making it selective

Ex.
(1) MacConkey
 S: only gram (-) will grow
 D: lactose vs non-lactose fermenters

Mixed bacterial colonies in MacConkey agar

(2) EMB

E coli colonies in Eosin Methylene Blue Agar (Note: Greenish Metallic Sheen)

(3) Oxidase Positive culture

Black colonies = oxidase positive
Enterics are oxidase negative bacteria, therefore culture media should not produce black colonies

TSI Medium
 To determine following characteristics in a GNB
o Fermentation of Glucose, Sucrose & Lactose
o Gas production during glucose fermentation
o H2S production
 Medium:
o Triple Sugar Iron agar containing Glucose, Sucrose, Lactose (1:10:10), Phenol red indicator, Iron salt, Sodium
thiosulfate (H2S source)

o A: Uninoculated tube
o B: K/A Tr(ace) H2S: glucose fermented, lactose & sucrose not fermented (Salmonella Typhi)
o C: K/Ag++: Glucose fermentation but no sucrose or lactose fermentation. (Non-typhoidal Salmonella spp.)
o D: K/A: Glucose fermented, no lactose or sucrose fermented, no gas produced (Shigella flexneri)
 Enterics
o E: A/A: Glucose and lactose and/or sucrose fermented gas produced. (E. coli.)
 Should present as red in MacConkey
 Crack in the butt is indicative of glucose is metabolized to CO2
 Enterics
o F: No fermention, no gas production (Pseudomonas aeruginosa)

Lysine Iron Agar

 To demonstrate the Lysine decarboxylation, deamination and H2S production by enteric organisms
 Proteus mirabilis: Slant Red; Butt Yellow; H2S negative
 Salmonella Typhimurium: Slant Purple; Butt Purple; H2S positive
 Shigella flexneri: Slant Purple; Butt Yellow; H2S negative
 o A: uninoculated tube
o B: LDC negative, lysine deaminase negative/H2S negative (C freundii)
o C: LDC positive, lysine deaminase negative/H2S negative (Salmonella serotype typhi)
o D: LDC negative, lysine deaminase positive/H2S negative (P mirabilis)
o E: LDC positive, lysine deaminase negative/H2S positive (salmonella serotype Newport)
 Result interpretation
o H2S Production
 Positive: black colour along the streak or throughout medium
 Negative: No Colour change
o Lysine Decarboxylation/LDC (detected in butt of the tube)
 Positive: Purple
 Negative: Yellow
o Lysine Deamination (detected on agar slant)
 Positive: Red
 Negative: Purple

KJA GAS H2S MR VP IND CIT PAD URE MOT LYS ARG ORN

Tribe 1: Eschericheae
Genus: Escherichia
 E coli A/A + - + - + - - - + + -/+ +/-
Genus: Shigella
 Groups A, B, C Alk/A - - + - -/+ - - - - - - -
 S sonnei Alk/A - - + - - - - - - - - +
Tribe II: Edwardsielleae
Genus: Edwarsiella
 E tarda Alk/A + + + - + - - - + + - +
Tribe III: Salmonelleae
Genus: Salmonella Alk/A + + + - - + - - + + +/- +
Tribe IV: Citrobactereae
Genus: Citrobacter
 C freundii Alk/A + + + - - + - +/- + - +/- -/+
Alk/A
 C diversus Alk/A + - + - + + - +/- + - +/- +
Tribe V: Klebsielleae
Genus: Klebsiella
 K pneumoniae A/A ++ - - + - + - + - + - -
 K oxytoca A/A ++ - - + + + - + - + - -