EVIDENC

E
 BIOCHEMISTRIES
Subject: Microbiology
Teacher: María Azucena Márquez
Group: 6º A
Career: Biochemical Engineering
Members

-Márquez González Imelda

-Montes Arias Dennisse
-Segoviano Quintana Paulina

What are they

Biochemical tests in microbiology are a set
of chemical tests that are carried out based
on the metabolic characteristics of bacteria
for their identification. They generally
determine the activity of a metabolic pathway from a substrate that is incorporated into the culture
medium and which, when this microorganism develops, modifies it or not.
The final objective of this type of tests is to identify microorganisms that are identical at the genus
or species level (Rojas, 2011).

Fig.1. Catalase test in po

Commonly performed biochemical tests
Enzymes linked to respiration: catalase and coagulase o Decomposition of carbohydrates,
Decomposition of simple sugars, organic acids and amino acids and others
others.
Indole
o Oxygen requirements: OF (Oxide-
H2S
Fermentation) Phenylalanine
o Production of acid or acid and gas: Urea
Fermentation of carbohydrates o Detection of Decarboxylation of Lysine,
enzymes and metabolic pathways Unique source of Arginine, Ornithine
carbon Combined trials
o Citrate or TSI (Triple Iron three Sugars)
or Malonato o LIA(Lysine-Iron Agar) o Esculin
o Coliform hippurate Bile
Use of nitrogenous compounds or Nitrate reduction Detection of exoenzymes
Assimilation o Lectinase
Denitrification o Proteases, Coagulase
o Amylases o Cellulases o
Hemolysis
BIOCHEMICAL TESTS
(Bacteria)

PRIMARY TESTS
• Staining of GRAM .
• Proof ofcatalase .
• Proof ofoxidase .
• Proof ofoxidation/fermentation known as OF .

(Peréz Martínez, 2012)

 GRAM stain
IDENTIFY: GRAM + or GRAM – bacteria

Reagents:
•Distilled water (for smears).
❖ Crystal violet also known as gentian violet.
❖ Safranin.
❖ A 1:1 alcohol-acetone mixture.

Make smears.
(Peréz Martínez, 2012)
Let air dry
 ■ Crystal violet
■ Iodine Alcohol
■ Safranin

Techniq
ue: (G ; Application of crystal Application of iodine
(mordant)
€ Alcohol wash
(decolorization)
violet (purple dye)
0 Application of safranin (counterstain)

Result:

(Albo Domínguez,
CATALASE test
IDENTIFY: Bacteria with the presence of the catalase enzyme, for
example Staphylococcus aureus, Escherichia coli, Pseudomonas sp .

Reagents:
• 30% peroxide.

Technique:
• Place a drop of peroxide on a slide and then place a
sample of the isolated colony with a toothpick.

Results:
• If there are bubbles it means that the enzyme is present.

(Albo Domínguez,
2014)
OXIDASE test
IDENTIFY: Bacteria that are similar to each other, for example
Neisseria and Moraxella.

Reagent:
• Chromogenic dye (Tetramethyl-p-phenylenediamine, Dimethyl-
p-phenylenediamine, Indophenol).

Technique:
• A sample is taken from the isolated colony with a sterile
toothpick.
• The sample is placed in contact with the impregnated strip or
disk.
• A purple color must be observed in a period of no more than 30
seconds for it to be positive, otherwise the result is negative.

(Albo Domínguez,
2014)
OF test
Identifies: oxidative bacteria, fermenters of certain carbohydrates and facultative
anaerobes.

Reagents:
• OF agar (agar, peptone and bromothymol blue as pH
indicator).

Technique:
• In two tubes, using a loop, you take some bacteria
from an isolated colony. Both tubes are inoculated
by the sting method.
• One tube will be closed with the intention of
allowing some oxygen to enter, 1.5-2ml of sterile
mineral oil will be added to the second tube and the
lid will be closed completely, they will be incubated
for 24 hours.

(Albo Domínguez,
• Results:
OPEN TUBE SEALED TUBE INTERPRETATION

Yellow sell Oxidation (aerobic)

Yellow (facultative anaerobe) Yellow Fermentation

Green (strict anaerobe) Yellow Fermentation

sell sell Negative (NH3)

(Albo Domínguez, 2014)

Secondary biochemical tests.
• Simple: Citrate, Malonate, MR-VP, Nitrate
and Litmus Milk, urea.
• Multiple: TSI, LIA and SIM.
• Specials: Coagulase, Hyaluronidase and
Acriflavin.

(Albo Domínguez, 2014)

Simple
Citrate.

Function: The use of citrate as the only carbon source is a

useful test in the identification of Enterobacteriaceae. It is
detected in this medium, through the growth and
alkalinization of the medium. This increase in pH is observed
with the bromothymol blue indicator, which turns to pH 7.6.
Reagents:
• Simmons citrate agar.

Technique:
• Inoculate by seeding by exhaustion on the surface of the
agar slant.
• Incubate at 37ºC for 24 hours.

(Albo Domínguez, (+) (-)

2014)
Malonato.
• Malonate is an organic salt used as the sole carbon source. At the same time
that it uses ammonium sulfate as a N source, an increase in pH occurs due to the
formation of NaOH.
Function: Used to rule out enterobacteria.

Reagents:
• Malonate broth.
• Simmons tube.

Technique:
• Sow a sample of the same type of colony in the malonate broth and
in the Simmons tube.
• Incubate at 37°C for 24 hours.
• Positive (+)-Alkaline reaction (blue)
• Negative(+)-No color change (green)
(Albo Domínguez, 2014)
Methyl Red-Voges Proskauer Test
MR-VP.

Function:
• VP: Capacity of a microorganism to produce acetylmethylcarbinol
(acetoin), from the fermentation of glucose.
• RM: Check the ability of a microorganism to produce and
maintain stable acidic end products of glucose fermentation.
Reagents:
• Methyl red.
• Glucose broth.
Technique:
• Inoculate strains in both tubes.
• Incubate at 37°C for 24 hours.
• Add drops of methyl red to the MR tube and do not shake.
Nitrate.

Identifies: the presence of nitrate reductase and nitrite reductase.

Reagents:
• Reagent A (0.8% sulfaminic acid in 5N glacial acetic acid)
• Reagent B (0.5% α-naphthylamine in 5N glacial acetic acid).
• Nutrient nitrate broth.

Technique:
• A culture medium is inoculated with a bacteriological loop by
direct sting.
• It is incubated at 37ºC for 24 hours and after incubation two
drops of reagents A and B are added.

Nitrates (-) (+)

Litmus Milk.

Identify: the ability of certain

microorganisms to produce certain metabolic
reactions in milk media.

Reagents:
• Iridescent milk pH = 6.8.

CLOT
Technique:
• Inoculate the tubes with a bacteriological loop.
• Incubate 35-37° C in aerobiosis or
anaerobiosis, depending on the microorganism
studied, for twenty-four to forty-eight hours.
Longer incubation periods may be necessary.
TSI (Triple Sugar Iron Agar)
It is a nutrient medium that allows studying the capacity for the production of acid and gas
from glucose, sucrose and lactose and for the production of hydrogen sulfide.
Thanks to its composition, it is one of the most used culture media for
the differentiation of Enterobacteriaceae.

Incubation at 35º for 18-24 hours

Results
• Acidic taco (yellow): Fermented glucose.
• Acid bevel (yellow): Fermented lactose and/or sucrose.
• Acidic taco (yellow): Fermented glucose.
• Alkaline bezel (red): Unfermented lactose and/or sucrose.
• Alkaline block (red) and alkaline bezel (red): None of the three
carbohydrates is fermented TSI test
a) G, S and L fermented, gas production
• The appearance of bubbles in the block indicates that the b)Fermented glucose
fermentation has been carried out with gas production. c) Production of hydrogen sulfide
• A blackening in the medium indicates the production of hydrogen d)No fermented sugar
sulfide . (Rojas,2011)
• This test allows us to differentiate microorganisms that produce decarboxylation
or deamination of lysine, as well as the production of H2S.

LIA (Lysine on
agar)
• In the first stages of incubation, the medium turns the pH indicator
to acidic (yellow), due to glucose fermentation. If the amino acid is
decarboxylated, amines will be formed that cause a return to the
original color or towards a basic color (violet).
• Results
• Alkaline bottom/alkaline bottom: Decarboxylated lysine
• Alkaline base/acid base (non-decarboxylated lysine)
• Red bevel/acid background (deaminated lysine)

A) Uninoculated medium
B) Positive decarboxylation (+)
C)Decarboxylation (+) and H2S
(+)
D)Decarboxylation (-)
E)Deaminated lysine
(Rojas,2011
)
SIM (Indole Mobility Sulfide)
• Indole is a metabolic degradation product of tryptophan.
Bacteria that have the enzyme tryptophanase are capable
of hydrolyzing tryptophan into indole, pyruvic acid and
ammonia.
• The formation of a red complex occurs when indole reacts
with Kovac's reagent.
• Medium used: Rich in tryptophan
• Incubation at 35º for 18-24 hours
Positive(+)-Cherry red ring formation
Negative(-)-No color change Indole test in Proof (-)
tryptophan broth. A(+) Test (+)
Mobility: The culture medium has a consistency B(-)
semisolid, mobility is observed by the growth of
microorganisms throughout the tube.
Coagulase
The coagulase test consists of knowing whether the bacteria analyzed
contains the enzyme coagulase or not.
Coagulase is an enzyme that converts fibrinogen to fibrin. For this we
use human plasma that contains fibrinogen. This test is widely used
when it is suspected that the bacteria you have is Staphylococcus
species.
Medium: Rabbit plasma
Incubation 37º for 4 h
 Proof Medium/reagent Planting procedure
Sulfide Production. SIM agar Sowing by puncture.
Indole production. SIM agar Sowing by puncture.
Mobility (motility:. SIM agar Sowing by puncture.
Fermentation AgarTSI. Mixed sowing.
Of carbohydrates. Dephenol + carbohydrate broth. Inoculation of the medium with a loop loop
Citrate. Simmons Citrate Agar. Mixed sowing.
RM-VP broth. Inoculation of the medium with a ring loop.
Mello Red - Voges Proskauer. RM-VP broth. Inoculation of the medium with a ring loop.
Urease. Urea Broth. Inoculation of the medium with a ring loop.
Lysine decarboxylation. LIA agar. Mixed sowing.
Nitrate reduction. Nitrate broth. Inoculation of the medium with a ring loop.
On a clean slide, smear a colony of the microorganism and add 2
Catalase. Hydrogen peroxide 3%.
drops of the reagent.
Rub a colony of the microorganism on the reagent and make
Oxidase.
Oxidase test strips (or ampoules). observations.
Gelatin hydrolysis. Jelly. Sowing by puncture.
Inoculate the microorganism in 0.5mL of EHI broth and incubate at
Coagulase. EHI broth and human or rabbit plasma. 37°C/18-24h; Once the incubation is completed, add 0.5mL of
plasma and incubate at 37°C/4hr (-observe until 18-24h).
Sow by exhaustion and incubate at 370C/24h.
Starch hydrolysis. Gram Starch and Lugol Agar. Once incubation is complete, flood the boxes with Gram's Lugol.
Sow the microorganism by exhaustion and incubate at 37°C/24h.
Hemolysis. Blood Agar.
Nutrient Agar added with Skim Milk. Sow by exhaustion and incubate at 37°C/24h.
Casein
Sow by exhaustion and incubate at 370C/24h.
Desoxi rri bon ucleas a. DNAse agar.
At the end of the incubation time, flood the box with 1M HCL.
Sow the microorganism massively with a sterile swab. Soak each
sensidisc with a different antibiotic and distribute them evenly on the
Müeller Hinton agar, Sensidisks and surface of the box, marking the antibiotic used on the back of the
Antibiogram.
antibiotics. box. Incubate at 37°C/24h.
19Z Microbiological diagnosis
TABLE 2-8. Biochemical reactions for I a Lmuaas
identification of enterobacteria (Edwards and Ewing)
Enterobacteriaceae

ColOv Cke\ mecLO

YDLOQUOIC ®UEO
1

-YO1 1 Thousand f
Tribe
Escherichieae Edwardsielleae Salmonellae
Co Vuo mnccto voetoclo of Klebsielleae
ColOv owcINA) d¿\ Klebsiellea e Proteeae

SerD ©Enterobacter Enterobacter be rati Pee tuff cterium Proteus. Providence

o COecl' o
Test or substrate
Escherichia Shigella Edwardsiella Salmonella Arizona Ci trobacter Klebsiella • hafniae^í / iquefaciens
cloacae aerogent". mirabilis morganii rettger and alcalifaciens stuartii
■ VOSC
37°C 22°C 37° < c 22°C 25°C

What Indo!
OleGsa
4- — • +• -
oeo broth
- — O.T.
OgocoD
- - - - - -- - either - - - Yo 4- + +

Methyl red + •+ + + - - + or - + or • — — or + — or + + or — + + 4- + + +

Voges-Proskauer - - - - - - + + or — + - either +- + or — + — or + - — or '+ - - - -

O Simmons Citrate
PocOCC\O©
- - I know
- d l - +-f Q oav1Wo
( + ) OR —
d
PG)O
+ + +- d d -+ or (+) - + + +
Hydrogen sulfide (TSI) Yes\ chopped (Or
SUcnco
- - - + - either - - - - - - -• + +- - .- - -

0 Urease —. - — - — • d- —' + or -- -—- —- d

- to.
d- d“ + 4- + +
AND —
KCN - - - - - + + + + + + + + or — 4- + + + + +
C Mobility

PeSC
+ or - - -- Yo + 4- - +" + +■ + d + + + or — •b + + *+ 4- +

Gelatin (22 C) - - - - t+) - - ( - ) either - — or ( + )

O OCINO - + + + OR ( + ) + OR ( + ) + -■ - -'. -

Lysine decarboxylase d
{oco • -
YE +
PCOCOVO
+ + + or — + + - - - - - .-

Arginine dihydrolase d - either () - (+) or + + O (4- ) d - + •

- - - - - — or + - - - - -’

Ornithine decarboxylase d d* + + d ++ O +mCwVO

+ oJC +- + + toxoncez OlvececOn
- Ce lo VcoCur
4- +. - - -

c Phenyalanine deaminase - - - • - - - - - - - + + + $ -+

Malonate Motility
- - - - $M + d - PcoCcco
+ or - + or + or — + or — - - - — or ♦- - - - -
a+
-

-o Gas from glucose Not Idac

* + + mo l + +
OICOCOCC
++ + + + + + or — or +
1ovOdo2+ oralCedO\
— +
0c the
d
DcoCUC
- either +. 4-0 —
-^Lactose
* -’ - d 'dz - ++ - either. ( + ) — or-(+) d (+)
OnWOOyo OK
- or (+) d
codwo Cc
-
(oOC- - - - -

G Sucrose

Mannitol
d

+
1nd) + or —
-♦

- +
Mlio -

+
d

+
OcoCOC
++
++ d

+
d

+
+

+
+

+
+

+
+

4-
+

-
d

-
-

-. + or
d

-
.d d

Dulcitol d d - d*'* - d —O + — or + - - - -
Se\c
-
Q+oc I•-CCO moved
- - - -

Salicin d - - - - d + D,COGvO p
+ or ( + ) + d d + + + 4- d d - d - -

Adonitol z - - -
CAvoo eke
- - - + or
KCCo
— or +
VC y + - -
Uevde d d d CICU) - - - -■
■
d + -

Inositol - - - d - - + d + - - + + d - - _--------------- + - 4-
—in+- eke CObOno Dodo
DcocoCO V
Sorbitol d - + + + + ++ - - + + + - - - d - d
Citoo Ce +
Arabinose

Raffinose
with+ CLQ
d
d

d -
-

- SivOD- 5
-+

d
c$r© YEc+C
+

+
++

++
+-

-
+

-
+

-+
+

+
-

-
++

+ OR ( + )
- -

-
-
-
-

.-
-

-
-

Ramnosa d covlaanin©
d - +
oiolonnO + + S5MO
+ ev +210-ZaO
+ + +- - - - U€XOed - - -• + or - -
Cscthino 20
Certain biotypes of S. flcxneri produce gas; ios cultures of S. sonnt^i ferment lactose and sucrose slowly and decarboxylate ornithine.
t The species of 5. typhosa, S. choleraesuis. S. enteritidis bioser. Paratyphi A and Pullorum, and a few others, usually do not rapidly ferment dulcitol. The 5. choteraesuis does not ferment arabinose. +.90% or more positive in 1 or 2 days; .90% or more negative; d, different biochemical types 1 + ,( + ), - 1; (+), late positive; +o . majority of positive cultures; — or +, negative majority; w, weakly positive

OmC1O or
reaction.
+ The volumes of gas produced by Serra crops (a. Proteus and Providencia are small.
Qcodovo estTO Courtesy of; Enteric Section, Enterobacteriaceae Branch, Bacteriology Division, CDC, Atlanta, Ga.
a separate entity, but has been regrouped with Salmonella as the genus Proteus with the designation Proteus inconstans. This is due to the utilization of carbohydrates by Yersinia through the first that in 1894 he identified this bac as the cause of the
subspecies Salmonella 4. The Yersinia t group only recently separated from the genus
eN210-Z00 fermentative rather than oxidative pathway, and the lack of cytochrome plague. Ea Yersinia) formerly Pasteurella pestis, is the type
<n i.'.onae. Pasteurella, it has now been incorporated into the Pmterobacteriaceae oxidase activity. species currently endemic in rodents, with sporadic human
3. The Providencia group, including Provi-Jcnce alcalifaciens family. In 1954, Fhal proposed this reclassification based on
in (O)O
The Yersinia group gets its name from the microphone French infections reported annually, particularly in the southwest.

the
locOdciosccovo>c
and Providozce stuartii, is no longer known as such, but is included in biologist Alexander Yersin, who was the

- PococcCD V\
Software for identification through biochemical
tests (ERIC-Electronic RapID Compnedium)
Bacterial identification gallery, where the code for the type of
bacteria comes
bacteria, product (chemical tests) and presentation (galleries).
You are here: Home i Bacterial Identification Galleries

Microbiology Menu

Start
€ Bacterial Identification Galleries
Waters Size: EEIE
Agars and Peptones

Allergens in Food and

Drinks

Anaerobiosis

An tibí ograms

Antisera

Prepared Bags

Prepared Bottles

Pattern Strains

Autoclave control One-Step Inoculation Results in 4 hours

Interlaboratory and Intercomparative
Tests Summary table of galleries in pdf List of identified microorganisms
Bacterial Identification Galleries
Download the "ERJC" identification program
Identification Latex

OXOID Manual ERIC Program User Manual

Dehydrated Media

Media with ISO Formulation

IDENTIFICATION OF RAPID ENTEROBACTERIA:
Media by Microorganism ISO Standard
Code Product Presentation
Methods USDA-FSIS Methods
RS311006
Parasitology Pathogens by PCR
RAPID ONE PANEL PACK OF 20 20 galleries
Peptones and Hydrolysis of Prepared
R8325106 RAPID INOC FLUID (2ML) PACK OF 20 TUBES 20 tubes
Plates and Prepared Tubes
R8309002 RAPID SPOT INDOLE RGT 15ML 15mL
Herds of Cnntarte and MB026BA MICROBACT OXIDASE STRIPS 50 strips
R20412 MCFARLAND TURBIDITY STD 2 Unit
Access to the online identification program.
SOFTWARE
IDENTIFICATION OF MICROBACT ENTEROBACTERIA:
Code Product Presentation
MB1082A MICROBACT REAGENT SET D (Indole, VPI, VPII. kit
TDA, NIT A/B)
SOFTWARE

Download the Microbact 2009 program (ZIP file). Access to the online identification program.
Color code of the gallery wells. Work protocol and list of microorganisms.

LISTERIA IDENTIFICATION:
Code Product Presentation
MB1128A MICROBACT 12L 20 IDENTIFICATIONS 20 galleries
MB1249A HAEMOLYSIN REAGENTS mL 5mL
SOFTWARE Download the Microbact 2009 program (ZIP file).
Access to the online identification program.
Color code of the gallery wells.

IDENTIFICATION OF ANAEROBIC:
Code Product Presentation
R8311002 RAPID ANA II PANEL PACK OF 20 20 galenas
RS325102 RAPID INOC FLUID (1ML) PACK OF 20 TUBES 20 tubes
R8309002 RAPID SPOT INDOLE RGT 15ML 15mL
R20413 MCFARLAND TURBIDITY STD 3 Unit
SOFTWARE Access to the online identification program.

IDENTIFICATION OF CORYNEBACTERIA
Code Product Presentation
R&311OO8 RAPID CB PLUS PANEL PACK OF 2D 20 galenas
RS325106 RAPID INOC FLUID (2ML) PACK OF 20 TUBES 20 tubes
R8309003 RAPID NITRATE A REAGENT 1 5ML 15mL
RS309004 RAPID NITRATE B REAGENT 1 5ML 15mL
R20414 MCFARLAND TURBIDITY STD 4 Unit
SOFTWARE Access to the online identification program.

IDENTIFICATION OF NEISSERIA, HAEMOPHILUS

Code Product Presentation
• References:
• Koneman. (2006). “ Bacteriological diagnosis, text and color atlas” 6ed.
Editorial Panamericana , Argentina.
• Albo Domínguez Jeannette. (20014). “Notes on identifying
microorganisms based on bacteriological techniques.” CBTis 65
• Peréz Martínez María De la Cruz. (2012). “Biochemical tests for the
identification of bacteria of clinical importance” 3ed. Panarmericana
Publishing House, Argentina.
• Rojas (2011) Concepts and practice of general microbiology. UNC
Palmira
• OXOID (2012) Bacterial identification. [place
web]( )
http://www.analisisavanzados.com/index.php/galerias-de- identification-bacteriana