Biochem tests in medical microbiology for college students

Medical microbiology related biochemical tests

1. BIOCHEMICAL IDENTIFICATION
OF BACTERIA

Dr Ravi Kant Agrawal, MVSc, PhD
Senior Scientist (Veterinary Microbiology)
Food Microbiology Laboratory
Division of Livestock Products Technology
ICAR-Indian Veterinary Research Institute
Izatnagar 243122 (UP) India

2. + Determining the nutritional and metabolic capabilities of a
bacterial isolate is the most common approach used for
determining the genus and species of an organism.

+ The methods available use a combination of tests to establish
the enzymatic capabilities of a given bacterial isolate as well as
the isolates ability to grow or survive the presence of certain
inhibitors (e.g. salts, surfactants, toxins and antibiotics)

3. A.Establishing Enzymatic Capabilities
A. Establishing Enzymatic Capabil

Enzyme based tests are designed to measure the presence of a single enzyme as well as a
complete metabolic pathway.

SINGLE ENZYME TESTS:
Catalase test

< Coagulase test

«+ Pyrase test

«+ Hippurate hydrolysis test
Oxidase test

“ Indole test

“= Dnase test

«+ ONPG (B-galactosidase) test
+ Urease test

Assays for Metabolic Pathways: Carbohydrate oxidation and fermentation
+ Oxidation fermentation tests

+ Carbohydrate fermentation in TSIA

Methyl red test

“+ Voges Proskauer test

Amino acid degradation

$ Decarboxylase-dihydrolase reactions

+ deamination reactions

«+ decarboxylation and deamination reactions in LIA

Single substrate utilization
Citrate utilization test

“ Acetate utilization test

“ Acetamide utilization test

4. B. Establishing Inhibitor Profiles

+ Bacitracin susceptibility test

“ Bacitracin and sulfamethoxazole-trimethoprim susceptibility test
* Novobiocin susceptibility test

“+ Vancomycin susceptibility test

+“ Antibiotic disks for presumptive identification of anaerobes

C. Other more specific tests

* Growth in various NaCl concentrations - Enterococci and Vibrio species

“+ Susceptibility to optochin and solubility in bile - Streptococcus pneumoniae

++ Ability to hydrolyze esculin in the presence of bile - Enterococcus spp. and
Group D streptococcus

+ CAMP - Streptococcus agalactiae

5. A.Establishing Enzymatic Capabilities
B.SINGLE ENZYME TESTS

CATALASE TEST

PURPOSE

To differentiate members of the family Microcococcaceae (including
Staphylococcus) which are catalase positive from Streptococcus species
which are catalase negative.

* To differentiate Listeria monocytogenes and corynebacteria (catalase
positive) from other Gram positive, non-sporeforming bacilli.

Principle:
+ The enzyme catalase catalyzes the release of water and oxygen from
hydrogen peroxide.
catalase
2 H202 - > 2 H20 + 02
bubbles or effervescence

6. — rapid and sustained appearance of bubbles or effervescence
— lack of bubble formation 30 seconds later

- Staphylococcus aureus; — Streptococcus pyoc

7. Coagulase Test

PURPOSE

“To determine the ability of the organism to produce coagulase which
clots plasma.

*“ To distinguish the pathogenic coagulase positive staphylococcus
from the nonpathogenic coagulase negative staphylococcus.

Principle:
u. is an enzyme that converts soluble fibrinogen into
insoluble fibrin.

Two forms of coagulase:

1. Bound coagulase (clumping factor) -

+ Detected in the coagulase slide test

+ Can directly convert fibrinogen to insoluble fibrin and causes the
staphylococci to clump together

2. Free coagulase

> Detected in the coagulase tube test

+ Reacts with a globulin plasma factor a reacting factor-CRF)
to form a thrombin like factor, staphylothrombin --> catalyzes the
conversion of fibrinogen to insoluble fibrin

8. Slide Coagulase test
- white fibrin clots in plasma; - smooth suspension

Tube Coagulase test
- formation of fibrin clot; —no clot is formed

Slide coagulase test Tube coagulase test

- Staphylococcus epidermidis - Staphylococcus aureus
- Staphylococcus aureus - Staphylococcus epidermidis

9. PYRASE (PYR) TEST

Purpose:

“To determine the ability of the organism to hydrolyze the substrate L-
pyrrolidonyl-beta-napthylamide.

+ To differentiate Enterococcus species from non-enterococcus species.
“ Useful for presumptive identification of Group A beta hemolytic
streptococcus (Streptococcus pyogenes)

hydrolysis
L-pyrrolidonyl-beta-napthylamide ---------- -> Beta napthylamide +
Pyrrolidonylarylamidase P-dimethylaminocinnamaldehyde
(color developer)
Interpretation: Pink to cherry red color

“Positive — pink to cherry red color (after the addition of color developer)
“Negative - no color change in inoculated portion of the disk

10. Interpretation

: Pink to cherry red color (after the addition of color
developer)
: No color change in inoculated portion of the disk

itive Enterococcus
. Negative Non-Enterococcus

11. Hippurate Hydrolysis Test

Purpose

“To determine the ability of the organism to produce hippuricase
which hydrolyzes the substrate hippurate.

Useful in the identification of Streptococcus agalactiae,
Campylobacter jejuni and Listeria monocytogenes.

Principle:

“The end products of hydrolysis of the substrate hippurate by a
constitutive enzyme hippuricase include glycine and benzoic acid.
+ Glycine is deaminated by the oxidizing agent, ninhydrin, which
is reduced during the process.

“ The end products of ninhydrin oxidation react to form a purple
colored product.

12. Hippurate hydrolysis test

Interpretation

* Positive — deep purple
color

+ Negative - slightly yellow
pink or colorless

J

A B

A. Positive Streptococcus
agalactiae

B. Negative Enterococcus

13. OXIDASE TEST
Purpose:
“ To screen colonies suspected of being one of
the Enterobacteriaceae (all negative).
“ To identify colonies suspected of belonging to
other genera such as Aeromonas, Pseudomonas,
Neisseria, Campylobacter and Pasteurella.

14. Principle

% The cytochrome oxidase test uses certain reagent
dyes, such as p-phenylenediamine dihydrochloride
that substitute for oxygen as artificial electron
acceptors. It is colorless in the reduced state.

“In the presence of cytochrome oxidase and
atmospheric oxygen, p-phenylenediamine is oxidized
forming indophenol blue.

P-phenylenediamine dihydrochloride --------------- --> Indophenol blue
cytochrome oxidase + atmospheric air

Tetramethyl-p-phenylene diamine hydrochloride ----------- > purple color

Dimethyl compound (1%) =. black color

15. Interpretation

Oxidase test

16. INDOLE TEST

Purpose:

“ To distinguish Enterobacteriaceae based on the
ability to produce indole from tryptophan.
“ To identify lactose fermenting members of
Enterobacteriaceae, Escherichia coli (indole positive)
from Klebsiella pneumoniae (indole negative).
* To speciate Proteus:

Proteus mirabilis: Indole negative

Proteus vulgaris : Indole positive

17. Principle

“ Bacteria that possess the enzyme tryptophanase are
capable of hydrolyzing and deaminating tryptophan
with the production of indole, pyruvic acid and
ammonia.

“ A red complex is formed when indole reacts with the
aldehyde group of p-dimethylaminobenzaldehyde, the
active chemical in Kovac’s and Ehrlich’s reagent.

Tryptophan --------------—> indole + pyruvic acid + NH3
tryptophanase
Indole + p-dimethylaminobenzaldehyde >red complex

18. Reagents used to detect indole

+ Ehrlich’s - to detect indole in anaerobic and
non-fermentative bacteria

+ Kovac’s - to identify members of Enterobacteriaceae

Media used with tryptophan

+ Sulfide indole motility (SIM)

+ Motility indole ornithine(MIO)

«- Indole nitrate

«* Rapid spot tests - filter paper strips impregnated
with p-didimethylaminocinnamaldehyde reagent -
useful in screening bacteria that are prompt indole
producers

19. INTERPRETATION

Positive Red ring at the interface of reagent
and broth (or reagent and xylene or chloroform)
“ Negative No color development

* Variable results Orange color, indicates
products of skatole, a methylated intermediate
that maybe a precursor to indole production

“Rapid spot test

Y” Paradimethylaminocinnamaldehyde: blue green

Y” Paradimethylaminobenzaldehyde: bright pink
color

21. A. Negative - Klebsiella pneumoniae
B. Positive - Escherichia coli

22. DEOXYRIBONUCLEASE TEST or
DNA HYDROLYSIS TEST

Purpose:

“To detect DNase activity in species of aerobic
bacteria.

*To differentiate non-fermenting Gram-negative
bacteria as well as Staphylococcus aureus and
Serratia marcescens.

23. Principle

Metachromatic dyes

“* Toluidine blue is complexed with DNA.

“Hydrolysis of DNA by the inoculated microorganism
causes changes of structure of the dye to yield a pink
color.

“ Methyl green is also complexed with DNA. If the
organism growing on the medium hydrolyzes DNA, the

green color fades and the colony is surrounded by a
colorless zone.

INTERPRETATION

Toluidine blue Methyl green

“Positive rose pink clear zone

“Negative no change no clearing

24. S. aureus

Serratia marcescens
S. epidermidis
Enterobacter cloacae

S. aureus
S. marcescens
S. epidermidis

25. ONPG (B-GALACTOSIDASE TEST)

Purpose:

* To determine the presence of late or slow
fermenting strains.
** To detect the late lactose fermenting strains of
Escherichia coli
To distinguish some Citrobacter species and
arizonae subspecies (ONPG positive) from
similar Salmonella subspecies (ONPG negative)
To speciate Shigella since Shigella sonnei is
the only ONPG-positive Shigella species.

26. Principle

Two enzymes required for lactose fermentation

‘“ Lactose permease actively transfers lactose
into the bacterial cell

“* Beta galactosidase: degrades lactose into
glucose and galactose

“Lactose fermenters possess both enzymes

“Slow or late lactose fermenters no permease;
only beta galactosidase

Non lactose fermenters: lack both enzymes

27. “ ONPG (o-nitrophenyl-beta-D-galactopyranoside) is
useful in detecting late lactose fermenters, since
ONPG molecule is structurally similar to lactose.

* It can enter the bacterial cell without a permease.
“ In the presence of galactosidase, ONPG (colorless)
is converted into galactose and o-nitrophenyl, which
is a yellow chromogen and the alkaline end
product.

Interpretation

“Positive yellow color within 20 minutes to 24 hours
** Negative: no color change / colorless after 24 hours

28. ONPG CA POR TIOS test

e Salmonella Typhimurium
ve Escherichia coli (EHEC)

29. UREASE TEST
Purpose
“To determine the ability of an organism to produce
the enzyme, urease, which hydrolyzes urea.
“To identify the rapid urease producers (Proteus
and Morganella) and weak urease producers
(Klebsiella pneumoniae and species of Enterobacter)

Principle

“Urease splits the urea molecule into ammonia (NH3),
CO2 and water(H20).

“ Ammonia reacts in solution to form an alkaline
compound, ammonium carbonate, which results in an
increased pH of the medium and a color change in the
indicator to pink red.

30. Urea + 2H20 ---mm > CO2 + H20 +2NH3
urease

Interpretation (NH4)2C03

Christensens Urea agar

Positive: Rapid urease activity; red throughout the medium
«+ Positive: Slow urease activity: red in slant initially gradually
converting the entire tube

«+ Negative: No urease activity; medium remains yellow

Stuart (urea) broth
“Positive: Red color in the medium
«+ Negative: No color change (buff to pale yellow)

31. A. Positive Proteus spp.
B. Positive Klebsiella spp.
C. Negative: Escherichia coli

Urease test
(Christensens Urea agar)

32. ction

sherichia coli

Urease test
Stuart Urea broth

33. ASSAYS FOR METABOLIC Pathways
CARBOHYDRATE FERMENTATION/
OXIDATION FERMENTATION TESTS

Purpose:

‘To determine whether a substrate utilization is an

oxidative or fermentative process for the

identification of several different bacteria.

** To separate organisms into two major groups:
Enterobacteriaceae fermentative

Pseudomonas oxidative

34. Composition:
+ High concentration of carbohydrates (1%)
** Small concentration of peptone (2%)
“Indicators
Bromcresol purple: purple to yellow
Andrade’s acid fuchsin: pale yellow to pink
Phenol red: red to yellow
Bromthymol blue: green to yellow

35. Mineral oil
overlay

Both tubes of O-F glucose
inoculated with test organism

Incubation
Oxidizer Fermenter Nonutilizer

(nonfermenter)

Principle of glucose oxidative fermentation test

36. INTERPRETATION

“ Glucose fermenter: When acid production is
detected on both tubes since fermentation can occur
with or without oxygen

« Glucose oxidizer: Acid is detected by the open
aerobic tube

“ Non-utilizer: Some bacteria do not use glucose as a
substrate

37. Open tube Closed tube Metabolism
Acid (yellow) alkaline (green) oxidative
Acid (yellow) acid (yellow) fermentation

Alkaline (green) alkaline (green) non-saccharolytic
(non-utilizer)

Oxidative-Fermentation Medium of Hugh and Leifson

38. Escherichia coli Pseud. aeruginosa Alcaligenes faecalis

aan

39. CARBOHYDRATE FERMENTATION IN TSI

Purpose: As an initial step in the identification of
Enterobacteriaceae.

Principle:

1. The action of many species of microorganisms on a
carbohydrate substrate results in the acidification of
the medium with or without gas formation.

2. Iron salts (ferrous sulfate and ferric ammonium
citrate) reacts with H2S to produce an insoluble black
precipitate (ferrous sulfide).

40. COMPOSITION

+ Protein sources - beef extract, peptone, yeast
extract, proteose peptone

*% Sugars (lactose, sucrose, glucose)

“ Indicators

a. phenol red - carbohydrate fermentation

b. ferrous sulfate - hydrogen sulfide production

“ Sodium thiosulfate - source of sulfur atoms

+ Sodium chloride - osmotic stabilizer

TSIA - two reaction chamber
+ Aerobic slant portion
“ Anaerobic deep portion

41. BIOCHEMICAL REACTIONS

«+ Carbohydrate fermentation
acid production

Yellow deep - glucose fermented

Yellow slant - lactose and/ or sucrose fermented

Gas formation

Bubble formation

Cracking or splitting of the agar

Upward displacement of the agar

Pulling away of the medium from the walls of test tube

#,

<<

4444

+

* H2S production
Y” Blackening of the butt (FeS - black precipitate)

D

42. + AI@H2S(-)
Acid slant; acid butt; gas
formation; no H2S

all sugars fermented; with =

gas formation;
no blackening of the butt
pesca ann kaart
Escherichia sae Dee
Klebsiella | À im
Enterobacter [Ts mao

Le mi al ms à da

Son, provided the tea Q
Jo a ato han ke: tain se. The formation of CO,
Pgo a hod ane fe read and, dogs gb indica

Come

43. + KI@H2S+
Y” alkaline slant; acid butt; with gas
formation with H2S
v glucose fermented; lactose and
or/sucrose not fermented; with gas
formation and black precipitate
Salmonella
Proteus
Citrobacter

44. “K/A H2S( -)
alkaline slant; acid butt; no gas; no H2S

glucose is fermented; lactose
and/or sucrose not fermented:
no gas formation; no black
precipitate

Shigella
Providencia
Serratia

anaerogenic Escherichia coli

45. + K/KH2S(-)
alkaline slant; alkaline butt; no gas;
no H2S

no sugars fermented; no gas;
no black precipitate in the butt

Pseudomonas
Alcaligenes

46. + A@H2S+
acid slant; acid butt; with gas; with
H2S

all sugars fermented; with gas

formation;
with black precipitate in the butt

Citrobacter freundii

47. METHYL RED-VOGESPROSKAUER TEST

Purpose:

To identify the lactose fermenting
Enterobacteriaceae such as Escherichia coli (MR
positive and VP negative) whereas most members of
the Klebsiella-Enterobacter-Serratia-Hafnia group are
VP positive.

48. Metabolism of glucose using MR and VP pathways

Glucose
Acetoin Pyruvic acid — Mixed acid fermentation

KOH + air pH less than 4.4 (red)

Diacetyl

a Napthol + creatine

pink red complex
Positive VP

49. “In the first pathway,
mixed acid products
(lactic, acetic, formic
and succinic) result,
leading to a decrease in
the pH of the medium
and a positive MR test.
+ The pH must drop to
4.4 or less for the MR
indicator to take on its
acidic red color.

Methyl Red test

- Escherichia coli
- Klebsiella pneumoniae

50. “In the second pathway,
acetylmethyl carbinol /acetoin
is an intermediate product to
butylene glycol.

“It is the neutral product
detected in the VP reaction.

“ In the presence of oxygen
and 40% potassium
hydroxide, acetoin is
converted to the diacetyl
form, which results in a red
color in the presence of
alpha-napthol.

Voges Proskauer test

— Klebsiella pneumoniae
re = Escherichia coli

51. Interpretation

Methyl red test

* Positive — distinct red color at surface of the medium
* Negative — yellow color at the surface of the medium

Voges Proskauer test

«+ Positive — pink red color at surface of the medium
“ Negative - yellow color at surface of the medium

52. AMINO ACID DEGRADATION
DECARBOXYLASE-DIHYDROLASE

Purpose: To determine the production of decarboxylase by
bacteria (Enterobacteriaceae).

Principle

“+ Decarboxylase enzyme: removes carboxyl groups from the
amino acids lysine and ornithine.

+ Dihydrolase enzyme: removes a carboxyl group from
arginine.

“ Glucose base without the amino acid and tubes
containing glucose & amino acid substrates are inoculated.

“ Decarboxylation and dihydrolation are anaerobic
reactions so inoculated tubes are overlayed with mineral oil
to exclude air.

53. Composition — Moeller decarboxylase medium

1. Glucose

2. Amino acid substrate (1% lysine, 1% arginine
1% ornithine)

3. pH indicator

a. bromcresol purple

v Alkaline pH - Purple

* Acid pH - Yellow

b. phenol red
v Alkaline pH - Red
v Acid pH - Yellow

54. Specific amine products

Lysine ==. > cadaverine

Arginine--------------- > citrulline---------- > ornithine
dihydrolase
reaction decarboxylation

putrescine

55. “+ Early incubation: both tubes yellow due to acidification of
the indicator (bromocresol purple) by the acid end products of
glucose fermentation.

“+ If amino acid is decarboxylated, alkaline amines are formed
and cause the indicator to revert to an alkaline pH.

INTERPRETATION

“ Control tube yellow- glucose fermentation; viable
organisms; pH of the medium has been lowered sufficient to
activate the decarboxylase

“ Positive test purple - decarboxylation; formation of the
alkaline amines from the decarboxylation

56. Moeller decarboxylase medium

\. Positive — purple; decarboxylation
. Negative - yellow; no decarboxylation; only glucose
fermentation

57. Decarboxylase-dihydrolase reactions - Enterobacter cloacae)

A. Control - without amino acid C. lysine-negative
B. Arginine - positive D. ornithine-positive

58. Enterobacter cloacae Klebsiella pneumoniae
Arginine +(purplejalkaline -(yellow) acid
Lysine -(yellow)acid +(purplejalkaline

Ornithine +(purple)alkaline -(yellow)acid

59. DEAMINASE REACTIONS

Purpose: To determine the deaminase activity using the amino
acids phenylalanine or tryptophan.

“Only Proteus, Providencia and Morganella species
possess the deaminase enzyme.

Principle:

“+ Deamination of the amino acid results in a colored
compound with the addition of 10% ferric chloride

Phenylalanine -----------------> PPA + 10% FeCl3
Phenylalanine deaminase green colour
Tryptophan------------- > Indole-pyruvic acid + 10% FeCI3

Tryptophan deaminase brown colour

60. NTERPRETATION Phenylalanine deamination test
deamination for
phenylalanine - intense
epoca]
+ | drehe for
tryptophan — brown color
tive — pres retains
its “original color after the
addition of ferric chloride

erichia coli

vulgaris

61. LYSINE IRON AGAR

Purpose: To determine the ability of the organism to
deaminate lysine, decarboxylate lysine and produce H2S.
“To identify Salmonella, Proteus, Providencia and

Morganella.

62. COMPOSITION

1. Proteins a

2. Sugar- Glucose Bon

3. Amino acid - Lysine
4. Sulfur

5. indicators IT

a. ferric ammonium citrate- >

H2S production

b. bromcresol purple - we

carbohydrate a — à

fermentation SR )

63. PRINCIPLE:

“As glucose fermentation occurs, deep of the tube turns
yellow.

“*Lysine decarboxylation produces alkaline cadaverine and
leads to reversion of the deep from yellow to purple.

« Lysine deamination occurs in the presence of oxygen (on
the slant) and results in the production of a red color.

+ H2S production is noted by a black precipitate in the deep
as H2S reacts with ferric ammonium citrate.

64. Interpretation

Lysine decarboxylation - butt
+ Positive — purple

“+ Negative - yellow

Lysine deamination - slant
Positive — red
% Negative -purple

65. un
UT 5524
y 10-31-97

KK alkaline slant/
alkaline butt

H2S(-) purple! purple
Negative deamination
Positive decarboxylation
No blackening of the
butt

KIK alkaline slant/alkaline
butt H2S + purple/purple

Negative deamination
Positive decarboxylation
With black precipitate in
the butt

urium

= remel

KIA alkaline slantíacid |
butt H25(-)
(purple/yellow)
Negative deamination
Negative
decarboxylation

No black precipitate

in the butt

higella flexneri

RIA red slant/acid butt
H2S(-) red/yellow

Positive deamination
Negative
decarboxylation

No black precipitate in
the

66. SINGLE SUBSTRATE UTILIZATION
CITRATE UTILIZATION TEST

Purpose:

“To determine if a member of the Enterobacteriaceae
is capable of utilizing citrate as the sole source
of carbon.

«Useful in the identification of the lactose fermenting
Enterobacteriaceae: Escherichia coli is citrate
negative Enterobacter and Klebsiella are positive

67. Principle

“Sodium citrate is the only carbon source in Simmons
citrate agar.

+ If the organism can utilize citrate, the sodium citrate is
converted to ammonia, which is then converted to
ammonium hydroxide.

+ The alkalinity of the compound formed raises the pH of the
medium, and the bromthymol blue indicator takes on its
alkaline color which is blue

Interpretation

+ Positive: Growth with an intense blue color on the slant or
solely the presence of growth

“> Negative: Absence of growth and no color change in the
medium (remains green)

68. A

LOT &acos
EXPOLFEB 95

Citrate Utilization test

A. Positive - Klebsiella pneumoniae
B. Negative - Escherichia coli

69. ACETATE UTILIZATION TEST

Purpose: To determine the ability of an organism to
use acetate as the sole source of carbon.

Principle:

«Breakdown of the sodium acetate causes the pH
of the medium to shift toward the alkaline range,
turning the indicator from green to blue.

70. Interpretation

* Positive - Medium
becomes alkalinized
(blue) because of the
nm À of the organism

ve — no growth
or enc with no
indicator change to blue

Acetate utilization test
(lebsiella pneumoniae
herichia coli

71. ACETAMIDE UTILIZATION TEST

Purpose
“To determine the ability of an organism to use acetamide as
the sole source of carbon.

Principle

“Bacteria that can grow on this medium deaminate
acetamide to release ammonia.

“ The production of ammonia results in a pH-driven color
change of the medium from green to royal blue.

Interpretation:

“Positive: Deamination of the acetamide resulting in a blue
color

“Negative: No color change

72. ACEI AMI
LOT si
EXP 131

ten
3:5

Acetamide utilization test

73. Establishing Inhibitor Profiles

Bacitracin Sensitivity. Test

Purpose:

“To differentiate Micrococccus and Stomatococcus
from Staphylococcus when combined with other
procedures such as the modified oxidase test.

Y For presumptive identification of Group A
streptococcus

Principle:

v Bacitracin (0.04 units) inhibits the growth of
Micrococcus and Stomatococcus and Group A
streptococcus while having no effect on
Staphylococcus which is resistant.

74. Interpretation

+ susceptible - zones of
inhibition greater than or
equal to 10 mm

“ resistant - zones of
inhibition less than or
equal to 9 mm.

Bacitracin susceptibility test

A. Susceptible: Micrococcus and
Stomatococcus

B. Resistant: Staphylococcus
epidermidis

75. Bacitracin and sulfamethoxazole-
Trimethoprim Susceptibility Test

Purpose: To identify the different species of Streptococcus
especially Group A and Group B beta hemolytic streptococci.

Principle:

* Group A beta hemolytic streptococci (Streptococcus
pyogenes) are susceptible to 0.04 units bacitracin but
resistant to 1.25 ug sulfamethoxazole-trimethoprim (SXT)

“ Group B beta hemolytic streptococci - resistant to both
bacitracin and SXT

76. Interpretation
* Susceptible: any zone of inhibition around either
disk
+ Resistant: growth up to the disk (no zone of
inhibition
Organism Bacitracin SXT
Group A susceptible resistant

Group B resistant resistant

Group C,F,G resistant susceptible

77. Novobiocin Susceptibility Test

Purpose
+ To differentiate the different species of coagulase negative
staphylococci.

Principle

+ After incubation with 5 ug of novobiocin, Staphylococcus
saprophyticus is not inhibited by the antibiotic whereas
Staphylococcus epidermidis are susceptible to novobiocin.

78. Interpretation:
“Susceptible - zone
greater than 16 mm

+ Resistant - zone
diameter less than or equal
to 16 mm

Novobiocin susceptibility test

A. Susceptible - Staphylococcus epidermidis
B. Resistant - Staphylococcus saprophyticus

79. Vancomycin Susceptibility Test

Purpose
“To differentiate Pediococcus from other alpha hemolytic
streptococcus.

Principle

“+ After incubation with 5 ug of vancomycin, Pediococcus is
not inhibited by the antibiotic whereas Viridans streptococcus
is susceptible to vancomycin.

80. Interpretation:
‘Susceptible - zone
of inhibition

— no
zone of inhibition

Vancomycin susceptibility test

A. Susceptible - Viridans streptococcus
B. Resistant - Pediococcus

81. ANTIBIOTIC DISKS FOR THE PRESUMPTIVE
IDENTIFICATION OF ANAEROBES

Purpose

«To determine an anaerobe's inhibition that can be used for
presumptive identification based on its characteristic
susceptibility pattern to colistin (10 ug), vancomycin (5 ug)
and kanamycin (1 mg).

82. Principle

“Most anaerobes have a
characteristic susceptibility
pattern to colistin (10 ug),
vancomycin ( 5 ug), and
kanamycin (1 mg) disks.

Kanamycin-inhibits facultative
Gram-negative bacilli

Vancomycin- inhibits facultative
and obligate Gram-positive
bacteria

Colistin- inhibits facultative
Gram-negative bacilli

10-7 Special patency antimicrobial disks for
Presumptive identification of anaerobes. The
pattern of susceptibility to the three antibiotic disks:
kanamycin (1000 pg), colistin (10 ye). and vancomycin (5

Microorganism type Kanamycin Vancomycin Colistin

Clostridium si?

Pept
anderobiu
Other gram-positive cocci § $
(1) Some strains are kanomycin-resistant
(2) V variable; R, resistant; $. susceptible
(3) Rare strains are susceptible

83. Interpretation

Antibiotic Disks for the Presumptive Identification of Anaerobes

Susceptible - zone greater than 10 mm
Resistant - zone of 10 mm or less

84. OTHER SPECIFIC TESTS

SALT TOLERANCE TEST

Purpose

“To classify bacteria based on their ability to grow in the
presence of 6.5% NaCl, a characteristic of certain species
of Gram positive and Gram negative bacilli.

“To differentiate the Group D (salt tolerant) from the non-
enterococci (intolerant).

85. Principle Nutrient broth or 6.5%NaCl

Trypticase broth-salt

free medium
+ Positive equal equal
+ Negative good very weak
Interpretation:

+ Positive : If growth is equivalent to both media - tolerant of salt
“Negative- growth on the salt containing medium is

very weak or absent

growth in the salt free medium is good

- intolerant of salt

Indicator: bromocresol purple
Positive: medium turns yellow from purple or the appearance of
growth

86. SALT TOLERANCE TEST

A. Positive - Enterococcus faecalis ( salt tolerant)
B. Negative - Streptococcus bovis (salt intolerant)

87. GROWTH IN BILE ESCULIN AGAR

Purpose:
“To distinguish Group D streptococci and Enterococcus
species from other Lancefield group of streptococci

Principle:

“Based on the organisms ability to grow in 40% bile and to
hydrolyze esculin to produce escultin

“+ Escultin reacts with ferric citrate to form a brown black
precipitate.

88. Wisrpestaos

«Positive:

¥ growth indicates
tolerance to 40% bile
(40% oxygall)

bie ac tkening y ind

Y

v lack of growth indicates
inability to grow in 40%
bile

Y” lack of color change
indicates inability to
hydrolyze esculin

DLJE

Bile esculin agar

A. Positive - Enterococcus faecalis
B. Negative - Streptococcus viridans

89. OPTOCHIN SUSCEPTIBILITY TEST

Purpose:
“To differentiate Streptococcus pneumoniae from other
alpha hemolytic streptococci

Principle:

“ In the presence of optochin, colonies of Streptococcus
pneumoniae are selectively lysed indicated by a zone of
inhibition after incubation under increased CO2.

“ Other alpha hemolytic streptococci are resistant to
optochin.

90. F zone of
inhibition at least 14 mm in
diameter using a 10 ug P
disk and at least 10 mm
using a 6 ug P disk

“ Negati growth up to
the disk or a zone of
inhibition
less than 14 mm with a 10
ug P disk or less than 10
mm with a 6 ug P disk

Optochin susceptibility
test

B, Negative - Viridans streptococci

91. BILE SOLUBILITY TEST

Purpose
«To differentiate Streptococcus pneumoniae (positive) from
other alpha hemolytic streptococci.

Principle

“Pneumococcal colonies are rapidly lysed by bile or a
solution of a bile salt such as sodium deoxycholate.

“+ Lysis depends on the presence of an intracellular
autolytic enzyme.

“+ Bile salts lower the surface tension between the
bacterial cell membrane and the medium thus accelerating
the organism’s natural autolytic process.

92. Interpretation mr
“Positive - colony
disintegrates; an

imprint of the Iysed

colony may remain

within the zone | A
I

«+ Negative - intact

colonies Bile solubility test

A. Positive - Streptococcus pneumoniae
B. Negative - Viridans Streptococci

93. CAMP REACTION

Purpose

“to demonstrate the phenomena of synergistic hemolysis
between group B streptococcus and beta hemolytic
Staphylococcus aureus.

Principle

“A characteristic “arrowhead” hemolytic pattern results
when the organism is streaked perpendicular to beta
hemolytic Staphylococcus aureus

94. Interpretation

* Positive - a zone of enhanced
hemolysis given by an
arrowhead appearance at the
junction of the

Staphylococcus and
Streptococcus - indicative of
en B streptococcus

* Negative - no zone of
sell anced hemolysis-

not indicative of Group B
streptococcus

CAMP REACTION

A. Positive - Streptococcus agalactiae
B. Negative - Streptococcus bovis

95. STARCH HYDROLYSIS

Principles:

>
>

>

Many bacteria produce enzymes called hydrolases.

Hydrolases catalyze the splitting of organic molecules

into smaller molecules in the presence of water.

The starch molecule consists of two constituents:

Y” Amylose, an unbranched glucose polymer (200 to
300 units)

v Amylopectin, a large branched polymer.

Both amylopectin and amylose are rapidly

hydrolyzed by certain bacteria,

Using their a-amylases, to yield dextrins, maltose, and

glucose.

96. Starch

; a-amylase
[Amylose + Amylopectin] >»
(Large polysaccharide) H20
Dextrins + Maltose + Glucose
(Intermediate (Disaccharide) (Monosaccharide)
polysaccharides)
Interpretation:

vGram’s iodine can be used to indicate the presence of
starch,
v When it contacts starch, it forms a blue to brown
complex.
y Hydrolyzed starch does not produce a colour change.
Y” If a clear area appears after adding Gram’s iodine to a
medium containing starch and bacterial growth:

> Amylase has been produced by the bacteria.
Y If there is no clearing, starch has not been hydrolyzed.

97. LIPID HYDROLYSIS

1. Determine the ability of bacteria to hydrolyze lipids by
producing specific lipases.

Principle:

> Lipids are high molecular weight compounds possessing large
amounts of stored energy.

+ The two common lipids catabolized by bacteria are the
triglycerides (triacylglycerols) and phospholipids.

> Triglycerides are hydrolyzed by the enzymes called lipases into
glycerol and free fatty acid molecules.

+ Glycerol and free fatty acid molecules can then be taken up by
the bacterial cell and further metabolized through reactions of:

y Glycolysis, B-oxidation pathway, and the citric acid cycle.

y These lipids can also enter other metabolic pathways where

they are used for the synthesis of cell membrane phospholipids.

> Since phospholipids are functional components of all cells, the
ability of bacteria to hydrolyze host-cell phospholipids is an
important factor in the spread of pathogenic bacteria.

98. y In addition, when lipase-producing bacteria contaminate food
products. the lipolytic bacteria hydrolyze the lipids, causing
spoilage termed rancidity.

H,COOC.CH,CH;CH, CH,OH
| lipase |

HCOOC-CH,CH,CH, ——»> HCOOC-CH;CH,CH, + CH,CH,CH,COOH
|

HoCOOC-CHsCH2CH3 HCOOC-CH,CH;CH;

(tributyrin) (butyric acid)

99. “The culture medium contains tributyrin as a reactant;
degradation of this compound gives rise to clear zones
surrounding the lipolytic colonies in the otherwise turbid
culture medium.

100. Nitrate reduction Test

elt is used to
determine if an
organism is capable
of reducing nitrate
(NO, to nitrite (NO,)
or other nitrogenous
compounds via the
action of the enzyme
nitratase (also called
nitrate reductase).

eThis test is important
in the identification
of both Gram-
positive and Gram-
negative species.

Biochemistry within bacteria

uftandiic achd + NN 1 -naphtheytanine + nitrite Kons
(rotorless) feolorless)

= water + ullobenzene aro-N.N-cemethyl-1 -naphiteytarine:
wed colon

101. *After incubation, these Wipe,
tubes are first do
inspected for the sulf. acid =
presence of gas in the :
Durham tube.

ein case of non
fermenters, this is
indicative of reduction
of nitrate to nitrogen
gas. However, in many
cases gas is produced
by fermentation and |
further testing is|
necessary to determine
if reduction of nitrate
has occurred.

102. Nitrate reduction Test
+.

* The reduction of nitrate to Fi À
nitrite was detected with er os

dimethyl-a-naphthylamine

(Wallace & Neave, 1927)

and sulphanilic acid.

* The reaction was rapid
with all the species tested;
at 30 min. the results were
consistent with the usual
cultural method

Ev
85
us
ae
à
SE
= 5
3
DE
AE]
KY
SIR
MR,

103. Purpose: This test is to determine an
organism's ability to produce proteolytic-
like enzymes and liquefy gelatin.
Principle: Gelatin is to large to enter a
bacterial cell wall and thus extracellular
enzymes must catabolize them into smaller
components before they can be utilized.
Possession of these extracellular
gelatinases can aid in the differentiation of
bacteria
v This test is used to differentiate between
species in the genera Staphylococcus and
Clostridium as well as aid in the
identification of other species and genra.

POSITIVE TEST =
medium liquefied.
NEGATIVE TEST =
medium remains
solid.

104. Thanks

Acknowledgement: All the material/presentations available online on the
subject are duly acknowledged.
Disclaimer: The author bear no responsibility with regard to the source
and authenticity of the content.