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Gram Staining
ƒ Introduced by Hans Christian Gram
ƒ Used to classify bacteria on the basis of their cellular morphologies, sizes and forms.
ƒ Permits the separation of all bacteria into two large groups, bacteria that retain the primary stain (Gram
positive) and those that take the counterstain (Gram negative).

Principle
ƒ Gram positive single layer of cell membrane with a thick cell wall
ƒ Gram negative double layer of cell membrane with a thinner peptidoglycan sandwiched between the two
cell membranes (see the image below)

Gram positive cell wall takes up the crystal violet and when followed by a mordant (iodine), it forms a
crystal violet complex within the cell. The crystal violet complex is larger than the crystal violet alone which
impedes it from being removed by the following step. Since the cell wall of the Gram negative is inside the
outer membrane, it cannot form a complex with the crystal violet even when there is already a mordant. The
primary stain stays on the outer membrane which is then washed by the alcohol allowing the safranin red to
counter stain it.

Procedure

Reagents:
Primary Stain: Crystal Violet
Mordant: Gram Iodine
Decolorizer: Ethyl Alcohol
Counterstain: Safranin Red

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Acid Fast Staining

ƒ Allows the detection of acid fast bacteria

Principle
The lipid capsule of the acid fast bacteria contains mycolic acid (long chain fatty acid) which is responsible for
its waxy characteristic that resists the penetration of an aqueous based solution (i.e. Crystal violet). The lipid
capsule takes up the carbolfuchsin and resists decolorization with an acid alcohol rinse. The acid fast bacteria

Other organisms that are acid-fast: Nocardia spp. and Cryptosporidium spp.

Procedure Interpretation
Number of AFB seen Report
(1000X magnification)
0 No AFB seen
1-2/300 fields Doubtful
1-9/100 fields 1+
1-9/10 fields 2+
1-9/ field 3+
>9/ field 4+

Seen in this picture are the

Acid fast bacteria
(Mycobacterium
tuberculosis) stained as
red (arrows) with the
surrounding blue
background

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Bacterial Cultivation
ƒ Usually required for a definitive identification and characterization of the etiologic agent (Gold
standard)

Purposes
ƒ To grow and isolate all bacteria present in a specimen
ƒ To determine which is the probable causative agent for the disease
ƒ To allow identification and characterization (therapy)

Principle
ƒ It is a process wherein the bacteria is taken from the infection site and planted into a medium that has its
nutritional and environmental requirements.
ƒ Cultivation somehow mimics the common niche of the organism.
(E.g. Vibrio spp. – Halotolerant; TCBS – contains high concentration of salt)

Classification and Functions

A. Enrichment
ƒ Contain specific nutrients required for the growth of a particular bacterial pathogens that may be
present alone or with other bacteria
ƒ Buffered charcoal-yeast extract agar (L-cysteine) – Legionella pneumophila
B. Supportive
ƒ Contain nutrients for the growth of different nonfastidious organisms without promoting any of
the other organism’s growth
ƒ Nutrient Agar
C. Selective
ƒ Contain agents that are inhibitory to all the organisms except those wanted microorganism
ƒ TCBS – Vibrio spp.
D. Differential
ƒ Contain certain factor that allows different strains of bacteria to exhibit certain metabolic or culture
characteristic when grown
ƒ sBAP (sheep’s blood agar plate) – differentiates Streptococcus spp.

Thiosulfate Citrate-Bile Salts Agar (TCBS)

ƒ Selective and differential for Vibrio spp.

Principle
ƒ Inhibitors of Gram Positive and negative bacteria
ƒ Bile salts
ƒ Citrate Æ Carbohydrate source

Yellow Colonies Green Colonies

(A) (B)
Vibrio cholerae Vibrio parahaemolyticus

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MacConkey Agar
ƒ Isolation and differentiation of lactose fermenting and non-lactose fermenting enteric bacilli
ƒ Selective (Gram negative bacteria)
ƒ Differential (Lactose fermenters and non-lactose fermenters)

Principle
ƒ Inhibitors of Gram Positive bacteria
Rapid Lactose Slow Lactose Non- Lactose
ƒ Bile Salts
Fermenters Fermenters Fermenters
ƒ Crystal violet
(Pink) (Pink – 48h) (Colorless)
ƒ Neutral Red
ƒ Lactose Æ only carbohydrate source Enterobacter spp. Serratia spp. Shigella spp.
ƒ Neutral red Escherichia spp. Citrobacter Salmonella spp.
ƒ Indicator Klebsiella spp. spp. Proteus spp.
ƒ Brown at pH 6.8-8.0
ƒ Pink-red at pH <6.8

Lactose Fermenters
Enterobacter aerogenes Klebsiella pneumoniae (mucoid colonies)

Eschericia coli

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Late Lactose Fermenters Non-Lactose Fermenters

(24hrs- NLF and 48hrs- LF) Proteus mirabilis (swarming colonies)

Citrobacter spp.

Serratia marcescens (red pigment production) Pseudomonas aeruginosa

Salmonella spp.

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Eosin Methylene Blue Agar (EMB)

ƒ Isolation and differentiation of lactose-fermenting and non-lactose fermenting enteric bacilli
ƒ For Escherichia coli identification

Principle Lactose Fermenters Non-Lactose Fermenters

ƒ Inhibitors of Gram Positive bacteria (Purple) (colorless)
ƒ Eosin
Escherichia coli Proteus spp.
ƒ Methylene blue
(Green metallic sheen) Pseudomonas aeruginosa
ƒ Lactose Æ only carbohydrate source
Klebsiella spp.
Serratia marcescens
Enterobacter aerogenes

Lactose Fermenters
Escherichia coli (green metallic sheen) Klebsiella pneumoniae

Non-Lactose Fermenters
Proteus spp.

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Salmonella Shigella Agar

ƒ Selective for Salmonella and Shigella spp.

Principle
ƒ Inhibitors of Gram Positive bacteria Red colonies Colorless w/o Colorless w/
ƒ Bile Salts black centers black centers
ƒ Inhibitors of other Gram Negative bacteria
ƒ Brilliant green
ƒ Bile Salts Coliforms Shigella spp. Salmonella spp.
ƒ Lactose Æ only carbohydrate source
ƒ Sodium thiosulfate Æ Sulfur source (H2S
production)
ƒ Ferric sulfide Æ black color
** H2S + Ferric Ammonium citrate = Ferric sulfide

Shigella spp.

Salmonella spp.

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Sheep’s Blood Agar Plate (BAP)

ƒ Allows the cultivation of fastidious microorganisms
ƒ Differential medium and NOT selective
ƒ Determines hemolytic capacity of different organisms

Principle
ƒ Fresh sterile sheep’s blood is added to the medium after autoclaving and just before the medium solidify
** Chocolate agar is made by adding the blood right after the autoclaving when the medium is still hot
ƒ Presence of blood determines the hemolytic capacity of an organism
ƒ Hemolysis is differentiated into Alpha(partial), Beta(Complete) and Gamma(None)

Alpha Beta Gamma

Alpha α Beta β Gamma γ

Streptococcus pneumoniae Streptococcus pyogenes Enterococcus faecalis

Optochin Test Bacitracin Test

S. pneumoniae(S) vs Viridian Strep (Resistant) S. pyogenes (S) vs. S. agalactiae (Resistant)

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Xylose Lysine Desoxycholate Agar (XLD)

ƒ Isolation and differentiation of Salmonella and Shigella spp. From other gram-negative enteric bacilli
ƒ Isolation and differentiation of stool pathogens
ƒ Differs from HE by the ability to detect Lysine decarboxylation

Principle
ƒ Inhibitors of Gram Positive bacteria
ƒ Sodium deoxycholate
- Partially inhibits E .coli
- Inhibits Proteus swarming
ƒ Xylose, Lactose and Sucrose (Carbohydrate source)
- Fermented by all the members of Enterobactericeae except for Shigella spp.
ƒ Phenol red Æ indicator
- Fermentation of Carbohydrate Æ yellow
ƒ Lysine
- Lysine positive turns yellow to red via decarboxylation
ƒ H2S Positive
- Reaction of H2S with ferric ammonium citrate Æ black centered colonies

Interpretation
Reaction Microorganisms
Red Colonies Shigella spp.
Red Colonies with black centers (H2S +) Salmonella spp
Yellow Colonies Escherichia coli
Yellow Colonies with black centers (H2S +) Citrobacter spp. and Proteus spp.

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Hektoen Enteric Agar (HE)

ƒ Differential, selective medium for Shigella and Salmonella spp. From other gram-negative enteric bacilli
ƒ Preferentially allows the growth of stool pathogens by inhibiting the enteric normal flora

Principle
ƒ Inhibitors of Gram positive and negative bacteria
- Bile salts
ƒ Carbohydrate source Lactose Non-lactose H2S
- Lactose fermenting fermenters Producers
- Sucrose Escherichia coli Shigella spp. Salmonella spp.
- Salicin Yersinia spp.
ƒ Bromthymol blue Æ indicator
- >7.6 Æ blue
- 6-7.6 Æ green
- <6 Æ yellow (fermentation of any sugar)
ƒ Sodium thiosulfate
- H2S production Æ black centered colonies

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Triple Sugar Iron Agar (TSI)

ƒ Initial step for the identification of Enterobactericeae
ƒ Used to determine whether a gram-negative rod utilizes glucose and lactose or sucrose fermentatively
and forms hydrogen sulfide H2S.
Principle
ƒ Fermentation of Glucose Æ makes slant and butt yellow, but the slant reverts to alkaline (red) due to the
oxidative decarboxylation. (K/A)
ƒ Fermentation of Glucose + Lactose or Sucrose Æ counteracts the alkalinity on the slant and yields a
yellow color (A/A)
ƒ Carbohydrate source (Triple Sugar)
o Lactose
o Sucrose
o Glucose
ƒ Ferrous sulfate Æ indicator for H2S gas production
ƒ Phenol red Æ indicator
C-- Control
1 – K/K
2 – K/A
3 – K/@ H2S +
4 – A/@
4A – K/@
5 – A/@ H2S +

**gas production is
observed by the presence of
bubbles or cracks

Reaction Fermented Carbohydrate Organisms

A/@ H2S + Glucose and Lactose and/or Sucrose Citrobacter freundii
A/@ H2S - Glucose and Lactose and/or Sucrose Escherichia coli
Klebsiella spp.
Enterobacter spp.
K/@ H2S + Glucose only Salmonella spp.
Proteus spp.
Citrobacter spp.
K/A H2S - Glucose only Shigella spp.
Providencia spp.
Serratia spp.
Anaerogenic E. coli
K/K H2S - None Pseudomonas spp.

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Lysine Iron Agar (LIA)

ƒ Determines the ability of the bacteria to deaminase, decarboxylase lysine and also to produce H2S
ƒ Useful in identification of Salmonella, Shigella, Proteus, Providencia and Morganella spp.
ƒ Proteus – only member of the Enterobactericeae Æ Deaminase lysine

Principle
ƒ Indicator Æ bromcresol purple
ƒ Glucose Æ Turns to yellow when fermented (seen in the butt)
ƒ Ability to produce H2S gas Æ
ƒ Deamination (slant) **A before C: deAmination before deCarboxylation : Slant before Butt
- Occurs in the presence of oxygen (Aerobic process)
- Ammonia produced will react with ferric ammonium citrate Æ dark red on slant
- Negative deamintaion Æ remain purple
ƒ Decarboxylation (butt)
- Occurs in the absence of Oxygen (anerobic process)
- Produces an alkaline environment ( yellow to purple: Æ )

A – K/K
B – K/K H2S +
C – K/A
D – R/A
E – Uninoculated

Slant (deamination)
(+) dark Red
(-) Purple

Butt (decarboxylation)
(+) Purple
(-) Yellow

Organism Lysine Lysine H2S

deamination decarboxylation Gas
Escherichia coli - + -
Proteus mirabilis + - -
Pseudomonas spp - - -
E. aerogenes - + -
Shigella spp. - - -
Salmonella spp. - + +

K/A: Negative deamination, Negative decarboxylation

Shigella spp.

K/K H2S+: Negative deamination, Positive decarboxylation

Salmonella spp.

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Simmon’s Citrate Agar (SCA)

ƒ Determines if the bacteria can utilize citrate as its own carbon source

Principle
ƒ Inhibitors of Gram Positive bacteria
ƒ Eosin
ƒ Methylene blue
ƒ Sodium Citrate Æ only carbohydrate source
- Sodium citrate Æ ammonia Æ ammonium hydroxide (alkaline) Æ Prussian blue

Positive reaction Negative reaction

(Prussian Blue or with Growth) (Green color or NO growth)
Klebsiella spp. Escherichia coli

Prussian blue color reaction is observed with the top test tube inoculated with K. pneumoniae.
The bottom test tube is uninoculated (normal color of green)

Indole Broth
ƒ Determine the ability of an organism to split tryptophan to form the compound indole

Principle
ƒ Tryptophan present in peptone is oxidized into indole by Tryptophanase

Tryptophan Æ indole + pyruvic acid + ammonia

ƒ Reagents used in detecting indole (dropped

o Erlich’s reagent (more sensitive) Æ used in Anaerobic and nonfermentative organisms
o Kovac’s reagent

Indole + p-dimethylaminobenzaldehyde (Kovac’s or Erlich’s) Æ red color between the rgt and broth

Indole Positive Indole Negative

Escherichia coli Enterobacter spp.
Proteus vulgaris Klebsiella spp.
Proteus mirabilis

Presence of red ring between the

reagent and the broth = Positive

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Motility Indole Ornithine Medium (MIO)

ƒ Determines the motility of the bacteria as well as its indole production and ornithine decarboxylation

Principle
ƒ Bromcresol purple Æ indicator
ƒ Agar used is semisolid (allows more movement of the bacteria)
ƒ Contains tryptophan for production
ƒ Contains Ornithine for detection of its decarboxylation

Interpretation
Test Reaction Organisms
Motility (+) diffused growth Proteus spp.
Salmonella spp.
(-) growth along the line Klebsiella spp.
Shigella spp.
Indole production (+) red ring Escherichia coli
(-) absence of red ring Klebsiella spp.
Ornithine decarboxylation (+) Purple ?
(-) Yellow ?

MIO medium

(+) Motility
(+) Indole
(+) Motility
(+) Motility and
(-) Ornithine decarboxylation
(-) Motility

Motility Agar w/o Bromcresol purple

1% triphenyltetrazolium chloride (responsible for the red color)

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Christensen’s Agar or Stuart Broth (Urease Test)

ƒ Determines the ability of an organism to produce urease

Principle
ƒ Urease catalyzes the hydrolysis of urea to produce ammonia and CO2
ƒ Phenol red Æ indicator
ƒ (+) for ammonia production reacts in the solution to produce ammonium carbonate that shifts the pH
from 6.8 (light orange or Salmon pink?) to 8.1 (magenta or pink-red or Fuchsia pink)

Urease Positive (Fuchsia Pink) Urease negative (Salmon pink?)

Rapid (w/in 4 hrs) Slow Escherichia coli
Proteus spp. Enterobacter spp.
Morganella spp. Klebsiella pneumoniae

Stuart Broth Christensen’s Agar

Positive
reaction

Oxidase Test
ƒ Determine the presence of bacterial cytochrome oxidase

Principle
ƒ Oxidation of tetramethyl-p-phenylenediamine dihydrochloride Æ indophenol (dark purple)
ƒ Production of intense dark purple color after the addition of the substrate Æ positive for oxidase

** Wires used for transferring organism to slide yield false positive result. So in the laboratory, a wooden
applicator stick is used to transfer the organism from the tube or plate to the slide.

Positive (Purple) Negative

Vibrio spp. Escherichia coli
Neisseria spp.

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Methyl Red / Voges-Proskauer (MRVP) Test

ƒ Determine the ability of an organism to produce and maintain stable acid and neutral end products from
glucose fermentation and to overcome the buffering capacity of the system.

Principle
ƒ Glucose is fermented to pyruvic acid by one of the two pathways Æ (+)MR or (+)VP
ƒ First pathway (detected by MR)
o Lactic acid, acetic acid, formic and succinic acid Æ decrease in pH Æ production of red color
ƒ Second pathway (detected by VP)
o Acetylmethyl carbinol (Acetoin)
ƒ Neutral end product
Acetoin + O2 + 40% KOH Æ Diacetyl Form
Diacetyl form + alpha napthol (added reagent) Æ production of red color
ƒ Must be incubated for 48 hrs
Methyl Red Voges-Proskauer

Positive Negative Positive Negative

Organism MR VP
Escherichia coli + -
Enterobacter spp. - +

ONPG (o-Nitrophenyl-B-D-Galactopyranoside) Test

ƒ Determine the ability of the organism to produce beta-galactosidase

Principle
ƒ Beta galactosidase hydrolyzes ONPG Æ orthonitrophenol (yellow color)

Positive Negative
Escherichia coli Salmonella spp.

Positive

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Notes:

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