A simple guide for the

identification of bacteria

Dr. Khalil Alkuwaity

 CONTENTS

Introduction 2
Abbreviations & shapes 3
The Family Stone 4
The Big Family 5
Gram Positive Cocci 6
Gram Negative Bacilli 8
Gram Positive Bacilli 9
Gram Negative Cocci 10
Gram Negative Coccobacilli 11
Anaerobic Bacteria 12
Acid Fast Bacilli 13
The Big Gathering 14
Member Profile 15
The Normal Flora 16
The Usual Suspects 17

Prepared by
DR. KHALIL ALKUWAITY
Department of Medical Laboratory Technology
Faculty of Applied Medical Sciences ‐ KAU
(December, 2012).

DR. ALKUWAITY 1 ONE BIG FAMILY

 INTRODUCTION

The purpose of this booklet is to provide a quick and easy

guide to the classification of clinically important bacteria.

The classification is principally based on the GRAM STAIN

result of the organism. Further diagnostic tests include:
BIOCHEMICAL reactions AND sensitivity to ANTIBIOTICS.
***
REMEMBER that good IDENTIFICATION always begins with
SEPARATED COLONIES and a clear GRAM STAIN result.

1 CULTURE

Gram‐negative
Gram‐negative coccobacilli
cocci in pairs
Gram‐positive
cocci in clusters

Gram‐positive
cocci in pairs
2 STAIN
Gram‐positive
cocci in chains

Gram‐negative
bacilli

Gram‐positive Yeast cell

bacilli (budding)

DR. ALKUWAITY 2 ONE BIG FAMILY

 ABBREVIATIONS & SHAPES

The following ABBREVIATIONS and SHAPES are used throughout this booklet:

GPB Gram Positive Bacilli

GPC Gram Positive Cocci
GNB Gram Negative Bacilli
GNC Gram Negative Cocci
GNCB Gram Negative Coccobacilli
AFB Acid Fast Bacilli

DNase positive

Bile Esculin positive

Lactose fermenter

Non‐lactose fermenter

Low virulence organism

S Sensitive
R Resistant
O2 Strict aerobic (O2 present)
CO2 Capnophilic (5% CO2)
MO2 Microaerophilic (Low O2)

Ø2 Strict anaerobic (O2 absent)

Spore‐forming
Motile
Diplococci
Likely to produce β‐lactamases
Curved bacilli

DR. ALKUWAITY 3 ONE BIG FAMILY

 THE FAMILY STONE

The following diagrams compare the CELL WALL structure of

Gram‐positive, Gram‐negative and Acid‐fast bacteria:

GRAM‐POSITIVE CELL WALL

Peptidoglycan (>60%)
Cytoplasmic membrane

GRAM‐NEGATIVE CELL WALL

Outer membrane → Lipopolysaccharide

Periplasmic space
Peptidoglycan (5‐10%)
Periplasmic space
Cytoplasmic membrane

ACID‐FAST CELL WALL

Glycolipids → Mycolic acid (60%)

Arabinogalactan
Peptidoglycan
Cytoplasmic membrane

Cell wall
Cell membrane
Cytoplasm

DR. ALKUWAITY 4 ONE BIG FAMILY

 THE BIG FAMILY

AFB

GPB GPC

Bacteria

GNB GNC

GNCB

NOTES
When stained using the Gram stain: GRAM‐POSITIVE bacteria appear PURPLE, while
GRAM‐NEGATIVE bacteria appear PINK.
ACID FAST BACILLI (AFB) are stained using special dyes such as the Ziehl‐Neelson
stain and they appear as RED bacilli against a blue background.
In addition, there are other types of bacteria that belong to different groups such as
the Spirochetes, Chlamydia and Mycoplasmas. These are not included in this guide,
as their diagnosis is based on different detection or culture techniques.

DR. ALKUWAITY 5 ONE BIG FAMILY

 Gram Positive Cocci

GPC

_
Catalase

Micrococcaceae
O2

Micrococcus

Staphylococci

Coagulase

+ _

S. aureus CoNS*

Methicillin Novobiocin

S R
R

MRSA S. epidermidis S. saprophyticus

Methicillin Resistant
Staphylococcus Aureus This distinction is usually made in
urinary tract infections.

*
Coagulase Negative Staphylococci

DR. ALKUWAITY 6 ONE BIG FAMILY

 Gram Positive Cocci

...continued

Catalase ̶

Streptococcaceae

6.5% NaCl +
Enterococci

Vancomycin

R
VRE
Streptococci

Colony on Blood Agar

Beta hemolysis Alpha hemolysis

OR
Bacitracin: S
Group A Optochin
Lancefield
Grouping

CAMP +
Group B
S
S. pneumoniae
6.5% NaCl ̶
Group D
R
Viridans Streptococci

Group A = Streptococcus pyogenes Enterococci include E. faecalis, E. faecium

Group B = Streptococcus agalactiae VRE: Vancomycin Resistant Enterococci
Group D = Non‐Enterococci Viridans Strept include S. mitis, S. mutans

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 Gram Negative Bacilli

Swarming, Urease +

Proteus spp.
Indole + XLD → Black center

Escherichia coli Salmonella spp.

ESBL**

Klebsiella spp. Shigella spp.

Enterobacteriaceae*
_

API 20E OR
Oxidase GNB Analytical VITEK
Profile Index

O2 Pyocyanin TCBS media

Pseudomonas aeruginosa Vibrio cholerae

MO2 MO2

Helicobacter pylori Campylobacter jejuni

Urease +

*
Other important
**
May produce ESBL enzymes Enterobacteriaceae include:
(Extended Spectrum Beta‐Lactamases)  Citrobacter freundii
=> Resistance to cephalosporins.  Enterobacter cloacae
 Morganella morganii
 Serratia marcescens
 Yersinia enterocolitica
Identify using API or VITEK

DR. ALKUWAITY 8 ONE BIG FAMILY

 Gram Positive Bacilli

B. cereus
Psychrophile

L. monocytogenes B. anthracis

Listeria Bacillus

All

Lactobacilli GPB Catalse +

Catalase 

Corynebacterium Diphtheroids

C. diphtheriae
Chinese letters
Tellurite‐blood agar
(Toxigenic) Elek test +

DR. ALKUWAITY 9 ONE BIG FAMILY

 Gram Negative Cocci

GNC

CO2 All
Neisseria spp Neisseriaceae Oxidase +

Neisseria gonorrhoeae Neisseria meningitidis

Glucose fermenter Glucose/maltose fermenter
Thayer Martin agar

Moraxella catarrhalis
Non‐fermenter

DR. ALKUWAITY 10 ONE BIG FAMILY

 Gram Negative Coccobacilli

XV

H. influenzae
V X

H. parainfluenzae H. ducreyi

V&X
Factors

CO2

Haemophilus

All

GNCB Catalase +
Oxidase +

Brucella Bordetella

Serology O2

B. pertussis
B. melitensis
Charcoal‐blood agar
B. abortus co2
B. suis

DR. ALKUWAITY 11 ONE BIG FAMILY

 Anaerobic Bacteria

C. tetani C. botulinum

C. perfringens C. difficile
Naglar reaction Toxin-A test

Clostridium

GPB

Ø 2

GNB

Bacteroides

B. fragilis
Metronidazole: S

DR. ALKUWAITY 12 ONE BIG FAMILY

 Acid Fast Bacilli

AFB
AFB

Mycobacterium*

O2

M. tuberculosis

TB rods stained using Ziehl‐Neelsen (ZN) stain and

Auramine‐Rhodamine (AR) fluorescent stain

TB colonies grown on
Lowenstein Jensen (LJ) medium

*
Mycobacterium species include:
Mycobacterium tuberculosis (MTB) => TB infection
Mycobacteria Other Than Tuberculosis (MOTT) => TB‐like infection

DR. ALKUWAITY 13 ONE BIG FAMILY

 THE BIG GATHERING

(Complete with the correct names of organisms)

B C

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 MEMBER PROFILE

(Growth and colony appearance)

ORGANISM MEDIA* INCUBATOR COLONY MORPHOLOGY

S. aureus BAP & MSA 37°C β‐hemolysis, yellow on MSA

S. pyogenes BAP Ø2 β‐hemolysis

GPC

S. pneumoniae BAP CO2 α‐hemolysis, doughnut shape

Enterococcus BAP 37°C Small, white

E. coli MAC 37°C Pink, dry

K. pneumoniae MAC 37°C Pink, mucoid

Proteus spp MAC 37°C Pale yellow

GNB

Salmonella spp DCA & XLD 37°C Pale yellow/pink, black center

Shigella spp DCA & XLD 37°C Pale yellow/pink

P. aeruginosa MAC 37°C Pale yellow, pyocyanin

V. cholerae TCBS 37°C Yellow (sucrose fermenter)

H. influenzae CAP CO2 Small, translucent

GNCB

Brucella spp BAP 37°C Gray‐white

B. pertussis Charcoal‐BAP 37°C Mercury‐like

C. diphtheriae Tellurite‐BAP 37°C Gray‐black

GPB

L. monocytogenes BAP 37°C β‐hemolysis

N. meningitidis BAP CO2 Shiny, grayish

GNC

M. catarrhalis BAP CO2 Crumble, slide across

C. perfringens BAP Ø2 Double β‐hemolysis

Others

B. fragilis BAP Ø2 Small, sensitive to MTZ disc

M. tuberculosis LJ medium 37°C Rough, tough and buff

* Shows characteristic morphology, but the organism may also grow on other media.
 ABBREVIATIONS:
BAP = Blood Agar Plate, CAP = Chocolate Agar Plate,
MSA = Mannitol Salt Agar, DCA = Deoxycholate Citrate Agar,
MAC = MacConkey agar, XLD = Xylose Lysine Desoxycholate,
LJ = Lowenstein Jenson, TCBS = Thiosulfate Citrate Bile salts Sucrose.

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 THE NORMAL FLORA

(Normally present in healthy individuals)

BODY SITE NORMAL FLORA

Skin, Eye CoNS*

Viridans Streptococci
& Urethra
Diphtheroids

CoNS*
Viridans Streptococci
Oropharynx
Diphtheroids
Neisseria spp

GBS
CoNS*
Vagina
Lactobacilli
Candida spp

Bacteroides
Clostridium
Large intestine Coliforms**
Proteus
Pseudomonas

CSF
BODY FLUIDS

Blood
Urine (in bladder) Normally Sterile
Synovial Fluid
Ascitic Fluid

*
CoNS: Coagulase Negative Staphylococci (e.g. S. epidermidis).
**
Coliforms: are lactose‐fermenting Enterobacteriaceae, such
as E. coli, Klebsiella, Citrobacter and Enterobacter.

DR. ALKUWAITY 16 ONE BIG FAMILY

 THE USUAL SUSPECTS

(Common causative agents)

Meningitis
N. meningitidis
Eye infection S. pneumoniae
H. influenzae H. influenzae Pharyngitis
S. pneumoniae E. coli Strept Group A,C,G
P. aeruginosa GBS H. influenzae (<6yrs)

Pneumonia
Septicemia S. pneumoniae
Staphylococci H. influenzae
Streptococci M. catarrhalis
GNB GNB
C. albicans
Anaerobes Skin & Wound
S. aureus
Gastroenteritis S. pyogenes
Salmonella GNB
Shigella Anaerobes
Campylobacter
Helicobacter
Urinary Tract
Vibrio
Infection
E. coli
Genital infection Other Body Fluids Klebsiella
N. gonorrhoeae Staphylococci Proteus
T. pallidum Streptococci Enterococcus
H. ducreyi GNB P. aeruginosa
G. vaginalis Anaerobes S. saprophyticus
C. albicans C. albicans

DR. ALKUWAITY 17 ONE BIG FAMILY