CLINICAL BACTERIOLOGY

BACHELOR OF SCIENCE IN MEDICAL LABORATORY SCIENCE

LECTURE Lecture By: Mr. Kristan Dela Cruz Transcribed By: Erysipelothrix rhusiopathiae
REVIEWER
Learning Objectives
BIOCHEMICAL DIFFERENTIATION
At the end of the discussion, the students shouldbe able to:
A/A A/A, K/A, K/A K/K
1. Demonstrate understanding of the clinically H2S+ H2S+
significant members of the Family Enterobacteriaceae Serratia Citrobacter Citrobacter Shigella Pseudomonas
based on their general characteristics and physiology.
Escherichia Arizona Arizona Citrobacter
2. Demonstrate and understand the laboratory culture
work-ups for the isolation and identification of the Enterobacter Proteus Salmonella Providencia
Family Enterobacteriaceae. Klebsiella Edwardsiella Proteus

Yersinia
GRAM-NEGATIVE BACTERIA FAMILY ENTEROBACTERIACEAE

Bacilli
Lactose Fermenter - color pink Non-
Lactose Fermenter - colorless  Rahn proposed the Family in 1936.
Oxidase Test Pseudomonas -
positive  Edwards and Ewing described the 11 Genera and 26
H2S Production - negative /medium color black. Species that are under the Family in 1972.
G(-) only. .
 Farmer noted 22 Genera and 69 Species under the
E.rhusiopathiae - G(+) produces H2S. Family in 1985.

CLASSIFICATION
OF ENTERICS
GENERA AND SPECIES TO BE CONSIDERED
 Due to the very large number of organisms in the
Family Enterobacteriaceae, species are grouped into Opportunistic Pathogens
Tribes, which have similar biochemical
characteristics. Citrobacter freundii Citrobacter
 Within each Tribe, species are further sub- grouped (diversus) koseri Citrobacter
under genera and species. braakii
Cronobacter sakazakii (prev. Enterobacter)
Tribe Genus Tribe Genus Edwardsiella tarda
Klebsiella Enterobacter aerogenes
Enterobacter Enterobacter cloacae
Pantoea
Escherichiaeae Escherichia Klebsielleae
Cronobacter Enterobacter gergoviae
Hafnia
Serratia
Enterobacter amnigenus
Enterobacter (cancerogenous) taylorae Escherichia
Proteus
Edwardsielleae Edwardsiella Proteeae Morganella coli (incl. Extraintestinal) Ewingella americana
Providencia Hafnia alvei Klebsiella
Salmonelleae Salmonella Yersinieae Yersinia pneumoniae Klebsiella
Citrobacteriaceae Citrobacter
oxytoca
Morganella morganii subsp. morganii

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 CLINICAL BACTERIOLOGY
BACHELOR OF SCIENCE IN MEDICAL LABORATORY SCIENCE
LECTURE Lecture By: Mr. Kristan Dela Cruz Transcribed By: Erysipelothrix rhusiopathiae
REVIEWER
Morganella psychrotolerans  All Yersinia are motile except Y.
Pantoea agglomerans (prev. Enterobacter) pestis at 25°C. (SKYPe)
Proteus mirabilis Proteus  All are Catalase Positive except Shigella
vulgaris Proteus penneri dysenteriae.
Providencia alcalifaciens  All are Cytochrome Oxidase Negative except
Providencia heimbachae Plesiomonas.
Providencia rettgeri  All are Nitrate Reducers except Erwinia, Pantoea
Providencia stuartii (Enterobacter) agglomerans, Photorabdus and
Serratia marcescens Xenorabdus.
Serratia liquefaciens group Serratia  Most are commensal flora of the intestinal tract except
odorifera Salmonella, Shigella, and Yersinia.
 Some are with pili or fimbriae.
Pathogenic Organisms  Grows well on McConkey Agar.
 DOC: Aminoglycosides, Trimethoprim-SXT, 3rd Gen
PRIMARY INTESTINAL PATHOGENS Cephalosporins
Escherichia coli (diarrheagenic)
Plesiomonas shigelloides Salmonella MNEMONIC: CPON NO GF FA
(all serotypes) Shigella dysenteriae  CPON - Catalase +, Oxidase -
(group A) Shigella flexneri (group B)  NO - Nitrate Reducers
Shigella boydii (group C) Shigella  GF - Glucose Fermenters
sonnei (group D)  FA - Facultative Anaerobes

PATHOGENIC YERSINIA SPP.

Virulence Factors
Yersinia pestis Pili
Yersinia enterocolitica subsp. enterocolitica Yersinia  Used for attachement; colonize the area and invade
frederiksenii tissues.
Yersinia intermedia Yersinia
psuedotuberculosis Plasmid
 Some may possess plasmid; resistance to
FAMILY ENTEROBACTERIACEAE antibiotics.
 ESBLs (Extended Spectrum Beta-Lactamases)
General Characteristics  E.coli, K.pneumoniae, K.oxytoca
Antigens
 Gram (-) enteric coccobacilli, short, plump bacilli.  Some may possess antigens that may be used to
 Non-spore formers. identify other groups.
 Facultative Anaerobes
 Antigenic Group Antigenic Structure
 Cell Wall (O Ag/Somatic Ag) - Somatic, Heat O/Somatic Antigen
Stable, Lipopolysaccharide.  Outer part of Cell Wall
 Flagella (H) - Flagellar, Heat Labile, Protein  For E. coli there are 164 types of O antigen and
 Capsule (K) - Capsular, Heat Labile, specific type maybe associated with a particular
Polysaccharide disease.
 K1 - E.coli; Vi - S.typhi  Heat Stable
 BAP/CAP: Large moist gray colonies except  Liposaccharide
Klebsiella and Enterobacter  Serotype 0111 - Diarrhea in Infants
 All are Gamma Hemolytic except E. coli.  Serotype 0157 - Verotoxin / Verocytotoxin Production
 All are Non-Encapsulated except Klebsiella  For E. coli and Shigella serotyping.
and Enterobacter.
 All are Glucose Fermenters and often with gas K/Enveloped Antigen
production aerogenic except Shigella.  Covers the O Antigen
 All are Motile (PERITRICHOUS) at 37°C except  Consists of Capsular Polysaccharide
SKY.  Heat Labile
 Shigella  K1 Antigen - E. coli
 Klebsiella  Vi Antigen - S. typhi
 Yersinia pestis

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 CLINICAL BACTERIOLOGY
BACHELOR OF SCIENCE IN MEDICAL LABORATORY SCIENCE
LECTURE Lecture By: Mr. Kristan Dela Cruz Transcribed By: Erysipelothrix rhusiopathiae
REVIEWER
H/Flagellar Antigen
 Found in the Flagella
 Protein in nature
 Heat Labile
 Found only among motile enteric
 For Salmonella serotyping

Media Inhibitory CHO

CULTURE
Indicator Colony Color
Non-
Fermenter Fermenter
Eosin Y and Eosin Y and
EMB Methylene Lactose Methylene Pink-purple Colorless
Blue Blue
Crystal
MAC Violet and Lactose Neutral Red Pink Colorless
Bile Salts
Xylose
Red/
XLD Bile Salts Lactose Phenol Red Yellow
Colorless
Sucrose
Salicin
Bromthymol Green/
HEA Bile Salts Lactose
Blue
Yellow
Colorless
Sucrose
Bile Salts
SSA Brilliant Lactose Neutral Red Red Colorless
Green
Brilliant Bismuth Salmonella - Black Black
BSA Green
Glucose
Sulfite with Metallic Sheen
Thymol Blue
Bile Salt and Green/
TCBS pH=8.6
Sucrose
Bromthymol
Yellow
Colorless
Blue

1. EMB - Eosin Methylene Blue

2. MAC - MacConkey Agar
3. XLD - Xylose Lysine Deoxycholate
4. HEA - Hektoen Enteric Agar
5. SSA - Salmonella Shigella Agar
6. BSA - Bismuth Sulfite Agar
7. TCBS - Thiosulfate Citrate Bile Salt Sucrose

MNEMONIC ACCORDING TO MEDIUM INDICATOR

Bromthymol Blue - CHOTS
 Citrate Utilization Test
 Hektoen Enteric Agar
 Oxidative Fermentative Medium
 Thiosulfate Citrate Bile Salt Sucrose
 Simmon Citrate Agar

Phenol Red - BMX-CUT

 Brilliant Green Agar
 Mannitol Salt Agar
 Xylose Lysine Deoxycholate
 Cystine Trypticase Agar
 Urease Test
 Tween 80 Agar/TSI

Neutral Red - SMASH-CIN

 Salmonella Shigella Agar
 MacConkey Agar
 ASHdown Medium
 CIN (Cefsulodin-Irgasan-Novobiocin) Media

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 CLINICAL BACTERIOLOGY
BACHELOR OF SCIENCE IN MEDICAL LABORATORY SCIENCE
LECTURE Lecture By: Mr. Kristan Dela Cruz Transcribed By: Erysipelothrix rhusiopathiae
REVIEWER
Bromcresol Purple - LIA
 Glycolysis - Glucose ferments into Pyruvic Acid.
 LIA (Lysine Iron Agar)  Turns Red to Yellow
 Deamination - Uses the protein Peptone to remove
OTHER CULTURE MEDIA the amino acid group forming NH2.
a) GN (Gram Negative) Broth - for Salmonella.  Turns Yellow to Red
b) Selenite Broth - enrichment broth for
Salmonella and Shigella.
c) Tetrathionate Broth - enrichment broth for
Salmonella
d) CIN (Cefsulodin Irgasan Novobiocin) - for
Yersinia
 Mnemonic - YerCINia
e) BGA (Brilliant Green Agar) - for other
Salmonella except S. typhi
 Inhibitory Agent - Brilliant Green
 pH Indicator - Phenol Red

BIOCHEMICAL TESTS
K/K
A. TSI (Triple Sugar Iron)
 Uses TSI dispensed as Slant (Lactose and
Sucrose) and Butt (Glucose). K/A
 Detects:
1. CHO Fermentation
2. Gas Production
A/A
3. H2S Production
 Composition:
Protein Composition Peptone
Fermentable CHO 1 part of Glucose 10
parts of Sucrose
10 parts of Lactose
pH Indicator Phenol Red
H2S Indicator Ferrous Sulfate/Sodium
Thiosulfate
Possible Results: H2S Production: Black
In acid pH: Yellow (A) Color
In alkaline pH: Red (K) Gas Production: Splitting
of Media; Pulling Away
or Cracks

FAMILY ENTEROBACTERIACEAE

Escherichia Klebsiella Enterobacter

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 CLINICAL BACTERIOLOGY
BACHELOR OF SCIENCE IN MEDICAL LABORATORY SCIENCE
LECTURE Lecture By: Mr. Kristan Dela Cruz Transcribed By: Erysipelothrix rhusiopathiae
REVIEWER
BIOCHEMICAL TESTS Deaminase
 An enzyme that removes the Amino (NH2)
B. LIA (Lysine Iron Agar) Group from the amino acids.

 Principle: Test to detect if the bacteria have the ability Decarboxylase

to degrade amino acid lysine by Deamination or  An enzyme that removes Carboxyl (COOH) Group
Decarboxylation. from the amino acids.
 Can also detect H2S Production.
 Description of Reading:
 Slant (Aerobic) is observed for Deamination
 Butt (Anaerobic) is observed for Decarboxylation
 Possible Result:
 pH Indicator: Bromcresol Purple
 H2S Indicator: Ferric Ammonium Citrate =
Black
 Lysine Deamination - Slant
 Postive - Red (R) (ACIDIC)
 Negative - Purple (K) (ALKALINE)
 Lysine Decarboxylation - Butt
 Positive - Purple (K) (ALKALINE) Proteus Providencia Morganella
 Negative - Yellow (A) (ACIDIC)

 Lysine Deamination is an Aerobic Process which

occurs on the Slant of the media.
 Principle: Lysine is deaminased to form a- Keto
Acid that will react with the Iron Salts found at
the surface of the medium which results in Red
Color Slant.
Mnemonics:
 Lysine Decarboxylase is an Anaerobic Process which  K/K - EKESEHES
occurs in the Butt of the media.  K/A - YES-Ci
 Principle: Lysine is decarboxylated results in  R/A - PPM
Cadaverine which gives an Acid End Product
that causes the Butt to turn Yellow. BACTERIAL METABOLISM
 Cadaverine has the ability to neutralize the Acid Mixed Acid Fermentation Test
End Products which makes the Butt, Alkaline  Lactic acid, succinic acid, formic acid, acetic acid.
and turns Purple.  Detected by Methyl Red Test.
 If Butt produces Cadaverine, Purple, if not,
Yellow. Butanediol Fermentation Test
 Acetoin (Acetyl methyl carbinol) and
butanediol which are neutral end products.
 Acetoin Production is detected by Voges-
Proskauer Test.

THE IMViC
K/K
INDOLE TEST
 Based on the ability of the organism to produce Indole
from Tryptophan.
 Media:
 Tryptophan Broth
 Peptone Broth
 SIM
 Result:
 (+) Red Ring
 (-) No Color Development

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 CLINICAL BACTERIOLOGY
BACHELOR OF SCIENCE IN MEDICAL LABORATORY SCIENCE
LECTURE Lecture By: Mr. Kristan Dela Cruz Transcribed By: Erysipelothrix rhusiopathiae
REVIEWER
 Reagent: KOVAC’s (pDAB) or Erlich’s Reagent

VOGUES-PROSKAUER TEST
 Detects Acetoin or Acetylmethyl Carbinol from
glucose fermentation.
 Media:
 MRVP Broth or Clark Lubb’s Broth
 Result:
 (+) Pink-Red
 (-) No Color Change
 Reagent: Barrit’s Method and Coblentz Method

METHYL RED TEST

 To detect Acid Production when glucose is
metabolized.
 Media:
 MRVP Broth or Clark Lubb’s Broth
 Peptone Glucose Broth
 Result:
 (+) Red Color (pH <4.5)
 (-) No Color Change
 Reagent: Methyl Red Indicator

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 CLINICAL BACTERIOLOGY
BACHELOR OF SCIENCE IN MEDICAL LABORATORY SCIENCE
LECTURE Lecture By: Mr. Kristan Dela Cruz Transcribed By: Erysipelothrix rhusiopathiae
REVIEWER
C
ITRATE UTILIZATION TEST
IMViC Reaction of Some Clinically Important
Bacteria
 Based on the ability of the organism to utilize
Citrate as a sole source of carbon.
 Media:
 Simmon Citrate Agar (Slant)
 Principle:
 Citrate, through the enzyme Citrate Permease,
Pyruvate is formed which can enter the
organisms metaboloc cycle for energy
production.
 Once Pyruvate enters the cycle, it will produce
Ammonium Phosphate.
 Ammonium Phosphate, through Citrate, is
converted into Ammonia and Ammonium
Hydroxide.
 Ammonia is Alkaline which increases the pH of
the media and turns Bromthymol Blue from
Green to Blue.
 Result:
 (+) Blue
 (-) Green/Yellow
 Reagent: Bromthymol Blue

MNEMONIC FOR INDOLE POSITIVE BACTERIA

 Moy PEKPEC Po?
 Morganella
 Proteus vulgaris
 Escherichia coli.
 Klebsiella oxytoca
 Providencia
 Edwardsiella tarda
 Citrobacter koseri
 Plesiomonas

Note: All “Utilization” Test will always have a (+)

Result Blue. MNEMONIC FOR VOGUES-PROSKAUER POSITIVE
 Acetate Utilization Test BACTERIA
 Acetamide Utilization Test  KEESH
 Citrate Utilization Test  Klebsiella
 Malonate Utilization Test  Enterobacter
 Ewingella
 Serratia
 Hafnia

MNEMONIC FOR CITRATE POSITIVE BACTERIA

 SaPro CHEEK
 Salmonella enteriditis
 Providencia
 Citrobacter
 Hafnia

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 CLINICAL BACTERIOLOGY
BACHELOR OF SCIENCE IN MEDICAL LABORATORY SCIENCE
LECTURE Lecture By: Mr. Kristan Dela Cruz Transcribed By: Erysipelothrix rhusiopathiae
REVIEWER
 Ewingella
 Enterobacter GELATIN HYDROLYSIS (GELATINASE TEST)
 Klebsiella
 Purpose:
OTHER BIOCHEMICAL TESTS  To determine the ability of organism to produce
proteolytic enzyme Gelatinase that liquefy
ONPG TEST gelatin.
 Reagent:
 O-nitrophenyl-beta-d-galactopyranoside  Gelatin Deep
 Purpose:  Incubated at 35°C or 25°C for up to 14 days.
 To determine the ability of organism to  Result:
produce the enzyme b-galactosidase.  (+) Result: Partial or Total Liquefaction
 To detect Late Lactose Fermenters  (+) Proteus vulgaris
 Reagent: O-nitrophenyl-beta-d-  (-) Enterobacter aerogenes
galactopyranoside (ONPG)
 Result: H2S PRODUCTION
 (+) Result: Yellow  Purpose:
 (-) Result: Colorless  Useful in differentiating Salmonella (+) from
 (+)Organism: Rapid and Late Lactose Shigella (-).
Fermenters and Hafnia alvei.  Detection of H2S can be made on several media:
 (-) Organism: Non-Lactose Fermenters SIM (most sensitive), LIA, TSI, SSA, HEA.
 (+)Result: Black
UREASE REACTION  (+) Organisms: SPACEd
 Salmonella
 Purpose:  Proteus
 To determine if organism is able to produce  Arizona
Urease; Useful in identification of PPM.  Citrobacter
 Reagent:  Edwardsiella
 Urea Agar/Slant (Christensen Urea Agar) with
Phenol Red as pH indicator.
 Result:
 (+)Result: Magenta (Bright Pink)
 (-) Result: Remains Yellow
 (+) Organisms: “PPM-H”
 Proteus
 Providencia
 Morganella
 Helicobacter pylori

MALONATE UTILIZATION
 Purpose:
 To determine the ability of an organism to use
MOTILITY TEST
Sodium Malonate as the sole source of carbon.
 Bacteria that can grow in the malonate broth can  Purpose:
also use Ammonium Sulfate as nitrogen source.  Useful in the identification of Non-motile Enterics
 To differentiate Salmonella from Shigella. (Shigella and Klebisella) the only non-motile
 Reagent: coliforms.
 Malonate Broth  Reagent:
 Result:  TTC (Triphenyl tetrazolium chloride) - a colorless
 (+) Result: Blue dye that turns red with organism’s growth.
 (-) Result: Green  Result:
 (+) Result: Motile if growth is Outside the line of
streak
 (-) Result: Non-motile if growth is At The Line of
streak.

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 CLINICAL BACTERIOLOGY
BACHELOR OF SCIENCE IN MEDICAL LABORATORY SCIENCE
LECTURE Lecture By: Mr. Kristan Dela Cruz Transcribed By: Erysipelothrix rhusiopathiae
REVIEWER  Ornithine ----decarboxylase---------->Putrescine
DEAMINATION REACTION (Ornithine Decarboxylase Test)
 Arginine ----decarboxylase-------->Citrulline
 Purpose: (Arginine Dihydrolase Test)
 To determine if organism is capable of
deaminating phenylalanine into phenyl pyruvic Note: Citruline decarboxylase into Ornithine
acid.
 Reaction: Phenylalanine >Phenylalanine
deaminase >Phenyl Pyruvic Acid
 Reagent:
 Phenylalanine Slant (0.2% phenylalanine) and
10% Ferric Chloride
 Modification: Use of Tryptophan Agar to Detect for
Tryptophan deaminase
 Reaction: Tryptophan--------------->Tryptophan
deaminase > Indole-Pyruvic Acid
 (+)PPM
 (-)Escherichia coli
 Result:
 (+) Result: Brown
 (-) Result: Green

KCN (POTASSIUM CYANIDE) BROTH TEST

DECARBOXYLATION REACTION (MOELLER’S
 Purpose:
METHOD)
 To differentiate Proteus (+) from Salmonella (-).
 Purpose:  Result:
 Determines the ability of organisms to use  (+) Result: Turbidity
amino acids as energy and carbon sources. Such  (-) Result: Clear
organism can decarboxylase (or hydrolyze) an  (+) Organisms: Enterobacter, Proteus,
amino acid to form amine, causing alkaline pH. Providencia, Morganella, Citrobacter
 Principle:
 Alkaline pH shift in the Medium MUG TEST
 Reagent:  Purpose:
 Decarboxylase Broth (Lysine,Ornithine and  To determine the ability of the organism (E. coli)
Arginine) to produce enzyme Beta-D- Glucoronidase
 pH Indicator: which hydrolyzes beta-D- glucopyranosid-uronic
 Bromcresol Purple and Cresol Red derivatives into a glycons and D-glucoronic acid.
 Uses Mineral Oil as an oxygen barrier; to make  Fluoremetric Method - more preferred.
environment Anaerobic.
 Reaction: LOA
 Lysine ----decarboxylase-------->Cadaverine
(Lysine Decarbosylase Test)

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 CLINICAL BACTERIOLOGY
BACHELOR OF SCIENCE IN MEDICAL LABORATORY SCIENCE
LECTURE Lecture By: Mr. Kristan Dela Cruz Transcribed By: Erysipelothrix rhusiopathiae
REVIEWER
 Reagent:
 Methylumbelliferyl-beta-D-glucoronide
(MUG DISK)
 (+) Result: Electric Blue Fluorescence
 (+) E. coli
 (-) Pseudomonas aeruginosa

TYPICAL COLONIAL MORPHOLOGY OF

ENTEROBACTERIACEAE

http://endless.horse/

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