Gram Stain-

Gram-Positive: Streptococcus, Staphylococcus, Corynebacterium, Listeria, Bacillus,

Clostridium, etc.

Gram-Negative: E. coli, Salmonella Typhi, Shigella spp, Pseudomonas aeruginosa, Neisseria

gonorrhoeae, Chlamydia trachomatis, Yersinia pestis, etc.

AGAR PLATES-
Enriched; blood agar, chocolate agar….

Selective; MacConkey, Eosin-methylene blue (EMB) mannitol salt (MSA)

contain inhibitory chemicals (antibiotics, dyes, salts…)

Differential; MacConkey, EMB, MSA (contain a substrate that differentiates the selective
growth, lactose, sucrose, mannitol …)

Selective & Differential; MacConkey agar, Mannitol Salt agar

1
LAB # 2 and # 3 Staphylococcus (Chapter 14)
Micrococcus luteus in BAP Staphylococcus aureus in BAP (beta-hemolysis)

Manitol Salt Agar Media Novobiocin Disk Test

Coagulase Tube test (Above pos/Bellow neg) Staphylococcus spp gram stain

2
Lab # 4 and # 5, Chapter 15 Streptococcus, Enterococcus, and other catalase negative GPC
S. pyogenes in BAP (beta hemolysis) Bacitracin disk (Taxo) S. pyogenes susceptible

S. pneumoniae in BAP (alpha hemolysis) Bile Solubility Test

Enterococcus faecalis in BAP (No Hemolysis) Bile Esculin test

3
Camp Test

Catalase Test Optochin disk test

Streptococcus spp Gram Positive Cocci

LAB # 6 and # 7, Chapter 17 Neisseria

4
Oxidase Positive Oxidase Negative Cystine Trypticase Agar (CTA)

Chocolate agar plate Neisseeria spp Gram Negative diplococci

Lab # 8 and # 9, Chapter 18 Haemophilus

ALA-Porphyrin Test Legionella in BCYE agar plate

X (hemin) and V (NAD) factors

5
Lab # 10, #11, #12, #13, CHAPTER 9 ENTEROBACTERIACEAE

Hektoen enteric (HE) Agar plate

E. coli in EMB agar plate Enterobacteria gram negative rods

Lab # 14, #15, Chapter 20 CAMPYLOBACTER

Campylobacter gram stain (Seagull shape) TCBS agar (yellow positive- green negative)

6
For O/129 Susceptibility test Aeromonas resistant (left)/Vibrio susceptible (right)

Phenylethyl alcohol agar(PEA)- selective media for gram stain positive, inhibits the growth of
gram-negative organisms, prevents proteus from swarming.

7
Sheep blood agar (SBA)- it contains general nutrients and 5% sheep blood. Useful for fastidious
organism and for determining the hemolytic capabilities of an organism. Some bacteria produce
exoenzyme that lyse the red blood cells and degrade hemoglobin(hemolysins).
Beta- hemolysis-breaks down rbcs and hemoglobin completely, it leaves clear zone around
bacterial growth.
Alpha-hemolysis- partially breaks down rbc and leaves greenish color behind.
Gamma- hemolysis- organism does not break down the blood cells, no clearing will occur.

Mannitol salt agar (MSA)- Selective and differential media for gram stain positive, high salt
tolerance organism grow like Staphylococcus. Contains sugar mannitol and pH indicator phenol
red.
If organism ferments mannitol, acidic byproduct is formed and causes phenol red to change color to
yellow. E.g.- pathogenic staphylococcus.
If organism does not ferment mannitol, no color change occurs. E.g.- non-pathogenic
staphylococcus.
8
Trypticase soy agar (TSA) - used as medium for maintaining or subculturing reference strains.
E.g. Enterobacteriaceae and Staphylococci. Tryptic soy agar with the addition of salt can be helpful
in determining the halotolerance level of microorganisms.

Organisms Growth

Aspergillus brasiliensis Positive

Candida albicans Positive

Staphylococcus aureus Positive; pigmented shiny round colonies

Staphylococcus epidermidis Positive

Bacillus subtilis Positive; flat, large irregular colonies

Pseudomonas aeruginosa Positive

Escherichia coli Positive; shiny round colonies

Salmonella Typhimurium Positive

9
Simmons Citrate agar-

Differentiates based on the utilization of

citrate as the sole source of carbon.

When the bacteria metabolize citrate,

the ammonium salts are broken down
to ammonia, which
increases alkalinity. The shift in pH turns
the bromthymol blue indicator in the
medium from green to blue above pH 7.6.
Ammonium Dihydrogen Phosphate is
the sole source of nitrogen.
Sodium Citrate is the sole source
of carbon in this medium.

Citrate utilization- alkaline reaction, color changes to blue color.

No Citrate utilization- no color change to blue, remains green color.
Differentiates gram negative bacteria on basis of citrate utilization. Simmons Citrate Agar is primarily
used to aid in the identification of Enterobacteriaceae-

 Escherichia coli (usually -) and Shigella spp. (-) from other commonly encountered

Enterobacteriaceae (variable +).

 Edwardsiella spp. (-) from Salmonella spp. (usually +)

 Serratia proteamaculans (+) from  Yersinia  pseudotuberculosis (-) and Yersinia enterocolitica

(usually -)

 Klebsiella-Enterobacter groups (usually +) from E.  coli (usually -)

 Proteus rettgeri (+) from Morganella morganii biogroups 1 and 2 (-)

 Yokenella regensburgei (+) from Hafnia alvei (-)

 Leminorella grimontii (+) from L. richardii (-)

 Acidovorax delafieldii (+) from A. facilis (-) and  A. temperans (-)

Chocolate agar- It is used in the isolation and

cultivation of fastidious microorganisms, particularly
Haemophilus and Neisseria species.
Chocolate agar is also considered as a variant of the
blood agar plate, containing red blood cells that have
been lysed by slowly heating to 80°C.
The heat inactivates enzymes which could otherwise
degrade NAD.

10
The added supplements provide the necessary X factor (from Hemoglobin) and V factor (from
Growth Supplement) required by the fastidious organisms.

Modified Thayer-martin agar-

Isolates pathogenic species.
Selective for N. gonorrhoeae, N. meningitidis an N. lactamica and inhibits saprophytic Neisseria
and Moraxella catarrhalis and other bacteria.
Four antimicrobial agents that have been added:
Vancomycin- inhibits gram positives.
Colistin- inhibits gram-negatives.
Nystatin- inhibits fungi.
Trimethoprim- inhibits the swarming of proteus.

Thayer-Martin (TM)-
Selective media. Lacks Trimethoprim (inhibits the swarming of proteus).

11
12
Xylose lysine deoxycholate (XLD) agar:

Selective and Differential

a. Selective: inhibits with sodium deoxycholate

gram-positive, some gram-negative.

b. Differential with 3 carbohydrates: sucrose,

lactose in excess, and xylose with a phenol red
indicator (acid = yellow, alkaline = pink)

c. Lysine present to detect lysine

decarboxylation d. Thiosulfate present to detect
hydrogen sulfide (H2S) = black precipitate

XLD chemical reaction-

selective/differential; bile salts/lactose/H2S

lactose, sucrose, xylose  glucose  pyruvic acid + indicator

thiosulfate  H2S + ferric ions  black precipitate

Hektoen enteric (HE) agar chemical reaction:

Selective: Bile salts inhibit gram-positive, some

gram-negative.

Differential: Lactose and sucrose fermentation

a. Non pathogens: Lactose fermentation with acid

production (low PH/ orange color).

b. Pathogens (salmonella and shigella) non

fermenters green to blue green color with H2S in
the form of black points in the agar.

c. PH Indicator: Bromothymol blue

13
14
15
16
BIOCHEMICAL TESTS-
Staphylococcus

Staphylococcus species- found in normal flora of skin and mucous membrane.

Staphylococcus aureus- white, cream or golden colony.

Micrococcus luteus- bright yellow colony.

M. luteus- yellow pigment, coagulase negative, bacitracin susceptible

S. saprophyticus- seen in UTI.

Gram stain positive –

Catalase Test-

To see if Bacterial isolate is able produce catalase enzyme.

Lack of bubbles indicate a negative catalase test.

Bubbles appear due to liberation of O2.

17
Bacitracin test-

Sensitive- any zone of inhibition around the

disk

Resistant- no zone of inhibition

Positive- Streptococcus pyogenes

Negative- Streptococcus agalactiae.

Coagulase positive- S. aureus

Bound-coagulase- located on the exterior cell wall.

Free-coagulase- secreted inside the cell into the extracellular environment.

Clumping under 10 seconds or less is positive. No clumping is negative. Is confirmed by tube test.

Coagulase negative- S. epidermidis, S. Saprophyticus

18
Novobiocin test-

Resistant < 16mm- S. Saprophyticus

Sensitive >16 mm- other coagulase-negative staphylococci, such as S. epidermidis

Oxidase-

Determines the presence of the cytochrome oxidase system that oxidizes reduced cytochrome with
molecular oxygen.

Oxidase negative- gram positive cocci (staph, strept), Enterobacteriaceae (except Plesiomonas)

Oxidase positive- Pseudomonads, Neisseria spp.

Positive result- lavender color

List the streptococci that give positive reactions for each of the following biochemical tests?
Sodium Hippurate Hydrolysis- Group B (S. agalactiae)

19
Bile Solubility – Group A (S. pnumoniae)
Bacitracin Disk- Group A (S. pnumoniae)
Optochin Disk- Group A (S. pnumoniae)
PYR- Group A or GDE
6.5% NaCl broth- Group D enterococci
Bile Esculin – Group D and enterococci
CAMP test- Group B (agalactiae) beta-streptococci

Streptococci and the type of hemolysis?

Group A streptococci (s. pyogenes)-BETA hemolysis
Group B streptococci (s. agalactiae)-BETA hemolysis
Group D enterococci (E. faecalis)-GAMMA hemolysis
Group D enterococci non-enterococcus (s. bovis)-GAMMA hemolysis
Viridans streptococci- ALPHA hemolysis
S. pnumoniae – ALPHA hemolysis

CTA sugars-
For Neiserria, gonorrhea and meningitidis species
Glucose, maltose, lactose, and sucrose
pH indicator- phenyl red
If sugar is oxidized acid is produced and medium turn from pink to yellow.
N. gonorrhoeae utilizes glucose only.
N. meningitidis utilizes glucose and maltose.
N. lactamica utilizes glucose, maltose, and lactose.

20
• Triple sugar iron (TSI) agar or Kligler iron agar (KIA) to determine glucose and lactose, or sucrose,
utilization (sucrose in TSI only) and hydrogen sulfide production

• Methyl red and Voges-Proskauer tests to determine end products of glucose fermentation

• Indole test to determine whether indole is formed from tryptophan by tryptophanase

• Urease test to determine hydrolysis of urea

• Simmons’ citrate to determine whether citrate can be used as the sole carbon source

• Carbohydrate fermentation

21
• Lysine iron agar (LIA) to determine lysine decarboxylase activity

• Sulfide-indole-motility (SIM) or motility-indole-ornithine (MIO) media

Triple Sugar Iron Agar

A=acid production; K=alkaline reaction; G=gas production; H2S=sulfur reduction

S.N. Result (slant/butt) Symbol Interpretation

Glucose and lactose and/or sucrose

1 Yellow/Yellow A/A
fermentation. They are being utilized

Glucose and lactose and/or sucrose

2 Yellow/Yellow with bubbles A/A,G
fermentation, Gas produced.

Yellow/Yellow with black Glucose and lactose and/or sucrose

3 A/A,H2S
precipitate fermentation, H2S produced.

Glucose and lactose and/or sucrose

Yellow/Yellow with bubbles
4 A/A,G,H2S fermentation, Gas produced, H2S
and black precipitate
produced.

Glucose fermentation only, peptone

5 Red/Yellow K/A
catabolized.

22
 Glucose fermentation only, Gas
6 Red/Yellow with bubbles K/A,G
produced.

Red/Yellow with black Glucose fermentation only, H2S

7 K/A,H2S
precipitate produced.

Red/Yellow with bubbles and Glucose fermentation only, Gas

8 K/A,G,H2S
black precipitate produced, H2S produced.

No fermentation, Peptone catabolized

9 Red/Red K/K under aerobic and/or anaerobic
conditions.

Test organism Slant Butt Gas production H2S production

Escherichia coli ATCC25922 Yellow Yellow + –

Pseudomonas aeruginosa ATCC27853 Red Red – –

Salmonella enterica  ATCC14028 Red Yellow + +

Shigella sonnei ATCC9290 Red Yellow – –

23
TSI agar and Kligler iron agar (KIA) are useful in the presumptive identification of gram-negative enteric
bacteria, particularly in screening for intestinal pathogens. The formulas for TSI agar and KIA are
identical except that TSI agar contains sucrose in addition to glucose and lactose. Lactose is present in a
concentration 10 times that of glucose (1% lactose and 0.1% glucose). In TSI agar, sucrose is also present
in a 1% concentration. Ferrous sulfate and sodium thiosulfate are added to detect the production of
hydrogen sulfide gas (H2S). Both TSI agar and KIA are useful in detecting the ability of the microorganism
to ferment carbohydrates (glucose and lactose in KIA and glucose, lactose, or sucrose in TSI agar); to
produce gas from the fermentation of sugars; and to detect the production of H2S.

24
pH indicator – phenol red

Uninoculated medium- red (pH 7.4)

Both TSI agar and KIA are poured on a slant. The slant portion is aerobic; the butt, or deep portion, is
anaerobic.

Hydrogen Sulphide (H2S) Production Test-

It is used for the detection of hydrogen Sulphide (H2S) gas produced by an organism.
H2S is produced by certain bacteria through reduction of Sulphur containing amino acids like
cystine, methionine or through the reduction of inorganic Sulphur compounds such as
thiosulfates, sulfates or sulfites.
Hydrogen Sulphide, a colorless gas, if produced reacts with the metal salt (containing iron or
lead) forming visible insoluble black precipitate of ferrous Sulphide.

Positive result: blackening on the medium

Negative result: no blackening on the
medium

Positive control: Proteus vulgaris

Negative control: Escherichia coli

H2S production may be inhibited on TSI for organisms that utilize sucrose and
suppress the enzyme mechanism that results in production of H2S.

SIM is more sensitive in the detection of H2S than either TSI or KIA, because
of its semisolid nature, its lack of interfering carbohydrates, and the use of peptonized iron as an
indicator.

SIM- Sulfur Indole Motility-

SIM medium also tests for indole production and motility. Sulfur reduction to H2S is an
anaerobic activity and can be accomplished by bacteria in two different ways, depending on the
enzymes present.
Differentiates gram-negative bacteria in family Enterobacteriaceae.

Positive- cloudiness spreading from the inoculation line.

Positive for indole- a pink to red color after the addition of Kovac’s reagent

25
26
Motility Test-
Motile bacteria move using flagella, thread like locomotor appendages extending outward from
the plasma membrane and cell wall either single flagellum or multiple flagella.
The non-motile bacteria will only grow in the soft agar tube and only the area where they are
inoculated.
The medium mainly used for this purpose is SIM medium (Sulphide Indole Motility medium)
which is a combination differential medium that tests three different parameters, Sulfur
Reduction, Indole Production and Motility. 
Observing growth in a semisolid medium. Incubation temperature is important. Some bacteria are
motile only at room temperature. two motility tubes be inoculated, one incubated at room temperature
and the other at 35° C.

27
Motile: organisms will spread out into the medium from the site of inoculation, diffuse zone

Non motile:  organisms remain at the site of inoculation

Positive: Escherichia coli 
Negative: Staphylococcus aureus 

Motility indole ornithine (MIO) agar-

Used to detect motility and indole and ornithine decarboxylase production.

Differentiates Klebsiella spp. From Enterobacter and Serratia spp.

Positive result- pink to red color

Ornithine decarboxylase and motility should be read first before the addition of Kovac’s reagent.

28
Lysine Iron Agar (LIA)-
It contains amino acid lysine, glucose, ferric ammonium citrate, and sodium thiosulfate.

LIA is used to determine whether bacteria decarboxylate or deaminate lysine.

pH indicator- bromocresol purple

lysine positive (dark purple butt) – Salmonella spp.

Lysine negative- Citrobacter spp.

Purple butt- positive, removal of COOH, became alkaline.

The medium has an aerobic slant and an anaerobic butt.

Lysine deamination is an aerobic process which occurs on the slant of the media. 
Lysine decarboxylation is an anaerobic process which occurs in the butt of the media.
The indicator in LIA is bromocresol purple. Bromocresol purple, the pH indicator, is yellow at or
below pH 5.2 and purple at or above pH 6.8.

Lysine Decarboxylation (detected in butt):

29
Positive Test: Purple slant/purple butt (alkaline), the butt reaction may be masked by H2S
production.
If the organism produces lysine decarboxylase, cadaverine is formed. Cadaverine neutralizes the
organic acids formed by glucose fermentation, and the butt of the medium reverts to the alkaline
state (purple)
(tube 4)- Yersinia enterocolitica

Negative Test: Purple slant/yellow butt (acid), fermentation of glucose only. If the

decarboxylase is not produced, the butt remains acidic (yellow). 
(tube 3)- Shigella spp.

Lysine Deamination (detected on slant):

Positive Test: Red slant
Organisms that deaminate lysine, form alpha – ketocarboxylic acid, which reacts with iron salt
near the surface of the medium under the influence of oxygen to form reddish-brown compound.

(tube 6) Providencia spp. (tube 8) Morgonella spp

Negative Test: Slant remains purple
If deamination does not occur, the LIA slant remains purple.

H2S Production:
Positive Test: Black precipitate (tube 2) Salmonella spp. (tube 7) Proteus spp.
Negative Test: No black color development

Gas production: demonstrated by the presence of bubbles or cracks in the medium.

1 2 3 4 5 6 7 8

30
Purple slant and yellow butt: H2S positive: Citrobacter freundii 
Purple slant and purple butt, H2S negative: Escherichia coli 
Purple slant and butt, H2S positive: Salmonella Typhimurium
Red slant and yellow butt, H2S negative: Proteus mirabilis 

Urease Test-
Urease test is used to identify organisms that are capable of hydrolyzing urea to produce
ammonia and carbon dioxide.
The ammonia combines with the carbon dioxide and water to form ammonium carbonate, which
turns the medium alkaline, turning the indicator from its original orange-yellow color to bright
pink.
The test also differentiates Proteus from the non-lactose-fermenting bacteria.

pH indicator- phenol red

positive for urea- bright pink color.

A positive test is demonstrated by an intense magenta to bright pink color in 15 min to 24 hours.
A negative test shows no color change.

31
Positive test: Proteus mirabilis.
Negative test: Escherichia coli.

32
Glucose Metabolism and Its Metabolic Products

Glucose metabolized via the Embden-Meyerhof pathway produces several intermediate byproducts,
including pyruvic acid. Further degradation of pyruvic acid can produce mixed acids as end products.
However, enterics take two separate pathways: the mixed acid fermentation pathway or the butylene
glycol pathway.

The methyl red (MR) test and the Voges-Proskauer (VP) test detect the end products of glucose
fermentation. Each test detects products from a different pathway. The MR and VP tests are part of the
IMViC reactions: indole, MR, VP, and citrate.

IMVC test series -

The Indole test looks for the production of indole, the chemical that gives feces its typical odor.
Bacteria that are indole positive have enzymes that can digest the amino acid tryptophan to produce
indole.

The Methyl Red test detects the products of glucose metabolism, particularly whether a mixture of
acids are produced that drop the pH of the growth medium to below pH 4.5 (known as mixed acids
fermentation).

Voges-Proskauer looks for production of a molecule called acetoin (acetylmethylcarbinol), a neutral

fermentation product from glucose. Because acetoin has a neutral pH, enteric bacteria are typically
positive for one or the other (Methyl Red or Voges-Proskauer but not both).

Citrate test determines whether the unknown can use citrate as its sole carbon source for growth. The
results of these four basic tests are central to identifying enteric organisms.

Indole Test-
To detect the formation of indole from tryptophan by the enzymatic action of tryptophanese.
It is a qualitative test that tests the conversion of tryptophan into indole.
33
It is still used as a traditional method to distinguish indole-positive E. coli from indole-
negative Enterobacter and Klebsiella.
Indole, if present, combines with the aldehyde in the reagent to produce a pink to red-violet
quinoidal compound (benzaldehyde reagent) or a blue to green color (cinnamaldehyde reagent). 

Positive result- The formation of pink-red coloration (cherry red ring) in the reagent at the point
of contact between the reagent and the medium.
Negative result-The absence of color or the appearance of slightly yellow color.
MR test along with VP test is performed simultaneously because they are physiologically
related and are performed on MRVP broth.

Methyl red (MR) test-

Glucose → Pyruvic acid →

Mixed acid fermentation (pH 4.
4)

→ (positive) red color with

methyl red indicator

MR test is used to determine

the ability of an organism to
produce and maintain stable
acid end products from
glucose fermentation.
All members of the
Enterobacteriaceae can
convert glucose to pyruvic
acid by the Embden-
Meyerhof pathway, but
bacteria can further
metabolize pyruvic acid by
two different pathways.
Positive Test- If the
organism produces a large
number of organic acids that

34
include formic acid, acetic acid, lactic acid, and succinic acid from glucose fermentation, the
broth medium will remain red after the addition of methyl red, a pH indicator.
Negative organisms further metabolize the initial fermentation products by decarboxylation to
produce neutral acetyl methyl carbinol (acetoin), which results in decreased acidity in the
medium and raises the pH towards neutrality (pH 6.0 or above). 
Negative Test- For those organisms which do not produce the acid end products, the broth
medium will change to yellow coloration indicating a negative test.
Positive: Bright red color

Weakly positive: Red-orange
color.
Negative: Yellow color
MR positive:  Escherichia coli 
MR negative: Enterobacter
aerogenes 

Voges-Proskauer (VP) test-

Glucose→ Pyruvic acid →Acetoin →Diacetyl + KOH + α Naphthol → Red complex → 2 3, -Butanediol

Bacteria tend to be positive for either MR or VP but not both. Some bacteria are negative for both tests.

35
Positive Test- If the organism produces a large number of organic acids that include formic acid,
acetic acid, lactic acid, and succinic acid from glucose fermentation, the broth medium will
remain red after the addition of methyl red, a pH indicator.
Negative organisms further metabolize the initial fermentation products by decarboxylation to
produce neutral acetyl methyl carbinol (acetoin), which results in decreased acidity in the
medium and raises the pH towards neutrality (pH 6.0 or above). 

Positive: Crimson to ruby pink (red) color

Negative: No change in coloration

Positive Result:
 
Glucose ------Glucose Metabolism-------> Pyruvic Acid.
Pyruvic acid ---------------> Acetoin.
Acetoin + added alpha-naphthol + added KOH = red color.
 
Negative Result:
 
Glucose ------Glucose Metabolism-------> Pyruvic Acid.
Pyruvic acid -----------------> No Acetoin.
No acetoin + added alpha-naphthol + added KOH = copper color.

VP positive: Enterobacter aerogenes 
VP negative:  Escherichia coli 

ORGANISM VP POSITIVE/NEGATIVE
Enterobacter cloacae Positive
Klebsiella pneumoniae Positive
Proteus mirabilis Negative
Escherichia coli Negative
Enterobacter aerogenes Positive

36
Citrate Utilization Test-
Simmons’ citrate-

Determines whether an organism can use sodium citrate as a sole carbon source. Medium contains
ammonium salts as the sole nitrogen source. Bacteria able to use citrate will use the ammonium salts,
releasing ammonia.

pH indicator- bromothymol blue, green to blue – positive test

Salmonella,  Edwardsiella, Citrobacter, Klebsiella, Enterobacter, Serratia,
and Providencia usually give a positive reaction.
Escherichia, Shigella, Morganella, and Yersinia give a negative reaction.
The growth of the organism is indicative of the utilization of citrate as it is an intermediate
metabolite in the Krebs cycle. The enzyme citrase breaks down citrate into oxaloacetate and
acetate, where oxaloacetate is further broken down to form pyruvate and carbon dioxide.

The release of carbon dioxide induces the metabolism of

ammonium salts, causing the formation of ammonia or sodium
carbonate, both of which increase the alkalinity of the
medium.

Positive test- growth with a color change from green to

intense blue along the slant.
Negative test- no growth and no color change, and the color
of the slant remains green.

37
Oxidation Fermentation Test-
Oxidation fermentation test is used to determine whether an organism uses carbohydrate
substrates to produce acid byproducts.
Two tubes are required for interpretation of the OF test. Both are inoculated, and one tube is
overlaid with mineral oil, producing an anaerobic environment.

38
 Growth of microorganisms in this medium is either by utilizing the tryptone which results in an
alkaline reaction (dark blue color) or by utilizing glucose, which results in the production of acid
(turning bromothymol blue to yellow).

Organism Nature of organism Aerobic fermentation Anaerobic fermentation

Acid production (Yellow), Acid production (Yellow),

Escherichia coli  Facultative anaerobe
positive reaction positive reaction

unchanged (green) or
Acid production (Yellow),
Pseudomonas aeruginosa  Oxidative alkaline (blue), negative
positive reaction
reaction
The metabolic end products of carbohydrate fermentation can be either organic acids (lactic acid,
formic acid, or acetic acid) or organic acid and gas (hydrogen or carbon dioxide).

Fermentation- Production of acid in the overlaid tube and open tube results in a color change
Oxidation- Acid production in the open tube and color change.

Oxidative: yellow coloration in open tube only

Fermentative: yellow coloration on both open and closed tube

O/F basal medium (OFBM)

that contains the same

concentration of carbohydrates
(1%) found in the TSI medium but a
lower concentration of peptones
(0.2%).

pH indicator -bromothymol blue.

Uninoculated medium- green

acid environment- turns yellow.

alkaline environment- turns blue.

Oxidation requires oxygen (aerobic

respiration) or another inorganic
molecule (anaerobic respiration),
such as nitrate (NO3), as a terminal
electron acceptor.

39
OF OPEN OF CLOSED GLUCOSE UTILIZATION

Yellow (+) Yellow (+) Facultative anaerobe

Yellow (+) Green (-) Oxidative

Green (-) Green (-) Asaccharol

Carbohydrate Fermentation Test-

Some bacteria have the ability to degrade complex carbohydrates like lactose, sucrose or even
polysaccharides. 
Such bacterium should possess the enzymes that should cleave the
glycosidic bonds between the sugar units and the resulting simple
carbohydrate can be transported into the cell.
Acid production: Changes the medium into yellow color- organism ferments the given
carbohydrate and produce organic acids there by reducing the ph of the medium into
acidic.

Acid and Gas production: Changes the medium into yellow color-organism ferments
the given Carbohydrate and produce organic acids and gas. Gas production can be
detected by the presence of small bubbles in the inverted durham tubes.

Absence of fermentation: The broth retains the red color. The organism cannot utilize
the carbohydrate, but the organism continues to grow in the medium using other energy
sources in the medium.

Amino Acid Utilization

Decarboxylase and Dihydrolase Tests

Decarboxylase tests determine whether the bacterial species possess enzymes capable of
decarboxylating (removing the carboxyl group, COOH) specific amino acids in the test medium. Lysine
and ornithine are two amino acids commonly used to test for decarboxylase activity.

Degradation of amino acids and their specific end products Lysine (amino acid) →Lysine decarboxylase→
Cadaverine (amine) + CO2

Ornithine →Ornithine decarboxylase → Putrescine

Arginine →Arginine dihydrolase → Citrulline → Ornithine → Putrescine

Organism possess decarboxylase- purple color (positive result)

Organism does not possess decarboxylase- remains yellow (negative result).

40
 Phenylalanine deaminase (PAD) test

Deaminase Test

Phenylalanine deaminase (PAD) test

Amino acids can be metabolized by deaminases that remove an amine (NH2) group. The phenylalanine
deaminase (PAD) test determines whether an organism possesses the enzyme that deaminates
phenylalanine to phenylpyruvic acid.

Only Positive for Enterobacteriaceae - Proteus, Morganella, and Providencia organisms.

Deamination of Phenylalanine

Phenylalanine → Phenylalanine deaminase → Phenyl pyruvic acid → (FeCl3) green

41
 Malonate utilization-

Determines whether the organism is capable of using sodium

malonate as its sole carbon source. They also use ammonium sulfate
as a nitrogen source.

pH indicator- bromothymol blue

positive test- alkaline, green to blue

Nitrate and Nitrite reduction-

Determines whether an organism has the ability to nitrate to nitrite and reduce nitrite to nitrogen gas
(N2).

No color develops- no reduction of nitrate to nitrite and then nitrite to nitrogen gas.

Adding Zinc dust- reduces nitrate to nitrite

Zinc dust- true- negative test

Nitrate reduction test reaction

Nutrient broth with 0 1. %potassium nitrate →

Nitrate reductase → Nitrite → Sulfanilic acid + N,N-Dimethyl-α -naphthylamine → Diazo red dye

Vibrio, Aeromonas, Campy, Helicobacter

Biochemical Tests done for enteric like species-

Catalase test-

Oxidase test-

Indole test-

O/129 (2,4-diamino-6,7 diisopropylteridine)

disk:

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Susceptibility test used in the Microbiology lab to differentiate Vibrio (susceptible) from Aeromonas
(resistant).
Results:
a.
Resistant: no zone of inhibition (Aeromonas)
b.
Susceptible: zone of inhibition (Vibrio, with the exception of isolates from India and
Bangladesh)

String Test: A definite test for the identification of Vibrio is the

String Test:
Results:
a.
Positive: Formation of a sting as the loop drawn away from the slide (Vibrio)
b.
Negative: No string formation.

Salt Tolerance 6.5% NaCl broth:

It is a test used in the microbiology lab to differentiate halophilic

spp (salt tolerant), from non-halophilic spp (not salt tolerant).
Positive result: Turbidity or growth (Halophilic Vibrio spp, except Vibrio cholera)
Negative result: No turbidity or growth (Non halophilic Aeromonas spp)

Hippurate test:

It is a rapid colorimetric test that detects the production of the

enzyme hippuricase. Group B Streptococci (S. agalactiae, Listeria
monocytogenes and Campylobacter jejuni) produce the enzyme
hippuricase which hydrolyzed Sodium Hippurate into two end
products: benzoic acid and glycine.
Positive result: A deep blue/violet color in 30 minutes.

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Negative result: No color in 30 minutes
Substrate: Sodium Hippurate

End Products: Sodium Benzoate and Glycine

Indicator: Ninhydrin (detects glycine

MEDIAS:

Thiosulfate-citrate-bile salt-sucrose agar (TCBS):

Selective and differential media that allows

the growth of Vibrio’s spp. It contains
sodium thiosulfate and sodium citrate to
inhibit the growth of Enterobacteriaceae,
bile salts to inhibit the growth of gram-
positive bacteria. Sucrose is added to aid in
the differentiation of the Sucrose Fermenting
Vibrio’s and the Non-Sucrose fermenting
Vibrio’s. Bromothymol blue is included as a
PH indicator.
Sucrose Fermenters: Yellow Color (V. cholera, V. alganolyticus)
-
Non-Sucrose Fermenters: Green color (V. parahaemolyticus, V. vulnificus

Shooting star Vibrio cholerae

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 Campy Agar (CAMPY):

Selective media used in the Microbiology lab to

support the growth of Campylobacter Jejuni. It is made
with a base of Brucella agar and 10% of sheep RBCs, and
supplemented with trimethoprim, vancomycin, polymyxin B,
cephalothin and Amphotericin B.

Darting Motility Campylobacter (sudden movement from

one side to another side)

Biochemical reactions for Enterobacteriaceae Lactose Fermenters Exercices:

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Biochemical reactions for Enterobacteriaceae Non-Lactose Fermenters Exercices:

47
48
49
CHAPTER 19 ENTEROBACTERIACEAE

Shigella colorless no H2S Salmonella with H2S (black colonies)

50
Hektoen enteric (HE) Agar plate
Shigella colorless no H2S Salmonella with H2S (black colonies)

E. coli in EMB agar plate Enterobacteria gram negative rods

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Virulence factors of Helicobacter pylori (Lophotrichous flagella)

Gram Stain of Helicobacter pylori

Gram stain of Campylobacter jejuni

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Chemical Reactions-

1- INDOLE (SIM TEST = Sulfide, Indole and Motility Test)

a) Indole Production

Triptophanase

Triptophan -------------------→ Indole + Kovac’s Reagent ----→ Pink to Red Color (Positive)

Triptophan = Substrate

Triptophanase = Enzyme

Indole = Product

Kovac’s Reagent = Indicator (Pink to Red Color Positive for Indole Test)

b) Production of H2S (Hydrogen Sulfide)

Sodium Thiosulfate ------→ H2S + Ferrous Ammonium Sulfate------→FeS (Black precipitate)

Sodium Thiosulfate = Substrate

Thiosulfate Reductase/ Sulfite Reductase= Enzyme

H2S (Hydrogen Sulfide) = Product

Ferrous Ammonium Sulfate (NH4FeSO4) = Indicator

Enteros positive for H2S production: C. freundii, E.tarda, P. mirabalis, P. vulgaris, Salmonella.

c) Motility: Movement away from the initial stab in the agar positive for motility. All Entero

motile, except K. pneumoniae, Shigella , (Y.enterocolitica non motile at 35 C).

2. METHYL RED- MR (Mixed Acid Pathway)

Glycolisis

a) Glucose ---------------→ Pyruvic Acid ------→ acid end products (succinic, formic, lactic,

acetic acid)

b) Acid end products + 2 drops of Methyl Red -------→ Red Color (Positive)

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Methyl Red = Indicator (Acid PH turns the medium red, which is positive for MR)

All Entero positive for MR except genus Kleibsella, Enterobacter and, Serratia

3. VOGES-PROSKAEUR-VP (Butylene Glycol Pathway or Butanediol Pathway)

Glycolisis

a) Glucose --------------→ Pyruvic Acid ------→ Acetoin or 2,3 Butanediol

b) Acetoin + 6 drops of alpha-naphthol + 40% NaOH --------→Diacetyl (Red Complex, +VP)

Reagent A = Alpha-naphthol = Indicator

Reagent B = 40% NaOH = Indicator

Only positive for VP genus Kleibsella, Enterobacter and, Serratia

NOTE: Just for your Information Serratia Liquefaciens is both MR and VP positive

4. CITRATE (Simmons’ citrate medium)

Citrase

a) Sodium Citrate + Ammonium salts (NH4H2PO4) ---------→ NH3 (Ammonia) + NH4OH

b) NH3 (Ammonia) + NH4OH + Bromothymol Blue ---------→ Blue Color (Positive)

Sodium Citrate = Substrate

NH4H2PO4 (Ammonium Salts) = Co-Substrate

Citrase/ Citrate-Permease = Enzyme

Bromothymol Blue = PH Indicator (Yellow = Acid; Blue = Alkaline)

Positive for Citrase/Citrate-Permease

enzyme: genus

Klebsiella, Enterobacter, Serratia,

Citrobacter, Providencia, and species P. mirabalis.

5- UREA

Urease

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Urea --------------→ NH3 + H2O + CO2 ------------→ Pink color (Positive)

Urea = Substrate

Urease = Enzyme

Phenol red = PH Indicator (Yellow = Acid; Pink = Alkaline)

Positive for Urease enzyme: genus Kleibsella, Citrobacter (delayed), Proteus, Morganella, Yersinia, and
species E. cloacae, P. retgeri, and P. stuarti (delayed).

6. DECARBOXYLASE TEST (Moeller decarboxylase base medium)

Lysine decarboxylase

Lysine (amino acid) ---------------------------------→ Cadaverine (Amine) + CO2

Ornithine decarboxylase

Ornithine (amino acid) ---------------------------------→ Putrescine (Amine) + CO2

Medium has glucose, which is utilized first by bacteria that turns

the medium acid (yellow

color) in order to activate decarboxylase enzymes. Once the

decarboxylase enzymes are

activated, they attack the COOH part of the Amino acid and
produced an amine, changing

the color from yellow to purple.

Lysine, Ornithine = Substrate

Lysine, Ornithine Decarboxylase = Enzyme

Bromocresol purple = PH Indicator (Purple Color Positive for Decarboxylase Test)

Cresol red = PH Indicator (Purple Color Positive for Decarboxylase Test)

PH indicator (yellow = acid; purple = alkaline)

7. PHENYLALANINE DEAMINASE TEST

Phenylalanine deaminase

Phenylalanine ----------------------------------→ Phenyl Pyruvic Acid + FeCl3

------→ Green Color

Phenylalanine = Substrate

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Phenylalanine Deaminase = Enzyme

Phenyl Pyruvic Acid = Product

FeCl3 (Ferric Chloride) = Indicator

Genus: Proteus, Morganella, and Providencia only ones positive for this test.

8. TSI (TRIPLE SUGAR IRON TEST), or KIA (KLIGLER IRON

TSI contains:

Three saccharides (sugars): Glucose 0.1%, Lactose 1%, and Sucrose 1%, for detection of

fermentation of glucose only, or fermentation of glucose, lactose and sucrose.

Sodium Thiosulfate for the detection of H2S production.

Phenol Red, which is used as a PH Indicator (yellow=acid; pink = alkaline; red = no change).

Slant (aerobic); Butt (anaerobic).

KIA contains:

Two saccharides (sugars): glucose and lactose, for the detection of fermentation of glucose only, or
fermentation of glucose and lactose.

Sodium Thiosulfate for the detection of H2S production.

Phenol Red, which is used as a PH Indicator (yellow = acid; pink = alkaline; red = no change)

Slant (aerobic); Butt (anaerobic).

9. ONPG (Ortho-Nitrophenyl-Beta-D Galactopyranoside test – Delayed Lactose fermenters)

Beta- Galactoside

ONPG ---------------------→ Galactose + alpha-nitrophenol (Yellow compound)

Delayed lactose Fermenters lacks Beta-galactoside permease, which is an enzyme that allows lactose to
enter the cell wall or cell membrane of the bacteria. Once lactose enters the cell membrane of the
bacteria, lactose is fermented by the use of the enzyme Beta- galactoside.

ONPG = Substrate

Beta- Galactoside = Enzyme

Delayed Lactose Fermenters: S. marcescens, Citrobacter.

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10. DNase TEST (Deoxyribonuclease)

DNase

DNA + Methyl Green complexes-----------→ Degraded DNA + Clear Zone (Positive)

DNA - Four Nitrogen Bases (Adenine (A), Thymine (T), Guanine

(G), Cytosine (C)) bonded

by Phosphodiester = Substrate

DNase = Enzyme

Methyl Green Complexes = Indicator

DNase positive Serratia marcescens.

11. Nitrate Broth Test

Nitrate reductase

a) Nitrate --------------------------→Nitrite + Sulfanilic Acid + N, N- Dimethyl-alpha-naphtylamiane ------→

Diazo red dye (Positive for Nitrate reduction to Nitrite.)

Nitrate reductase

b) Nitrate ------------------------Nitrite ----→ N2+ Sulfanilic Acid + N,N- Dimethyl-alpha-naphtylamiane

------→No Color (Possible further reduction to N2 or not reduction of Nitrate); add Zinc dust

And:

a. No change in color – presence of Nitrate reductase, Nitrate reduced to nitrate and

then to N2 gas

b. Change in color to red color- no Nitrate reductase present, no reduction at all (true

negative result).

Nitrate = Substrate

Nitrate reductase = Enzyme

Sulfanilic and N,N-Dimethyl-alpha-naphtylamine = Indicators

To test the ability of Enterics to reduce nitrates to nitrites.

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12) XLD chemical reaction-

selective/differential; bile salts/lactose/H2S

lactose, sucrose, xylose  glucose  pyruvic acid + indicator

thiosulfate  H2S + ferric ions  black precipitate

13) Hekteon agar chemical reaction

selective/differential; bile salts/lactose/H2S

lactose, sucrose, xylose  glucose  pyruvic acid + indicator

thiosulfate  H2S + ferric ions  black precipitate

YEAST

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Yeast- Unicellular, asexual reproduction by formation of buds
Molds- multicellular, filamentous (true hyphae), reproduce by production of condia (spores)

Laboratory growth-

59
Temperature- 25C, 35C
Humidity. Time- 48 hours to 30 days

Blastoconidium- a spore or conidium that detaches at a predetermined point of budding, leaving

a scar. Blastoconidia server as the most common form of reproduction in yeasts, recognized as
budding.
Pseudohyphae-

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Yeast cells and Pseudohyphae in smear of heart tissue.

Candida albicans: colonies with feet Candida albicans- germ tube test

Smooth, white, pasty colonies frowing

on SDA(sabouraud’s dextrose agar)
Feet-like extensions.

61
on Sabouraud glucose agar.

62
 Chlamydospore-
A reproductive unit derived from a pre-
existent hyphal cell, most commonly seen in
cornmeal mounts of candida albicans. They
may form at the apex (terminal) the side
(sessile) or within (intercalary) the hypha.

Chlamydospores and Blastoconidia:

Chlamydospores on cornmeal agar, (aniline blue) large, spherical, blue-staining spores.

Biochemical identification-
Fermentation; carbohydrate tubes- gas production, pH changes.
Assimilation- identifies which carbohydrates a yeast can utilize as a source of carbon for growth.
Auxanographic technique, disk diffusion

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Gram stain of C. glabrata-

65
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67
Cryptococcus neoformans-
Pigeons spread the fungus that causes cryptococcal meningitis.

Gram stain on Cryptococcus neoformans-

68
Encapsulated yeast cells in CSF fluid using india ink.
Cryptococcus neoformans- yeast cells with prominent capsules as seen in CSF fluid.

Cryptococcus neoformans- Yeast cells with capsules in brain tissue, mucicarmine stain.

Yeasts and other Opportunists-

69
70
Opportunists -

Mycology Robert Harr Questions-

Specimens for the recovery of fungi- Tissue biopsy, CSF, aspirate of exudate

Specimens rejected for the recovery of fungi- swabs

swabs are inadequate for the recovery of fungi because they are easily contaminated by surrounding
skin flora.

KOH direct mount technique-

A solution of 10% KOH is used for contaminated specimens such

as skin, nail scrapings, hair, and sputum to clear away background
debris that may resemble fungal elements.

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 India ink stain-

A positive India ink test shows yeast cells in CSF with a

surrounding clear area (the capsule) because the capsule of C.
neoformans is not penetrated by ink particles.

Meningitis caused by C. neoformans is diagnosed through

culture, biochemical reactions, and rapid agglutination tests for
cryptococcal antigen.

The India ink test is not diagnostic for cryptococcal meningitis because positive staining results are
demonstrated in less than 50% of confirmed cases.

Cutaneous disease involving skin, hair, and nails usually indicates an infection with a?

Dermatophyte- Superficial dermatophytes rarely invade the deeper tissues and are the cause of most
cutaneous fungal infections.

First step to be performed in the identification of an unknown yeast isolate?

Germ tube test

Germ tubes represent true hyphae without constriction.

Species that Do not form Hyphae-

Cryptococcus neoformans, candida glabrata, Rhodotrula rubra

Candida tropicalis-

Forms pseudohyphae, that resemble true germ tubes by producing a constriction at the point of origin
of the yeast cell.

Germ tubes represent true hyphae without constriction, and therefore the test should have been
repeated along with carbohydrate tests before making a presumptive identification.

Cornmeal agar with tween 80 (polysorbate) identifies-

Hyphae (true and pseudo)

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Blastoconidia and arthroconidia

Chlamydospores

Cornmeal agar with Tween 80 (polysorbate) reduces the surface tension and allows for enhanced
formation of hyphae, blastospores, and chlamydospores.

Pseudohyphae are the result of a pinching-off process, blastoconidiation, with the growth of

filaments with constrictions.

Germ tubes are the beginning of true hyphae (no constrictions).

Arthrospores are the result of a breaking-off process of true septate hyphae resulting in square

conidia.

Continue from qus 9 page 463

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